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British Pharmacopoeia Veterinaria PDF
British Pharmacopoeia Veterinaria PDF
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British Pharmacopoeia
(Veterinary) 2013
see Natices
NOTICES
PREFACE
BRITISH PHARMACOPOEIA COMMISSION
EXPERT ADVISORY GROUPS, Pl\NELS OF EXPERTS AND
WORKING PARTIES
CODE OF PRACTICE
MEMBERSHIP
BP Commission, EAGs, Panels and W orking Parties
STAFF
British Pharmacopoeia, BP Laboratory, Publisher
INTRODUCTION
Additions, Omissions, Technical Changes, Reference Substances
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs
Immunological Products
Surgical Materials
INFRARED REFERENCE SPECTRA
APPENDICES
INDEX
Notices
Patents In this Pharmacopoeia certain drugs and preparations have been included
notwithstanding the existence of actual or potential patent rights. In so far
as such substances are protected by Letters Patent their inclusion in this
Pharmacopoeia neither conveys, nor implies, licence to manufacture.
Effective dates New and revised monographs of national origin enter into force on
1 January 2013. Monographs of the European Pharmacopoeia have
previously been published by the Council of Europe and have been
brought into effect by means of N otices published in the Belfast,
Edinburgh and London Gazettes.
vi
Preface
vii
British Pharntacopoeia
Contntission
(a) the preparation under section 99(1) of the Act of any new edition of
the British Pharmacopoeia;
(b) the preparation under section 99 (1) of the Act, as given effect by
section 102(1) thereof, of any amendments of the edition of the
British Pharmacopoeia published in 1968 or any new edition of it;
(c) the preparation under section 100 of the Act (which provides for the
preparation and publication of lists of names to be used as headings
to monographs in the British Pharmacopoeia) of any list of names
and the preparation under that section as given effect by section
102(3) of the Act of any amendments of any published list;
(d) the preparation under section 99(6) of the Act, of any compendium,
or any new edition thereof, containing information relating to
substances and articles which are or may be used in the practice of
veterinary medicine or veterinary surgery;
viii
Expert Advisory Groups, Panels
of Experts and W orking Parties
IX
Code of Practice
x
Metnbership of the British
Phartnacopoeia Cotntnission
The list below ,¡includes those members who served during the period 2011
to 2012" \
Xl
Dr Brian R Matthews BPharm PhD FRPharmS FTOPRA MRI
Consultant on pharmaceutical and medical device regulatory affairs)' former Senior
Director) BC Registration) Aleon Laboratories
xii
Metnbership of Exp~rt Advisory
Groups, Panels of E,tperts and
Working Parties
xiii
PANELS OF EXPERTS
BIO: Biological and L Tsang (Chair), M A Dow (Vice-Chair), A F Bristow, D H Calam,
Biotechnological J Cook, L Findlay, A Onadipe, B Patel, A M Pickett, C Ponsar, 1 Rees,
Products D Sesardic, P Sheppard, W J Tarbit, J N A Tettey, AH Thomas,
R Thorpe
BLP: Blood K. Chidwick, A R Hubbard, S Jenkins, P Varley
Products
IGC: Inorganic and C T Goddard (Chair), A C Cartwright, N Fox, P Henrys, D Malpas,
General Chemicals C Mroz, D Riches
MIC: Microbiology V Fenton-May (Chair), S Denyer, D P Hargreaves, B R Matthews,
P Newby
RAD: Radioactive J Ballinger, J Brain, D Graham, S R Hesslewood, G Inwards, P Maltby,
Materials A M Millar, R D Pickett, R Smith, S Waters
VET: Veterinary E Williamson (Chair), A Cairns, S Cockbill, D Evans, E Flahive, P Lees,
Medicines B Ward
VIP: Veterinary A M Brady, K. Redhead, J Salt, P W Wells
Immunological
Products
WORKING PARTIES
CX: Excipients G Buckton (Chair), C Mroz (Vice-Chair), E Anno, R Cawthorne,
B R Matthews, M 1 Robertson, K. Slevin
IP: Inhaled Products S C Nichols (Chair), y Adjibade, M Dagli Alberi, J Lim, J Qiu, 1 Vaughan
XIV
Current British Pharrnacopoeia
/
Staff ~
ISO 9001
F527268
xv
Current British Phannacopoeia
Laboratory Staff
ISO 9001
( F527613
xvi
Current Staff of the Publisher of
the British Phartnacopoeia
1509001
F522428
xvii
Introduction xix
Introduction
This edition, together with the British Pharmacopoeia 2013, contains all the
monographs of the 7th Edition of the European Pharmacopoeia as amended
by Supplements 7.1 to 7.5. U sers of the British Pharmacopoeia and British
Pharmacopoeia (Veterinary) therefore benefit by finding within these two
compendia all current pharmacopoeial standards for veterinary medicines
used within the United Kingdom.
Effective Date The effective date for this edition is 1 }anuary 2013.
The British Pharmacopoeia General N otices (Part II) have been amended
as follows.
Labelling
The statement concerning Veterinary Medicines Regulations 2007 in this
General Notice has been amended to refer to current Veterinary Medicines
Regulations. The statement concerning best practice guidance has been
amended to delete reference to "Veterinary Medicines Guidance Note 26:
Marketing Authorisations - Legislative updates to SPCs and Product
Literature" .
Part III
The British Pharmacopoeia General N otices (Part III) have been amended
to harmonise with the changes published in Supplement 7.5 of the 7th
edition of the European Pharmacopoeia.
Additions A list of monographs included for the first time in the British
Pharmacopoeia (Veterinary) 2013 is given at the end of this Introduction.
It includes 1 new monograph reproduced from the 7th Edition of the
European Pharmacopoeia and 2 new national monographs for formulated
preparations.
Revisions The requirement for Specific optical rotation has been amended in the
monograph for Deltamethrin. The monograph for Cefalotin Sodium has
been revised to refer to the use of British Pharmacopoeia Chemical
Reference Substances.
Labelling Requirements
Infrared Reference As with the previous edition, the reference spectra are placed in alphabetical
Spectra order withiri. this edition.
European All monographs of the 7th Edition of the European Pharmacopoeia, which
Pharmacopoeia are used in veterinary practice but not normally in human medicine in the
United Kingdom, are reproduced in this edition of the British
Pharmacopoeia (Veterinary). Each of these monographs is signified by a
European chaplet of stars alongside its title. Additionally, reference to the
European Pharmacopoeia monograph number is included immediately
below the title in italics in the form 'Ph Eur monograph xxxx'. Where the
title in the British Pharmacopoeia is different from that in the European
Pharmacopoeia, an approved synonym has been created (see Appendix
XXI B (Vet)) and the European Pharmacopoeia title is included before the
monograph number. The entire Europeart Pharmacopoeia text is delineated
by two horizontallines bearing the symbol 'Ph Eur'.
Code of Practice Members of the British Pharmacopoeia Commission and its supporting
Expert Advisory Groups, Panels of Experts and W orking Parties are
required to comply with aCode of Practice on Declaration of Interests in
the pharmaceutical industry. Details of the Code are published on the
website (www.mhra.gov.uk/pharmacopoeia).
xxii Introduction
Additions The following monographs are new addi~ions to the British Pharmacopoeia
(Veterinary) 2013. '
Technical Changes The following monograph in the British Pharmacopoeia (Veterinary) 2013
has been technically amended since the publication of the British
Pharmacopoeia (Veterinary) 2012. This list does not include revised
monographs of the European Pharmacopoeia. An indication of the nature
of the change or the section of the monograph that has been changed is
given in italic type in the right hand column.
Cefalotin Sodium
General N otices 1
General Notices
2 General N otices
General N otices
Partl
,PartII
Definition ofTerms Where the term 'about' is included in a monograph or test it should be
taken to mean approximately (fairly correct or accurate; near to the actual
value).
Where the term 'corresponds' is included in a monograph or test it
shouldbe taken tornean similar or equivalent in character or quantity.
Where the term 'similar' isincludedin a monograph or test it shouldbe
taken to mean alike thoughnot necessarily identical.
Further qualifiers (such as numerical acceptance criteria) for the above
terms are. not includedin the BP (Vet). The acceptance criteria for any
individual case are set based on the range of results obtained from known
reference· samples, the level of precision of the equipment or apparatus used
and the .levelof accuracy required for the particular application. The user
should determine the variability seen in his/he! own laboratory and set in...
house acceptance criteria that he/she judges to be appropriate based on the
local operating conditions.
6 General N otices
Weights and The metric system of weights and mea8ure8 is employed; SI Units have
Measures generally been adopted. Metric ll1easures are required to have been
graduated at 20° andall mea8ure1l1entsinvolved in the analytical operations
ofthe Phatmacopoeiaare .intended~unlessotherwisestated;. to bemade at
that temperature.Graduateqglassapparatus usedin analyticaloperations
shouldc01l1plywithClas8 Arequirements.oftheappropriate IntemationaI
Standard issued bythe International. Organ.ization forStandardization.The
abbreviation for litre is.'L' ·throughout .thePharll1acopoeia.. In ·line. with
European Directive 801181/EEC, the abbreviation '1' i8 also perll1ittedfor
U8e~
Atomic· Weights The atomic. weightsad()ptedare the values . given in the Table ofRelative
Atomic Weights 2001.·published by thelnternational.Union ·Qf Pureand
Appl1ed Chemistry(Appendix XXV).
The term 'water bath'means a bath of boiling water, unless water at sorne
other temperature isindicated in the texto An alternative form of heating
may. be employed providing that the required temperature is approximately
maintained but notexceeded.
The reagents required for the assays and tests of the Pharmacopoeia are
defined in appendices~ The descriptions set out in the appendices do not
imply that the. mate'rials aresuitable for use in medicine.
Indicators, the coloursof which change over approximately the same range
ofpH,may be substituted forone another but in the event of doubt or
dispute as to. the equivalenceof indicators for a particular purpose, the
indicator specifiedjl1the text is alone authoritative.
The·quantity ofanindicatorsolution appropriate for use in acid-base
titrations. described in assays or· tests is 0.1 mL unless otherwise stated in
the texto
An,y solventrequired in an assay or test in which an indicator is· specified
is previously neutralisedtÓ theindicator, unless a blank test is prescribed.
Titles Subsidiary tides,where inc1uded, have the same significance as the main
tides. An abbreviated title constructed in accordance with the directions
givenihAppelldixXXI Ahasthe same significance as the main titie.
Titles thatate derivedby thesuitable inversion of words of amain or
subsidiary title,withthe additionof a preposition if appropriate, are also
officiaLtitles. Thus, the Iollowing are aH official tides: Acepromazine
Tahlets,· Tablets of Acepro111azine; Catechu Tincture, Tincture of Catechu;
Levamis(}le Injection, Injectionof Levamisole.
A title ofa formulatedpreparation that includes the full nonproprietary
nameofthe activeingredient or ingredients, where this is not included in
the titleof the monograph,· is also an official title. F or example, the title
Acepro111azine Maleate. Injection· has the same significance as Acepromazine
8 General Notices
Chemical Formulae When the chemical composition of an official substance is known or generally
accepted,the graphic andmolecular formulae, themolecular weight andthe
Chemical Abstracts Service RegistryNumber are normalIy givenat the
beginning ofthe monographforiríforn:ultion. Thisinformationrefers to the
chemically Ptlresubstanceandis119ttO· be regardedas· an indicationof the
puri~ofthe •.• official . material.Elsewl1ere,in.statement$.ofstandardsofpurity
andstren~fhand indescfipti~~80f processes ofa8say, it isevident from the
contextthat. theformulae del1otedlec~emieallypure .substances.
Wheretheabsolute.stereoche~icalconfiguration is specified, ·.the
International Unio~ofPureandAl'pliedChemistry (IUPAC}R/S and E/Z
systems of designationhavebeetl used. If the substance. is an enantiomer of
1,lnlmQ wn.ahsolll.te •stereQ(;hel11istrythesign oí the. opticalrotation, as
determined in the solvent~ndunderthe conditions .specified in the
monograph,· has beenattacp.edtothe systematicname. An indication of
signof rotation hasalso beengiven where this 18 iricorporated in a trivial
námethat appearson anIUPACpreferred listo
Allaminoadds; exceptglydne,havethe L-configuratíon Unless otherwise
indicated. The three;.letterandone.-Ietter symbols used for amino acids in
peptide andprotéirisequences arerhose recommended by the Joint
Cornmission on BiochetnicalNomenclatttre ofthe International· Uníon of
Pure. andAppliedChemistryandthe Intemational Union ofBiochemistry
andMolecularBiology.
Inthe graphic fomiuláe the foUowing abbreviations are used:
Me -CH) BuS -.:CH(CH 3)CH2 CH3
Et -CH2 CH3 Blln -CH 2 CH2 CH 2 CH3
Pri -"-CH(CH3 h But -C(CH3h
Prn -.:CH2 CH2 CH3 Ph -C 6Hs
Bui .,..CH2 CH(CH 3)2 Ac -COCH3
Colouring Agents If in a monograph for a formulated preparation defined by means ofa full
formula a specific colouring agent or agents is prescribed, suitable
alternatives approved in the country concemed may be substituted.
Identification The tests described or referred tounder the side-heading Identification are
not necessarily sufficient to establish absolute proof of identity. They
12 General N otices
Assays and Tests The assays and tests described are the official methods upon whieh the
standards of the Pharmacopoeia dependo The analyst is not prec1uded from
employing alternative methods, inc1uding methods of micro-analysis,in any
assay or testif itis known that the method used will give a result of
equivalent accura(2y~ Localreference materials may be used for routine
analysis, provided that these are calibrated against the official reference
materials~ In the eventof doubt or dispute, tbe methods of analysis, the
reference materials andthereferencespectra of the Pharmacopoeia are
alone authoritative.
Where the solvent used for a solution isuot named, the solvent is
Purified.Water.
Unless otherwise· prescribed, the assays and tests are carried out at a
temperaturebetween 15° and 25°.
A temperature.in a test for Loss on drying, where no temperature range
is given, implies· a range of ± 2° about the stated value.
Visual comparative tests, unless otherwise prescribed, are carried out
using identical tubes of colourless, .transparent, neutral glass with a fIat base
and· an internal diameter of 16· mm; tubes with a larger internal diameter
may be used but· the volume of liquiq_examined must be· increa.sed so tbat
the depthof liquidin thetubeis not less than that obtained when tbe
prescribed volume. of liquid and tubes 16 mm in internal diameter are. used.
Equal vdlumes of the liquids to be compared are examined down tbe
vertiealaxis of the tubes againstawhite background or, if necessary, against
a black background. The examination is carried out in diffuse light.
Where a direction is. given that an analytical operation is to. be carried out
'in subdued light', precautionsshould be taken to avoid exposure to direct
sunlight or other· strong light. Where a direction is given that an analytical
operation is to be carried out 'protected from light', precautions should be
taken to exc1udeactinic light by the use of low-actíníc glassware, workingin
a dark roomor similar procedures.
For preparations other than those of fixed strength, the quantity to be
taken for an assay or test is usualIy expressed in terms of the active
ingrediente This· means that the quantity of the active ingredient expected to
be present and the quantity ofthe preparatíon to be taken are calculated
from the strength stated on the labe!'
General N atices 13
Biological Assays Methods of assay described as Suggested methods are not obligatory, but
and Tests when another method is used its precision must be not les s than that
required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological
assay the potencyrequirement· is expressed in the monograph in
International Units (IU) or other Units per milligram. The material is not
of pharmacopoeial quality if the upper fiduciallimit of error is less than the
stated potency. For such antibiotics the required precision of the assay is
stated in the monograph in terms of the fiducial limits of error about the
estimated potency.
For other substances and preparations for which the monograph specifies
a biological assay,unless otherwise stated, the precision of the assay is such
that the fiduciallimits of error, expressed as a percentage of the estimated
potency, are within a range not wider than that obtained by multiplying by
a factor of ten the square roots of the limits given in the monograph for the
fiduciallimits of error about the stated potency.
In aIl cases fiducial limits of error are based on a probability of 95% (P =
0.95).
Where the biological assay is being used to ascertain the purity of the
material, the stated potency means the potency stated on the labe1 in terms
of International Uníts (IV) or other Units per gram, per milligram or per
millilitre. When no such statement appears on the labe1, the stated
potencymeans the fixed or mínimum potency required in the monograph.
This interpretation of stated potency applies in aH cases except where the
monograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in
the container, the stated potency means the total number of Intemational
Units (IU) ototherUnits stated on the label or, if no such statement
appears, the total activity ca1culated in accordánce with the instructions in
the monograph.
Wherever possible the primary standard used in an assay or test is the
respective International Standard or Reference Preparation established by
the W orld Health Organization for international use and the biological
activity is expressed in International Units (IU).
In other cases, where Units are referred to in an assay or test, the Unit
for a particular substance or preparation is, for the United Kingdom,the
specific biological. activity contained in such an amount of the respective
primary standard as the appropriate international or national organisation
indicates. The necessary information is provided with the primary standard.
Unless útherwise· directed, animals used in an assay or a test are healthy
animal s, drawn from a uniform stock, that have not previously been treated
with any material that will interfere with the assay or test. Unless otherwise
stated, guinea-pigs weigh not less than 250 g or, when used in systemic
toxicity tests, not less than 350 g. When used in skin tests they are white or
General N otices 15
light coloured. Unless otherwise stated, miee weigh not less than 17 g and
not more than· 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are sueh
that in .the U nitedKingdom theymay be .earried out only in accordance
withthe Animal s (Scientific Procedures). Act 1986. Instructions included in
such assaysand tests in the PharmacQpoeia, with respect to the handling of
animals, are· thereforé corif1ned· to·· those concerned with· the accuracy and
reproducibility of theassayor test.
Action and Use The statements given under this side-heading in monographs are intended
only. as .information on the principal pharmacological actions or the uses of
thematerials in veterinary medicine or pharmacy. It should not be. assumed
that the substance has no other action or use. The. statements are not
intended to be binding on prescribers or to limit their discretion.
General N otices 17
Antibiotics Intended Where a monograph for an antibiotic in the British Pharmacopoeia 01' in the
for Use in the British Pharmacopoeia (Veterinary) contains specific requirements relating
Manufacture of to sterility 01' toabnormal toxicity for material intended .for use in the
Intramammary manufacture of a parenteral dosage form,these requirements, together with
Infusions any qualification, apply also to any material intended for use in the
manufactureof an intl'amammaryinfusion.
Crude Drugs; Herbal and complementary medídnesare classed as medicines under Buropean
Traditional Herbal Directive· 2001/83/BC as amended. It is emphasised that, although requirements
and. Complementary forthequalítyofthe material are provided in the monograph to assíst the
Medicines registratíon schemcby theUK LicensingAttthority,the British Pharmacopoeia
Commissionhas not assessed the sáfety orefficacy ofthe material intraditional
use.
Monógraph Títle For tl'aditional hel'bal medicines,. themonograph title
i8 a combination of thebinomial name togethel' with a description ofuse.
Monographs Jol' the materialthat has not· beenprocessed (the herbal drug)
and the processedmaterial(the herbaldrug preparation) arepublished
wherepossible .. To distinguishbetween the two, the \VOl'd'Processed' is
included in the<relevantmonograph title.
DefinítionUnder the headingDefinition,the botanical name together
withanysynonyrnis given.Whereappropriate, formaterial that has not
beenprocessed, information On thecol1ectionlharvesting .and/ol' treatmentl
dryingof tl1ewholeherhaldrugmaybe gíven.For processed materials, th~
method ofprocessing,wh~reaPPtopriate, wiU normallybe givenin a
separate.s~ction.
Characteristícs References. to· odourare .included only where this is
highly characteristic.References totastearenot inc1uded.
ControlmethodsWl1ere applicable,thecontrol methods to be used in
mqnographs· are:
Ca) macroscopical·· and .11licroscopical~e8criptionsand chemical/
chromatographic testsforidentification
(b) tests forabsence of anyre1atecispecies
Cc) microbíal testtoass4re microbialquality
(d) tests for inorganic impuritiesandIlon-specific puríty tests, íncluding
extractíve tests Sulfaterlash and Heavy metals where appropriate
j
Homoeopathic
Medicines
appropriate.
General N atices 19
Part III
Quality systems The quality standards represented by monographs are valid only where the
artides in question are produced within the framework of a suitable quality
system.
Validation of The test methods given in monographs and' general chapters have heen
pharmacopoeial validated in·accordance with accepted scientific practice and current
methods recommendationsonanalyticalvalidation.Unless otherwise stated' in the
monograph or general chapter, validation of the test methodsbythe ana.lyst
is not required.
The•.·• terms . .·.'dried.·. •~()·• •.•·c~nstant• ·.·.m~§·s' ··and .•. 'igpited·· ·to.•.·constant. mass'.·· •.·m~an
that2sonsect1tivY.\\1eighipg~d9 not .differby l11,ore.than· 0.5 11lg;theZ
nd
Where !he name ()f the solvent is notstated, the term 'solution' impliesa
solution.·in.water.
Whel'e fue use of waterisspecified or i11lPliedinthe analytital
procedures .described· inthePharmacopoeiaor for fuepreparation· of
reagents, water complying with therequitements of themonograph Purified
water (0008) is used.;except thatfor many purposes the requirements for
bacterialendotoxins (Purifiedwaterin bulk)an4 microbial contamil1ation
(Purified water in containers) are n()t relevant. The terro 'distilled water'
indicates purified waterprepared by distillation.
General N otices 23
cold orcool;8°Cto15?C;
rQoil1tempera'tl.lte:15°C tÚ 25 oC.
1~4oi··MONOGRAPHS
RelánveAtóiriicand The relátiveat0111ic ·ll1ass(Ar) ·or the te1ative molecular mas s (Mr ) is shown,
Molecl1.1a:tMásses asandwheteapptopriate,atthe beginning of eaeh monograph. The relative
atomic andmolecular massesand the molecular and graphic formulae do
nofconstitute anaJYtical· staridardsfotthe stibstances described.
24 General N otices
products and finished products, where relevant). The absence of this section
do es not imply that attention to features such as those referred to aboYé i8
not required.
Characters The statements under the heading Charactersare not to be interpreted in. a
strictsense and are not requirements.
Solubility In statements of solubility in the Characters section, the tenns
used have the following significance, referred . toa· temperature· between
15 oC and 25 o,C.
Descriptive tetm Approximate vohl1tie of solventin millilitres
pef gram of solute
Very soluble less than 1
Freely soluble from 1 to 10
Soluble from 10 to 30
Sparingly soluble from 30 to 100
Slightly soluble from 100 to 1000
Very slight1y soluble from 1000 to 10000
Practically insoluble more than 10000
Identification Scope The tests given in the Identification section are notdesigned t() giv~
afull confirmation of the chemical structure or compositiún()f theproduc1:;
they are intended to give conf1rmation,withanacceptable degree of
assurance, that the article confonns to the descríptíononthe labe!'
First and second identifications Certain monographs have
subdivisions entitled 'First identification'and'Second identiñcátion'.The
test ot tests that constitutethe 'First identification'maybe used· inall
drcumstances. The test or tests that constitute the 'Second· identification'
may be used inphannacies provided it can be demonstrated that the
substance or preparation is fully ttaceable tú a batch certified lb. c011lply
with all the other requirementsof the mon()graph.
Certain monographs give two or more sets of tests for the purpose of the
first identification, which are equivalentand may be used independently.
Qne or more of these sets usually contain a cross-reference to a test
prescribed in the Tests section of the monograph. It may be used to
simplify the work of the analyst carrying outthe identification and the
prescribed tests. For example, one identificatiQn set cross':'refers to a test for
enantiometicpurity whilethe other set gives a test for specific optical
rotation: the intended purpose of the twO is the same,· tha.t i8, verification
that the correct enantiomer is. presento
Powdered herbal drugs Monographs on herbal drugs· may contain
schematic drawings of the powdered drug. These drawings complement the
description given in the relevant identification. test.
Tests and Assays Scope The requirements are not framed to take account of all possible
impurities. It is not to be presumed, for example,thatan impurity that is
not detectable by means of the prescribed tests is tolerated if common sehse
and good pharmaceutical practice require that it be absent. See also below
under ·Impurities.
26 General Notices
Storage
General N atices 27
Impurities A list of a11 known and potential impurities that have been shown to be
detected by the tests in a monograph may be given.See also chapter 5. JO .
Control of impurities in substances for pharrnaceutical use. The impurities are
designated by a letter or letters of the alphabet.Wherea lettet appears to be
missing, the impurity designated·by this letter has been de1eted from·the list
during monograph development prior to publication orduringmonograph
revision.
LDso The statistically determined quantity· of a Lo/lO dose The largest quantity of a toxin that, in the
substance that, when administered by the conditions of the test, when mixed with 0.1 IU
specified route, may be expected tocause the of antitoxin and administered by the specified
death of 50 per cent of the test animals within route, does not cause symptoms of toxicity in
a given period the test animals within a given period
MLD Mínimum lethal dos e Lf dose The quantity of toxin or toxoid that flocculates
L+/IO dos e The smaIlest quantity of a toxin that,in the in the shortest time with 1 IU of antitoxin
conditions of the test~ when mixed with 0.1· IU CCID so The statistically determined quantity of virus
of antitoxin and administered by the specified that may be expected to infect 50 per cent of
route, causes the death of the test ani11lals the cell cultures to which it is added
within a given period The statistically determined quantityof virus
EIDso
L+ dose The smallest quantity of a toxin that, in the that may be expected to infect 50 per cent of
conditions of the test, when mixed with 1 IU fertilised eggs into which it is inoculated
of antitoxin and administered by the specified The statistically determined quantity of a virus
IDso
route, causes the death of thetest animals that may be expected to infect 50 per cent of
within a given period the animals into which it is inoculated
lr/l00dose The smallest quantity of a toxin that, in the PD so The statistically determined dose of a vaccine
conditions of the test, when mixed with that, in the conditions of the test, may be
0.01 IU of antitoxin and injected expected to protect 50 per cent of the animals
intracutaneously causes a characteristic against a challenge dose of the micro-
reaction at the site of injection within a organisms or toxins against which it is active
given period
EDso The statistically determined dose of a vaccine
Lpll O dose The smallest quantity of toxin that, in the that, in the conditions of the test, may be
conditions of the test, when mixed with 0.1 IU expected to induce specific antibodies in
of antitoxin and administered by the specified 50 per cent of the animals for the relevant
route, causes paralysis in the test animals vaccine antigens
within a given period
PFU Pock-forming units or plaque-forming units
SPF Specified-pathogen-free.
30 GeneralNotices
Collectionsof micro-organisms
ATCC American Type. Culture Collectíon NCTC National Collection of Type Cultures
10801 University Boulevard Central Publie Health Laboratory
Mánassas,· Virginia 20110-2209, USA Colindale Avenue
C.LP. Collectionde Bactéríes de l'InstitutPasteur London NW9 5HT, Great Britain
B.P.52, 25 rue du DocteurRoux: NCYC National ColIection of Yeast Cultures
75724Paris Cedex: .15,·Franee AFRCFood Research Institute
IMI International Myeological Institute ColneyLane
Bakeham Lane Norwich NR4 7UA, Grea! Britain
Surrey TW20 ·9TY, .Great Britain NITE Biological Resouree Center
T.P. CoUeetion Nationalede Culture de Department of Biotechnology
Microorganismes (eN.C .M.) National Institute of Teehnology and
InstitutPasteur Evaluation
25,· rue du DocteurRoux: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,
75724 Paris Cedex 15, Franee 292-0818
Japan
NCIMB National Collection of Industrial and Marine
Bacteria Ltd S.S.r. Statens Serum Institut
23 St Machar Drive 80 Amager Boulevard, Copenhagen, Denmark
Aberdeen AB2 1RY, Great Britain
NCPF National Collection of Pathogenic Fungí
Loudon School of Hygiene and Tropical
Medicine
Keppel Street
LondonWClE7HT, Great Britain
1· The definitions 01 the units usedin the Internatzonal System are given in the booklet "Le Systeme International d'Unités (SI)" published by
theBureauInternational des Poids et Mesures, Pavillion de BreteuiZ,F-92310 Sevres.
General N otices 31
Table 1.6.-2. - SIunitsused in the European Pharmacopoeia and equivalence wifh other units
Quantity Unit
Name Symból Name Symból ExpressiÓl1 in SI Expression in other Conversión of ótber units into SI uníts
baseunits Slunits
Wave number v one pet metre l/m m-"
:. COricentration e mole 'Del' cubie Illetre moVm3 mol'm- 3 1 moljL" 1 M = 1 moljdm3 ", 103 mol'm- 3
(of amount of
substaneekinolar
eoncentration
Mass P kilogram per cubie kg/m 3 kg'm- 3 1 gIL "" 1 g/dm3 =1 kg-m- 3
concentration metre
..
General Notices 33
Monographs
******
where applicable, the substance also complies with the
Substances for Pharmaceutical
*~** *
requirements of the general monograph Products of
Use ** recombinant DNA technology (0784);
(Ph Eur monograph 2034) -- is obtained from animals susceptible to transmissible
~E~ _____________________________________________ spongifonn encephalopathies other than by experimental
chaHenge, where applicable, the substance also complies
DEFINITION with the requirements of the general monograph Products
Substances for phannaceutical use are any organic or with risk oJ transmitting agents of animal spongiform
inorganic substances that are used as active substances or encephalopathies (1483);
excipients for the production of medicinal products for -- is a substance derived from a fennentation process,
human or veterinary use. They may be obtained from natural whether or not the micro-organisms involved are modified
sources or produced by extraction from raw materials, by traditional procedures or recombinant DNA (rDNA)
fennentation or synthesis. technology, where applicable, the substance also complies
This general monograph does not apply to herbal drugs, with the requirements of the general monograph Products
herbal drugs for homoeopathic preparations, herbal drug of fermentation (1468).
preparations, extracts, or mother tinctures for homoeopathic If solvents are used during production, they are of suitable
preparations, which are the subject of separate general quality. In addition, their toxicity and their residuallevel are
monographs (Herbal drugs (1433), Herbal drugs for taken into consideration (5.4). If water is used during
homoeopathic preparations (2045), H erbal drug preparations production, it is of suitable quality.
(1434), Extracts (0765), Mother tinctures for homoeopathic If substances are produced or processed to yield a certain
preparations (2029)). It does not apply to raw materials for form or grade, that specific form or grade of the substance
homoeopathic preparations, except where there is an complies with the requirements of the monograph. Certain
individual monograph for the substance in the functionality-related tests may be described to control
non-homoeopathic part of the Phannacopoeia. properties that may influence the suitability of the substance
Where a substance for phannaceutical use not described in and subsequently the properties of dosage forms prepared
an individual monograph of the Phannacopoeia is used in a from it.
medicinal product prepared for the special needs of Powdered substances may be processed to obtain a certain
individual patients, the need for compliance with the present degree of fineness (2.9.35).
general monograph is decided in the light of a risk Compacted substances are processed to increase the partic1e
assessment that takes account of the available quality of the size or to obtain partic1es of a specific fonn and/or to obtain
substance and its intended use. a substance with a higher bulk density.
Where medicinal products are manufactured using Coated active substances consist of partic1es of the active
substances for phannaceutical use of human or animal origin, substance coated with one or more suitable excipients.
the requirements of chapter 5.1.7. Viral safety apply.
Granulated active substances are partic1es of a specified size
Substances for phannaceutical use may be used as such or as and/or form produced from the active substance by
starting materials for subsequent formulation to prepare granulation direct1y or with one or more suitable excipients.
medicinal products. Depending on the fonnulation, certain
If substances are processed with excipients, these excipients
substances may be used either as active substances or as
comply with the requirements of the relevant monograph or,
excipients. Solid substances may be compacted, coated,
where no such monograph exists, the approved specification.
granulated, powdered to a certain fineness, or processed in
other ways. A monograph is applicable to a substance Where active substances have been processed with excipients
processed with an excipient only where such processing is to produce, for example, coated or granulated substances, the
mentioned in the definition section of the monograph. processing is carried out under conditions of good
manufacturing practice/and the processed substances are
Substance for pharmaceutical use of special grade Unless
regarded as intermediates in the manufacture of a medicinal
otherwise indicated or restricted in the individual
producto
monographs, a substance for phannaceutical use is intended
for human and veterinary use, and is of appropriate quality CHARACTERS
for the manufacture of aH dosage forms in which it can be The statements under the heading Characters
used. (e.g. statements about the solubility or a decomposition
Polymorphism Individual monographs do not usuaHy specify point) are not to be interpreted in a strict sense and are not
crystaHine or amorphous forms, unless bioavailability is requirements. They are given for information.
affected. AH forms of a substance for phannaceutical use Where a substance may show polymorphism, this may be
comply with the requirements of the monograph, unless stated under Characters in order to draw this to the attention
otherwise indicated. of the user who may have to take this characteristic into
PRODUCTION consideration during formulation of a preparation.
Substances for phannaceutical use are manufactured by IDENTIFICATION
procedures that are designed to ensure a consistent quality Where under Identification an individual monograph
-and comply with the requirements of the individual contains subdivisions entitled 'First identification' and
monograph or approved specification.
38 General Monographs
'Second identification', the test or tests that constitute the synthetic products derived therefrom, to crude products of
'First identification' may be used in all circumstances. animal or plant origin or herbal products.
The test or tests that constitute the 'Second identification' Residual solvents
may be used in pharmacies provided it can be demonstrated Are limited according to the principIes defined in chapter
that the substance or preparation is fully traceable to a batch 5.4, using general method 2.4.24 or another suitable method.
certified to comply with aH the other requirements of the Where a quantitative determination of a residual solvent is
monograph. carried out and a test for loss on drying is not carried out,
Certain monographs give two or more sets of tests for the the content of residual solvent is taken into account for
purpose of the first identification, which are equivalent and calculation of the assay content of the substance, the specific
may be used independent1y. One or more of these sets optica! rotation and the specific absorbance.
usuaHy contain a cross-reference to a test prescribed in the Microbiological quality
Tests section of the monograph. It may be used to simplify Individual monographs give acceptance criteria for
the work of the analyst carrying out the identification and the microbiological quality wherever such control is necessary.
prescribed tests. For example, one identification set cross- Table 5.1.4.-2. - Aeeeptanee eriteria for mierobiologieal quality of
refers to a test for enantiomeric purity while the other set non-sterile substanees for pharmaeeutieal use in ehapter 5.1.4.
gives a test for specific optical rotation: the intended purpose Mierobiologieal quality of non-sterile pharmaeeutieal preparations
of the two is the same, that is, verification that the correct and substanees for pharmaeeutieal use gives recommendations
enantiomer is presento on microbiological quality that are of general relevance for
TESTS substances subject to microbial contamination. Depending on
Polymorphism (5.9) the nature of the substance and its intended use, different
If the nature of a crystalline or amorphous form imposes acceptance criteria may be justified.
restrictions on its use in preparations, the nature of the Sterility (2.6.1)
specific crystalline or amorphous form is identified, its If intended for use in the manufacture of sterile dosage forms
morphology is adequately controlled and its identity is without a further appropriate sterilisation procedure, or if
stated on the label. offered as sterile grade, the substance for pharmaceutical use
Related substances complies with the test for sterility.
U nless otherwise prescribed or justified and authorised, Bacterial endotoxins (2.6.14)
organic impurities in active substances are to be reported, If offered as bacterial endotoxin-free grade, the substance for
identified wherever possible, and qualified as indicated in pharmaceutical use complíes with the test for bacterial
Table 2034.-1 or in Table 2034.-2 for peptides obtained by endotoxins. The limit and test method (if not gelation
chemical synthesis. method A) are stated in the individual monograph. The limit
is calculated in accordance with the recommendations in
TabIe 2034.-1. - Reporting, identification and qualification of general chapter 5.1.10. Guidelines for using the test for bacterial
organic impurities in active subsfances endotoxins, unless a lower limit is justified from results from
Use Maximum Report- Identification Qualification production batches or is required by the competent authority.
daily ing threshold threshold Where a test for bacterial endotoxins is prescribed, a test for
dose threshold pyrogens is not required.
Human :;; 2 gjday > 0.05 per > 0.10 per > 0.15 per
use or cent cent or a cent or a Pyrogens (2.6.8)
human and daily intake daily intake If the test for pyrogens is justified rather than the test for
veterinary of> 1.0 mg of> 1.0 mg bacterial endotoxins and if a pyrogen-free grade is ofIered,
use (whichever is (whichever is
the lower) the lower) the substance for pharmaceutical use complies with the test
Human > 2 gjday > 0.03 per > 0.05 per cent > 0.05 per cent for pyrogens. The limit and test method are stated in the
use or cent individual monograph or approved by the competent
human and
veterinary authority. Based on appropriate test validation for bacterial
use endotoxins and pyrogens, the test for bacterial endotoxins
Veterinary Not > 0.10 per > 0.20 per cent > 0.50 per cent may replace the test for pyrogens.
use only applicable cent
Additional properties
Control of additional properties (e.g. physical characteristics,
functionality-related characteristics) may be necessary for
individual manufacturing processes or forrn.ulations. Grades
(such as sterile, endotoxin-free, pyrogen-free) may be
Reporting Identification Qualification produced with a view to manufacture of preparations for
threshold threshoJd threshold parenteral administration or other dosage forms and
> 0.1 per cent > 0.5 per cent : > 1.9 per cent appropriate requirements may be specified in an individual
monograph.
those statements that are necessary to demonstrate C. Dissolve 0.2 g in a mixture of 3 mL of water and 2 mL of
compliance or non-compliance with the monograph are 5M sodium hydroxide and shake with three 3-mL quantities of
mandatory. Any other labelling statements are inc1uded as ether. Add to the aqueous solution 2 mL of bromine solution,
recommendations. When the term 'label' is used in the warm in a water bath for 10 minutes, heat to boiling, cool
Pharmacopoeia, the labelling statements may appear on the and add 0.25 mL to a solution of 10 mg of resorcinol in 3 mL
container, the package, a leaflet accompanying the package or of sulfuric acid. A bluish black colour develops on heating for
a certificate of analysis accompanying the artic1e, as decided 15 minutes in a water bath.
by the competent authority.
TESTS
Where appropriate, the label states that the substance is: Acidity
- intended for a specific use; pH of a 1.0% w/v solution, 4.0 to 4.5, Appendix V L.
- of a distinct crystalline form;
- of a specific degree of fineness;
Melting point
- compacted; 136° to 139°~ Ap;pendix V A.
- coated; Related substances
- granulated; Complies with the test for related substances in phenothiazines,
- sterile; Appendix nI A, but using a mixture of 75 volumes of
- free from bacterial endotoxins; n-hexane, 17 volumes of butan-2-one and 8 volumes of
- free from pyrogens; diethylamine as the mobile phase.
- containing gliding agents. Loss on drying
Where applicable, the label states: When dried to constant weight at 105°, loses not more than
- the degree of hydration; 1.0% of its weight. Use 1 g.
- the name and concentration of any excipient. Sulfated ash
__________________________________________ ~E~
Not more than 0.2%, Appendix IX A.
ASSAY
Dissolve 0.4 g in 50 mL of acetic anhydride and carry out
Method I for non-aqueous titration, Appendix VIII A, using
crystal violet solution as indicator. Each mL of O.lM perchloric
Acepromazine Maleate acid VS is equivalent to 44.25 mg of C19H22N20S,C4H404.
Alfadolone Acetate
OAe
442.5 3598-37-6
~ Me
Appendix VI.
Me TESTS
Picoline
A. 2,4-dimethylaniline (2,4-xylidine), Dissolve 1.5 g in 30 mL of water in a distillation flask,
add 20 mL of a saturated solution of potassium carbonate
~NHCHO sesquihydrate, connect the flask to a coarse-fritted aerator
~ Me
extending to the bottom of a 100-mL graduated cylinder
Me containing 50 mL of 0.05M hydrochloric acid, and pass air,
which has previously been passed through sulfuric acid and
B. form-2',4'-xylidide, glass wool, through the system for 60 minutes. To 5 mL of
the hydrochloric acid solution add sufficient
0.05M hydrochloric acid to produce 200 mL. The absorbance
~N~NHMe of the resulting solution at 262 nm is not greater than 0.52,
Me ~ Me Appendix 11 B.
Loss on drying
0
When dried to constant weight at 100 at a pressure not
C. N-methyl-N'-(2,4-xylyl)formamidine, exceeding 0.7 kPa, loses not more than 1.0% of its weight.
Use 1 g.
H Me
Sulfated ash
~N~N~ Not more than 0.1 %, Appendix IX A.
Me ~ Me U Me
ASSAY
Carry out Method 1 for non-aqueous titration,
Appendix VIII A, using 0.3 g and 1-naphtholbenzein solution
D. N,N'-bis(2,4-xylyl)formamidine. as indicator. Each mL of O.lM perchloric acid VS is equivalent
to 15.77 mg of C14H19CIN4,HCl.
Amprolium Hydrochloride
-Apramycin Sulfate
Apramycin Sulphate
CI- ,HCI
~
HO o
H2 N
315.3 137-88-2
HO OH ~NHMe
OH
Action and use O O O HO OH
Antiprotozoa1; prevention and treatment of coccidiosis
(veterinary) .
R ~NH,
R =H NH 2 O NH 2
DEFINITION
Amprolium Hydrochloride is 1-(4-amino-2-propylpyrimidin-
5-ylmethyl)-2-methylpyridinium chloride hydrochloride.
It contains not les s than 97.5% and not more than 101.0% 784.8 41194-16-5
of C14H19CIN4,HCI, calculated with reference to the dried
substance. Actionand use
Aminoglycoside antibacterial.
CHARACTERISTICS
A white or almost white powder; odourless or/almost Preparations
odourless. Apramycin Veterinary Oral Powder
Freely soluble in water; slightly soluble in ethanol (96%); very Apramycin Premix
slightly soluble in ether; practically insoluble in chloroform.
DEFINITION
IDENTIFICATION Apramycm Sulfate is the sulfate of
A. The infrared absorption spectrum, Appendix 11 A, is 4-0- [(2R,3R,4aS,6R, 7S,8R,8aR)- 3-amino-6-(4-amino-
concordant with the reference spectrum of amprolium 4-deoxy-tX-D-glucopyranosyloxy)-8-hydroxy-
hydrochloride (RSV 07). 7-methylaminoperhydropyrano [3 ,2-b] pyran-2-yl]-
B. The light absorption, Appendix 11 B, in the range 230 to 2-deoxystreptamine. It is produced by the growth of certain
350 nm of a 0.002% w/v solution in O.lM hydrochloric acid strains of Streptomyces tenebrarius or obtained by any other
exhibits two maxima, at 246 nm and 262 nm. The means. The potency is not less than 450 Units per mg,
absorbances at the maxima are about 0.84 and about 0.80, calculated with reference to the anhydrous substance.
respectively.
Aprarnycin Sulfate 43
the sum of the areas of a11 the secondary peaks is not greater IMPURITIES
than 6 times the area of the principal peak in the
chromatogram obtained with solution (3) (15%).
Disregard any peak with an area less than 0.04 times the area
OMe
of the principal peak inthe chromatogram obtained with
solution (3) (0.1%).
Sulfated ash
Not more than 1.0%, Appendix IX A, Method 11. Use 1 g.
I
Water
OH
Not more than 14.0% w/w, Appendix IX C. Use 0.2 g and
20 mL of a mixture containing 1 volume of methanol and
2 volumes of formamide as the solvento The solvent mixture A. caerulomycin,
must be prepared at least 12 hours before use and should be
stored in an airtight container.
ASSAY
Carry out the microbiological assay of antibiotics,
Appendix XIV A, Method B. The precision of the assay is
such that the fiduciallimits of error are not les s than 95%
and not more than 105 % of the estimated potency.
Apramycin Sulfate intended for use in the manufacture of a
parenteral dosage form is decolourised and complies with the B. lividamine,
above requirements with the following modijications.
Preparation
Apramycin Injection
DEFINITION
The potency is not les s than 576 Units per mg, calculated
with reference to the anhydrous substance.
Related substances C. 2-deoxystreptamine
Carry out the method as described above. D. 3-hydroxyapramycin; R = OH,
LIMITS E. 'compound A',
In the chromatogram obtained with solution (1): F. 'compound B'.
the areas of any peaks corresponding to 3-hydroxyapramycin,
lividamine/2-deoxystreptamine (combined) and compound A
(identified using the reference chromatogram supplied with
apramycin BPCRS) are not greater than twice the area of the
principal peak in the chromatogram obtained with solution
Azaperone
(3) (5% each); (Azaperone for Veterinary Use~
the area of any peak corresponding to compound B is not Ph Eur monograph 1708)
greater thari 0.8 times the area of the principal peak in the
chromatogram obtained with solution (3) (2%);
the area of any other secondary peak is not greater than
0.8 times the area of the principal peak in the chromatogram
obtained with solution (3) (2%);
the sum of the areas of a11 the secondary peaks is not greater
than 4.8 times the area of the principal peak in the
chromatogram obtained with solution (3) (12%).
Disregard any peak with an area less than 0.04 times the area 327.4 1649-18-9
of the principal peak in the chromatogram obtained with
solution (3) (0.1%). Action and use
Dopamine receptor antagonist; neuroleptic (veterinary).
STORAGE
Preparation
If the substance is sterile, the container should be sterile,
Azaperone Injection
tamper-evident and sealed so as to exclude inicto-organisms.
~E~ __-+_______________________________________
LABELLING
The label states (1) that the material is suitable for parenteral DEFINITION
use; (2) where applicable, that it is sterile. 1-(4-Fluorophenyl)-4- [4-(pyridin-2-yI)piperazin-l-yl] butan-
Apramycin Sulfate intended for use in the manufacture of a l-one. /
parenteral dosage form without a further appropriate sterilisation Content
procedure complies with the following additional requirement. 99.0 per cent to 101.0 per cent (dried substance).
Sterility CHARACTERS
Complies with the test for sterility, Appendix XVI A. Appearance
White or almost white powder.
Azaperone 45
Solubility - sum of impurities B and C: not more than 3 times the area
Practically insoluble in water, freely soluble in acetone and in of the principal peak in the chromatogram obtained with
methylene ch1oride, soluble in ethanol (96 per cent). reference solution (b) (0.75 per cent);
It shows polymorphism (5.9). - total: not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
IDENTIFICATION (1.0 per cent);
Infrared absorption spectrophotometry (2.2.24). - disregard limit: 0.2 times the area of the principal peak in
Preparation Discs. the chromatogram obtained with reference solution (b)
Comparison azaperone CRS. (0.05 per cent).
If the spectra obtained show differences, dissolve the Loss on drying (2.2.32)
substance to be examined and the reference substan~e Maximum 0.5 per cent, determined on 1.000 g by drying
separately in acetone R, evaporate 10 dryness and record new in vacuo at 60 oC for 4 h.
spectra using the residues. Sulfated ash" (2.4.14)
TESTS Maximum O.l\per cent, determined on 1.0 g.
Appearance of solution ASSAY
The solution is c1ear(2.2.1) and not more intense1y coloured Dissolve 0.130 g in 70 mL of a mixture of 1 volume of
than reference solution Y6 (2.2.2, Method JI). anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R.
Dissolve 1.0 g in 25 mL of a 14 gIL solution of tartaric Titrate with 0.1 M perchloric acid, using 0.2 mL of
acid R. naphtholbenzein solution R as indicator.
Related substances 1 mL of 0.1 M perchloric acid is equivalent to 16.37 mg of
Liquid chromatography (2.2.29). C 19 HzzFN3 0.
Test solution Dissolve 0.100 g of the substance to be STORAGE
examined in methanol R and dilute to 10.0 mL with the same Protected from light.
solvento
IMPURITIES
Reference solution (a) Dissolve 5.0 mg of azaperone CRS and
Specified impurities A, B, C.
6.0 mg of benperidol CRS in methanol R and dilute to
200.0 mL with the same solvento
Reference solutio'IJ (b) Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 5.0 mL of the solution to
20.0 mL with methanol R.
Column:
- size: l = 0.10 m, (2) = 4.6 mm;
- stationary phase: base-deactivated octadecylsilyl silica gel for
ij¡
chromatography R (311m); ~R
- temperature: 25 oc. F o
Mobile phase:
- mobile phase A: dissolve 1.4 g of anhydrous sodium sulfate R A. 1-(2-fiuorophenyl)-4-[4-(pyridin-2-yl)piperazin-1-yl]butan-
in 900 mL of water R, add 16.0 mL of 0.01 M sulfuric l-one,
acid and dilute to 1000 mL with water R;
- mobile phase B: methanol R;
Time Mobile phase A Mobile phase B R~
1# R
(min) (per cent Vil? (percent Vil?
0-15 95 ~ 20 5 ~80 O
15 - 20 20 80
B. 4-[4-(pyridin-2-yl)piperazin-1-yl]-1- [4-[4-(pyridin-
2-yl)piperazin-l-yl]phenyl] butan-l-one,
Flow rate 1.5 mUmin.
Detection Spectrophotometer at 230 nm. R~
Jnjection 10!-LL.
Relative retention With reference to azaperone
~OH O
(retention time = about 9 min): impurity A = about 0.9;
impurity B = about 1.1; impurity C = about 1.15. C. 4-hydroxy-1-[4-[4-(pyridin-2-yl)piperazin-
System suitability Reference solution (a): l-yl]phenyl]butan-1-one.
- resolution: minimum 8.0 between the peaks due to ___________________________________________ PhE~
Loss on drying
Calcium Copperedetate When dried to constant weight at 105°, loses not more than
2.0% of its weight. Use 1 g.
ASSAY
For copper
0
Ignite 4 g at 600 to 700°, cool and heat the residue with
Ca2+ 12 mL of a mixture of equal volumes of hydrochloric acid and
water on a water bath for 15 minutes. Add 10 mL of water,
filter and dilute the filtrate to 100 mL with water (solution
A); reserve a portion for the Assay for calcium. To 25 mL of
solution A add 25 mL of water and 10 mL of bromine
solution, boil to remove the bromine, cool and add dilute
sodium carbonate solution until a faint permanent precipitate is
Action and use
produced. Add 3 g of potassium iodide and 5 mL of 6M acetic
Used in the treatment of copper deficiency.
acid and titrate the liberated iodine with O.IM sodium
Preparation thiosulfate VS, using starch mucilage as indicator, until only a
Calcium Copperedetate Injection faint blue colour remains; add 2 g of potassium thiocyanate
and continue the titration until the blue colour disappears.
DEFINITION Each mL of O.IM sodium thiosulfate VS is equivalent
Calcium Copperedetate is the dihydrate of calcium to 6.354 mg of Cu.
[ethylenediaminetetra-acetato( 4- )-N,NI ,0,0 1] copper(n).
For calcium
It contains not les s than 9.1 % and not more than 9.7% of
To 5 mL of solution A add 10 mL of water and 10 mL of a
calcium, Ca, and not less than 14.4% and not more than
10% v/v solution of mercaptoacetic acid, allow to stand until
15.3% of copper, Cu, both calculated with reference to the
the precipitate has coagulated, dilute to 100 mL with water,
dried substance.
add 5 mL of 5M sodium hydroxide and titrate with
CHARACTERISTICS 0.05M disodium edetate VS, using methyl thymol blue mixture as
A blue, crystalline powder. indicator, until the solution becomes a full purple colour,
Freely soluble in water, the solution gradually precipitating adding the titrant slowly as the end point is approached.
the tetrahydrate; practically insoluble in ethanol (96%). Each mL of 0.05M disodium edetate VS is equivalent to
2.004 mg of Ca.
IDENTIFICATION
A. Dissolve 0.2 g in 5 mL of water and add 1 mL of
6M acetic acid and 2 mL of dilute potassium iodide solution.
The solution remains c1ear and deep blue.
B. Ignite 0.2 g, dissolve the residue in 3 mL of
Carprofen
2M hydrochloric acid, neutralise the solution with 5M ammonia (Carprofen for Veterinary Use)
and add 1 mL of 6M acetic acid and 2 mL of dilute potassium Ph Eur monograph 2201)
iodide solution. A white precipitate is produced and iodine is
liberated, colouring the supernatant liquid brown. H eH3
C. Dissolve 0.5 g in 10 mL of water, acidify with
2M hydrochloric acid, add 25 mL of a 10% v/v solution of
ri~rre02H
mercaptoacetic acid and filter. Make the filtrate alkaline with
5M ammonia and add 5 mL of a 2.5% w/v solution of
ammonium oxalate. A white precipitate is produced which is
~
el and enantiomer
IDENTIFICATION ASSAY
Infrared absorption spectrophotometry (2.2.24). Dissolve 0.200 g in 50 mL of ethanol (96 per cent) R.
Companson carprofen CRS. Add 1.0 mL of 0.1 M hydrochlonc acid. Titrate with 0.1 M
If the spectra obtained in the solid state show differences,
sodium hydroxide, determining the end-point
potentiometrically (2.2.20). Read the volume added between
dissolve the substance to be examined and the reference
the 2 points of infiexion.
substance separately iIi acetone R, evaporate to dryness and
record new spectra using the residues. 1 mL of 0.1 M sodium hydroxide is equivalent to 27.37 mg of
ClsH12ClN02.
TESTS
Appearance of solution STORAGE
The solution is clear (2.2.1) and not more intensely <:;oloured Protected from light.
than reference solution BY3 (2.2.2J Method JI). ' IMPURITIES
Dissolve 1.0 g in methanol R and dilute to 25 mL with the Other detectable impunties (the following substances would, if
same solvento present at a sufficient level, be detected by one or other of
Related substances the tests in the monograph. They are limited by the general
Liquid chromatography (2.2.29). Carry out the test protected acceptance critdjon for other/unspecified impurities and/or
from light. -- by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these
Test solution Dissolve 50 mg of the substance to be
impurities for demonstration of compliance. See also 5.10.
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
Control of impunties in substances for pharmaceutical use): A J B J
CJ DJ EJ F J GJ H.
Reference solution (a) Dissolve 2.5 mg of carprofen for system
suitability CRS (containing impurity C) in the mobile phase
and dilute to 10.0 mL with the mobile phase.
Reference solution (b) Dilute 1.0 mL of the test solution to
100.0 mL with the mobile phase. Dilute 1.0 mL ofthis
solution to 10.0 mL with the mobile phase.
Column:
- size: 1 = 0.25 m, 0 = 4.6 mm; CI
- stationary phase: end-capped polar-embedded octadecylsilyl
amorphous organosilica polymer R (5 ~m). A. R = H: 2-(6-chloro-9H-carbazol-2-yl)-
Mobile phase Mix 30 volumes of a 1.36 gIL solution of 2-methylpropanedioic acid,
potassium dihydrogen phosphate R adjusted to pH 3.0 with F. R= C 2H s : diethy12-(6-chloro-9H-carbazol-2-yl)-
phosphonc acid R and 70 volumes of methanol R2. 2-methylpropanedioate,
Flow rate 1.3 mUmin.
Detection Spectrophotometer at 235 nm.
Jnjection 20 ¡..tL.
Run time 4 times the retention time of carprofen.
Retention time carprofen = about 10 min.
System suitabz1ity Reference solution (a):
- resolution: minimum 1.5 between the peaks due to
impurity C and carprofen. B. R = H, R' = C0 2H: (2RS)-2-(9H-carbazol-
Limits: 2-yl)propanoic acid,
- unspecijied impunties: for each impurity, not more than
C. R = Cl, R' = OH: (lRS)-1-(6-chloro-9H-carbazol-
twice the area of the principal peak in the chromatogram
2-yl) ethanol,
obtained with reference solution (b) (0.20 per cent);
- total: not more than 5 times the area of the principal peak G. R = Cl, R' = CO-O-C 2H s: ethyl (2RS)-2-(6-chloro-
in the chromatogram obtained with reference solution (b) 9H-carbazol-2-yl)propanoate,
(0.5 per cent);
r\~ÚR
- disregard limit: the area of the principal peak in the
yl~
chromatogram obtained with reference solution (b)
(0.1 per cent).
Heavy metals (2.4.8)
Maximum 20 ppm. CI
Catechu Cefalonium
Pale Catechu
COO-
Action and use
Intestinal astringent.
DEFINITION
Catechu is a dried aqueous extract prepared from the leaves
~N#S
S
O
°YOy NH,
H H H
N ~ N ~
1#
O
and young shoots of Uncaria gambier (Hunter) Roxb.
CHARACTERISTICS 494.5 5575-21-3 (anhydrous)
Odourless or almost odourless.
Macroscopical Catechu usually occurs as cubes, which are Action and use
sometimes more or less agglutinated and mixed with Cephalosporin antibacterial.
fragments of broken cubes; the cubes are friable and porous Preparation
and measure about 2.5 cm in each direction; larger cubes Cefalonium Intramammary Infusion (Dry Cow)
and brick-shaped pieces, up to 4 cm long, also occur and are
sometimes broken. Their colour is dull, pale greyish brown to DEFINITION
dark reddish brown externally and pale brown internally. Cefalonium is 3-(4-carbamoyl-l-pyridiniomethyl)-
Microscopical The diagnostic characters are: the abundant 7- [(2-thienyl)acetamido] -3-cephem-4-carboxylate dihydrate~
yellowish brown mas ses of acicular catechin crystals, soluble It contains not les s than 95.0% and not more than 103.5%
in hot water; varying amounts of fragments from the leaves of C20HlSN40SS2, ca1culated with reference to the
and flowering shoots of the plant including: unicellular anhydrous substance.
covering trichomes, 250 to 540 ).lm long with lignified walls, CHARACTERISTICS
pitted at the base, sorne with one or two thin transverse
A white or almost white crystalline powder.
septa; fewer smaller trichomes, 25 to 45 ).lm long, conical,
with warty, unlignified walls, epidermal cells of the leaves Very slightly soluble in water and in methanol; soluble in
thin-walled with a finely striated cuticle and paracytic dimethyl sulfoxide; insoluble in dichloromethane, in ethanol
sto mata, Appendix XI H, on the lower epidermis only; (96%) and in ether. It dissolves in dilute acids and in
reddish brown corolla segments with numerous covering alkaline solutions.
trichomes and characteristic pitted and lignified cicatrices in IDENTIFICATION
the epidermis; parenchymatous cells containing calcium A. The infrared absorption spectrum, Appendix II A, is
oxalate as cluster crystals and crystal sand; subspherical concordant with the reference spectrum of cefalonium
pollen grains, 11 to 18 ).lm in diameter with three pores, (RSV09).
three furrows and a minutely pitted exine; occasional B. The light absorption, Appendix II B, in the range 220 to
fragments of cork. 350 nm of a 0.002% w/v solution in water exhibits two
IDENTIFICATION maxima, at 235 nm and at 262 nm. The absorbance at
Warm 0.3 g with 2 mL of ethanol (96%), cool and filter. 235 nm is about 0.76 and at 262 nm is about 0.62.
Add 2 mL of 5M sodium hydroxide to the filtrate, shake, add TESTS
2 mL of petroleum spirit (boiling rangeJ 40° to 60°), shake and Specific optical rotation
allow to separate. A brilliant greenish fluorescence is Dissolve 0.25 g with the aid of gentle heat in sufficient
produced in the upper layer. dimethyl sulfoxide to produce 50 mL. Allow the solution to
TESTS stand for 30 minutes before measurement of the optical
Matter insoluble in ethanol (96%) rotation. The specijic optical rotation of the resulting solution
Not more than 34.0%, calculated with reference to the dried is -50 to -56, calculated with reference to the anhydrous
material, when determined by the following method. substance, Appendix V F.
Macerate 5 g, in coarse powder, with 100 mL of ethanol Related substances
(96%), allow to stand for 6 hours shaking frequently and Carry out the method for thin-Iayer chromatography,
allow to stand for a further 18 hours. Filter, wash the residue Appendix III A, using the following solutions in 8.3M acetic
with ethanol (96%) and dry to constant weight at 100°. acid.
Starch (1) 2.5% w/v of the substance being examined.
The residue obtained in the test for Matter ihsoluble in (2) 0.05% w/v of the substance being examined.
ethanol (96%) contains not more than an occ~sional starch
(3) 0.025% w/v of the substance being examined.
granule.
(4) 0.005% w/v of the substance being examined.
Water-insoluble matter
Not more than 33.0%, calculated with reference to the dried (5) O.O~% w/v of each of cefalotin sodium EPCRS and
material, when determined by the method for Maner isonicotihamide.
insoluble in ethanol (96%), but using water in place of the CHROMATOGRAPHIC CONDITIONS
ethanol (96%). (a) Use as the coating silica gel F254 •
Loss on drying (b) U se the mobile phase as described below.
When dried to constant weight at 105°, loses not more than (c) Apply 4 ).lL of each solution.
15.0% of its weight. Use 1 g.
(d) Develop the plate to 12 cm.
Ash (e) After removal of the plate, allow it to dry in air and
Not more than 8.0%, Appendix XI l examine under ultraviolet light (254 nm).
Clazuril 49
MOBILE PHASE
10 volumes of glacial acetic acid, 10 volumes of 1M sodium
acetate and 30 volumes of propan-2-01.
SYSTEM SUITABILITY
The test is not val id ul).less the chromatogram obtained with
solution (5) shows two clearly separated spots. D. isonicotinamide.
LIMITS
Any secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the
chromatogram obtained with solution (2) (2%), not more
than one such spot is more intense than the spot in the
Clazuril
chromatogram obtained with solution (3) (1 %) and not more (Clazurilfor Veterinary Use) Ph Bur monograph 1714)
than three such spots are more intense than the spot in the
chromatogram obtained with solution (4) (0.2% each). ./ H eN el
~o
Sulfated ash
Not more than 0.2%, Appendix IX A.
Water
el~ UNA NH
andenantiomer
n ~O:-~~OAC
and in methylene chloride.
lZ.."'~N\\\~Sj
IDENTIFICATION
A. Melting point (2.2.14): 199 oC to 203 oC.
S H H H
B. Infrared absorption spectrophotometry (2.2.24).
Comparison clazuril CRS.
A. cefalotin,
TESTS
Related substances
COOH
Liquid chromatography (2.2.29).
lZ.."'~N\\\~Sj
S H H H
Test solution Dissolve 20.0 mg of the substance to be
examined in the solvent mixture and dilute to 20.0 mL with
the solvent mixture.
Reference solution (a) Dissolve 5 mg of clazuril for system
B. 3-hydroxymethyl-7p-(2-thienylacetamido)-3-cephem-
suitability CRS (containing impurities A, B, C, D, E, F, G,
4-carboxylic acid,
H and 1) in the solvent mixture and dilute to 5.0 mL with
the solvent mixture.
n ~
Reference solution (b) Dilute 1.0 mL of the test solution to
"'~~N"'~)/
o:-;:t-J 100.0 mL with the solvent mixture. Dilute 2.0 mL of this
solution to 10.0 mL with the solvent mixture.
Column:
-- size: 1 = 0.10 m, 0 = 4.6 mm;
S H H H
-- stationary phase: octadecylsiljil silica gel for chromatography R
(3 ~m);
C. 3-hydroxymethyl-7 P-(2-thienylacetamido)-3-cephem- -- temperature: 35 oC.
4-carboxylic acid lactone,
50 Clazuril
N~o
Identification of impurities Use the chromatogram supplied
with clazuril for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due el UN)lNH and enantiomer
to impurities A, B, C, D, E, F, G, H and I.
I~
N"",
NH~
Relative retention With reference to cIazuril (retention
time = about 16 min): impurity A = about 0.6;
o
impurity B = about 0.78; impurity C = about 0.80;
impurity D = about 0.86; impurity E = about 0.9;
impurity F = about 0.95; impurity G = about 0.98; B. 2- [3-chloro-4- [(RS)-( 4-chlorophenyl)cyanomethyl]phenyl]-
impurity H = about 1.1; impurity 1 = about 1.2. 3,5-dioxo-2,3,4,5-tetrahydro-l ,2,4-triazine-6-carboxamide,
System suitability Reference solution (a):
- peak-to-valley ratio: minimum 1.5, where Hp = height
aboye the baseline of the peak due to impurity G and
H v = height aboye the baseline of the lowest point of the O
curve separating this peak from the peak due to cIazuril, and enantiomer
- the chromatogram obtained is similar to the el N)lNH
chromatogram supplied with clazuril for system ~0 O
suitability CRS.
Limits:
- correction factors: for the calculation of contents, multiply C. (2RS) -2-[2-chloro-4-(3, 5-dioxo-4,5-dihydro-l ,2,4-triazin-
the peak areas of the following impurities by the 2 (3H)-yl)phenyl] -2-(4-chlorophenyl) acetamide,
corresponding correction factor: impurity G = 1.4;
impurity H = 0.8;
- impurities A~ B~ C~ D~ E~ F~ G~ H~ 1: for each impurity,
not more than the area of the principal peak in the
chromatogram obtained with reference solution (b) and enantiomer
(0.2 per cent);
- unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.20 per cent);
- total: not more than 3 times the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.6 per cent); D. 2-[3-chloro-4- [(RS)-( 4-chlorophenyl)cyanomethyl]
- disregard limit: 0.25 times the area of the principal peak in phenyl]-N,N-dimethyl-3,5-dioxo-2,3,4,5-tetrahydro-
the chromatogram obtained with referenct:; solution (b) 1,2,4-triazine-6-carboxamide,
(0.05 per cent); disregard the peaks due tb the solvents.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g' by drying in
an oven at' 105 oC for 4 h.
and enantiomer
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve about 0.260 g in 35 mL of tetrahydrofuran R and
add 35 mL of water R. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.2.20). Carry E. methyl 2-[3-chloro-4-[(RS)-( 4-chlorophenyl)
out a blank titration. cyanomethyl] phenyl] -3 ,5-dioxo-2,3,4 ,5-tetrahydro-
1 mL of 0.1 M sodium hydroxide is equivalent to 37.32 mg of 1,2,4-triazine-6-carboxylate,
C17HlOClzN402'
Cloprostenol Sodium 51
DEFINITION
Cloprostenol Sodium is (± )-(5Z)-7 -(lR,3R,5S)-
2-[ (lE,3R)-4-(3-ehlorophenoxy)-3-hydroxybut-1-enyl]-
and enantiomer 3,5-dihydroxyeyc1opentylhept-5-enoate. It eontains not les s
than 97.5% and not more than 102.5% of C22H2sC1Na06,
ea1culated with referenee to the anhydrous substanee.
CAUTION Cloprostenol Sodium is extremely potent and
extraordinary care should be taken in any procedure in which it is
F. ethyl 2-[3-ehloro-4- [(RS)-( 4-ehlorophenyl) used.
eyanomethyl] phenyl] -3 ,5-dioxo-2,3 ,4,5-tetrahydro-
CHARACTERISTICS
1,2,4-triazine-6-earboxylate,
A white or almost white, amorphous powder; hygroseopie.
Free1y soluble in w,ater, in ethanol (96%) and in methanol;
praetieally insol:uble in acetone.
IDENTIFICATION
A. The infrared absorption spectrum, Appendix Il A, is
eoneordant with the reference spectrum of c1oprostenol sodium
(RSV 11).
G. 2-[3-ehloro-4-( 4-ehlorobenzoyl)phenyl] -1 ,2,4-triazine- E. Yields reaetion A eharaeteristie of sodium salts,
3,5 (2H,4H)-dione, Appendix VI.
TESTS
o~~ H eN el
Related substances
Carry out the method for liquid chromatography,
HNyN
o Appendix III D, using the following solutions in absolute
o N)lNH
ethanol.
(1) 2.0% w/v of the substanee being examined.
~0o
(2) 0.050% w/v of the substanee being examined.
CHROMATOGRAPHIC CONDITIONS
el
(a) Use a stainless steel eolumn (25 cm x 4.6 mm) paeked
with silica gel for chromatography (5 Ilm) (Partisil is suitable).
H. [2-ehloro-4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-
2 (3H) -yl)phenyl] [4- [[2-ehloro-4-(3, 5-dioxo-4,5-dihydro- (b) U se isoeratie e1ution and the mobile phase deseribed
1,2,4-triazin-2(3H)-yl)phenyl] below.
eyanomethyl]phenyl] (4-eh1orophenyl) aeetonitrile, (e) Use a fiow rate of 1.8 mL per minute.
(d) U se an ambient eolumn temperature.
H\ eN el
(e) Use a deteetion wavelength of 220 nm.
N~
and enantiomer
(f) Injeet 5 IlL of eaeh solution.
el V NH
I
NH
I 2
(g) Allow the ehromatography to proeeed for twiee the
retention time of the peak due to Cloprostenol.
N~O
MOBILE PHASE
1 volume of glacial acetic acid, 70 volumes of absolute ethanol
1. (Z)-2-[[3-ehloro-4- [(RS)-( 4-ehlorophenyl)eyanomethyl]
and 930 volumes of hexane.
phenyl] diaza~ylidene] aeetamide.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur LIMITS
In the ehromatogram obtained with solution (1):
the sum of the are as of any secondary peaks is not greater than
the area of the principal peak in the ehromatogram obtained
with solution (2) (2.5%).
Cloprostenol Sodium Water
Not more than 3.0% w/w, Appendix IX C. Use 50 mg
OH
dissolved in 1 mL of absolute ethanol.
H~§
H \,'......... /'-... /'-... ASSAY
,'" ~- "-/ ' COONa
Carry out the method for liquid chromatography,
H §=
OH
H
- ~
~
H OH
°Ú
~
:;--
I
C1 Appendix lIT D, using the following solutions in absolute
ethanol.
(1) 0.08% w/v of the substanee being examined.
(2) 0.08% w/v of cloprostenol sodium BPCRS.
446.9 55028-72-3 CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel eolumn (25 cm x 4.6 mm) paeked
Action and use
with silica gelfor chromatography (5 Ilm) (Partisil is suitable).
Prostaglandin (PGF2cJ analogue.
(b) U se isoeratie e1ution and the mobile phase deseribed
Preparation
be1ow.
Cloprostenol Injeetion
52 Closantel Sodium Dihydrate
H ~
OH
H
H~~ OH DI~
CI
Dissolve 0.50 g in ethanol (96 per cent) R and dilute to
50 mL with the same solvento
Related substances
.
H
Reference solution (a) Dissolve 10 mg of closantel for system
\\\\\\~COONa suitability CRS (containing impurities A to 1) in methanol R
and dilute to 1.0 mL with the same solvento
Reference solution (b) Dilute 1.0 mL of the test solution to
0yyCI
H
H OH V 100.0 mL with methanol R. Dilute 5.0 mL of this solution to
25.0 mL with methanol R.
Column:
B. (±)-(5B )-7-(lR,3R,5S)-2-[(lB,3R)- -- size: 1 = 0.10 m, 0 = 4.6 mm,
4-(3-chlorophenoxy)-3-hydroxybut-1-enyl]- -- stationary phase: base-deactivated octadecylsilyl silica gel for
3,5-dihydroxycyclopentylhept-5-enoate (trans-isomer). chromatography R (3 ¡.tm),
-- temperature: 35 oC.
Mobile phase:
-- mobile phase A: to 100 mL of a 7.7 giL solution of
ammonium acetate R previously adjusted to pH 4.3 with
elosantel Sodium Dihydrate acetic acid R, add 50 mL of acetonitrile R and 850 mL of
(Closantel Sodium Dihydrate for Veterinary Use, water R;
Ph Bur monograph 1716) -- mobile phase B: to 100 mL of a 7.7 gIL solution of
ammonium acetate R previously adjusted to pH 4.3 with
H ,CN acetic acid R, add 50 mL of water R and 850 mL of
oH3C~ acetonitrile R;
yONa
I and enantiomer
0-2
2 - 22 50
50
~ 20 50
50
~ 80
22 - 27 20 80
61438-64-0
Limits: ". OH
R3 and enantiomer
- correction factors: for the ca1culation of contents, multiply
the peak areas of the following impurities by the
corresponding coirection factor: impurity A = 1.5; C. R1 = H, R2 = C0 2H, R3 = 1: (2RS)-[2-chloro-
impurity B = 1.3; 4- [(2-hydroxy-3,5-diiodobenzoyl)amino]-
- impurity G: not more than 2.5 times the area of the 5-methylphenyl] (4-chlorophenyl)acetic acid,
principal peak in the chromatogram obtained with D. R1 = H, R2 = CONH2, R3 = 1: N-[4-[(lRS)-2-amino-
reference solution (b) (0.5 per cent); 1-(4-chlorophenyl)-2-oxoethyl] -5-chloro-2-methylphenyl]-
- impurities F, H, 1: for each impurity, not more than 2-hydroxy-3,5-diiodobenzamide,
1.5 times the area of the principal peak in the E. R1 = H, R2 = CN, R3 = Cl: 3-chloro-N-[5-chloro-
chromatogram obtained with reference solution (b) 4- [(RS)-( 4-chlorophenyl) cyanomethyl] -2-methylphenyl]-
(0.3 per cent); 2-hydroxy-5-iodobenzamide,
- impurities A, B, C, D, E, J: for each impurity, not more
F. R1 + R2 = O, R3 = 1: N-[5-chloro-4-(4-chlorobenzoyl)-
than the area of the principal peak in the chromatogram
2-methylphenyl] -2-hydroxy-3,5-diiodobenzamide,
obtained with reference solution (b) (0.2 per cent);
- any other impurity: for each impurity, not more than the G. R1 = H, R2 = C(=NH)OCH3, R3 = 1: methyl
area of the principal peak in the chromatogram obtained (2RS)-2-[2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]-
with reference solution (b) (0.2 per cent); 5-methylphenyl] -2-(4-chlorophenyl) acetimidate,
- total: not more than 7.5 times the area of the principal H. R1 = H, R2 = CO-OCH3, R3 = 1: methyl
peak in the chromatogram obtained with reference (2RS)-[2-chloro-4-[(2-hydroxy-3,5-diiodobenzoyl)amino]-
solution (b) (1.5 per cent); 5-methylphenyl] (4-chlorophenyl) acetate,
- disregard limit: 0.25 times the are a of the principal peak in 1. R1 = R3 = H, R2 = CN: N-[5-chloro-
the chromatogram obtained with reference solution (b) 4-[ (RS)-( 4-chlorophenyl)cyanomethyl] -2-methylphenyl]-
(0.05 per cent). 2-hydroxy-5-iodobenzamide,
Water (2.5.12)
»
4.8 per cent to 5.8 per cent, determined on 0.250 g. I
U se a mixture of 1 volume of dimethylformamide R and 0 ~
4 volumes of methanol R as the solvent.
eN
O 1.# I
ASSAY
Dissolve 0.500 g in 50 mL of a mixture of 1 volume of el NH
anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R.
Titrate with 0.1 M perchloric acid, determining the end-point HN el
potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 68.5 mg of
C22H13C1212N2Na02·
'Y00
yOH
f "eN
el
I
STORAGE
In an airtight container, protected from light.
J. N-[5-chloro-4-[[4-[[2-chloro-4-[(2-hydroxy-3,5-
IMPURITIES diiodobenzoyl) amino] -5-methylphenyl] cyanomethyl] phenyl]
Specified impurities A, B, C, D, E, F, G, H, 1, J. (4-chlorophenyl) cyanomethyl] -2-methylphenyl] -2-hydroxy-
3,5-diiodobenzamide.
l y y e o2 H _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
yOH
I
A. 2-hydroxy-3,5-diiodobenzoic acid,
54 Cloxacillin Benzathine
cf! o:tt
H Calculate the content of C16H20N2,(C19HlSC1N30SS)2 from
~ N ~ ~ S the difference obtained by carrying out the assay
-:;:? - Me
simultaneously using cloxacillin benzathine BPCRS and from
""- I
CI
O -i<Me
H COOH
the declared content of C16H20N2,(C19HlSCIN30SSh in
cloxacillin benzathine BPCRS.
2
For benzathine
To 1 g add 30 mL of a saturated solution of sodium chloride
32222-55-2 and 10 mL of 5M sodium hydroxide, shake well, and extract
with four 50 mL quantities of ether. Wash the combined
Action and use extracts with three 10 mL quantities of water, extract the
Penicillin antibacterial. combined washings with 25 mL of ether and add the extract
Preparations to the main ether solution. Evaporate the ether solution to
Cloxacillin Benzathine Intramammary Infusion (Dry Cow) low bulk, add 2 mL of absolute ethanol and evaporate 10
Ampicillin Trihydrate and Cloxacillin Benzathine dryness. To the residue add 50 mL of anhydrous acetic acid
Intramammary Infusion (Dry Cow) and titrate with O.lM perchloric acid VS, using 0.1 mL of
1-naphtholbenzein solution as indicator. Repeat the operation
DEFINITION without the substance being examined. The difference
Cloxacillin Benzathine is NJN' -dibenzylethylenediammonium between the titrations represents the amount of perchloric
bis [( 6R)-6-(3-0-chlorophenyl-5-methylisoxazole- acid required to neutralise the liberated base. Each mL of
4-carboxamido)penicillanate]. It contains not less than 92.0% O.lM perchloric acid VS is equivalent to 12.02 mg of
of C16H20N2,(C19HlSCIN30SS)2 and not less than 20.0% C16H20N2·
and not more than 22.0% of benzathine, C16H20N2, each STORAGE
calculated with reference to the anhydrous substance. Cloxacillin Benzathine should be kept in an airtight
CHARACTERISTICS container. If the material is sterile, the container should be
A white or almost white powder. sterile, tamper-evident and sealed so as to exclude micro-
Slightly soluble in water; freely soluble in methanol; slightly organisms.
soluble in ethanol (96%) and in propan-2-0l. LABELLING
IDENTIFICATION The label states, where applicable, that the material is sterile.
A. The infrared absorption spectrum, Appendix 11 A, is Cloxacillin Benzathine intended for use in the manufacturer of
concordant with the reference spectrum of cloxacillin either a parenteral dosage form or an intramammary infusion
benzathine (RSV 12). without a further appropriate sterilisation procedure complies with
B. Shake 0.1 g with 1 mL of 1M sodium hydroxide for the following additional requirement.
2 minutes, add 2 mL of ether, shake for 1 minute and allow Sterility
to separate. Evaporate 1 mL of the ether layer to dryness, Complies with the test for sterility, Appendix XVI A.
dissolve the residue in 2 mL of glacial acetic acid and add
1 mL of dilute potassium dichromate solution. A golden yellow
precipitate is produced.
C. Shake 50 mg with 10 mL of water and filter. To 5 mL of
the filtrate add a few drops of silver nitrate solution.
Cobalt Oxide
No precipitate is produced. Heat 50 mg with 2 mL of 240.8 1307-96-9
alcoholic potassium hydroxide solution on a water bath for
15 minutes, add 15 mg of activated charcoal, shake and filter. Action and use
Acidify the filtrate with 2M nitric acid. The solution yields Used in the prevention of cobalt deficiency in ruminants.
reaction A characteristic of chlorides, Appendix VI.
DEFINITION
TESTS Cobalt Oxide consists of cobalt(n,m) oxide (tricobalt
Water tetraoxide) with a small proportion of cobalt(m) oxide
Not more than 5.0% w/w, Appendix IX C. Use 0.5 g. (dicobalt trioxide). It contains not less than 70.0% and not
ASSAY more than 75.0% of Co, ca1culated with reference to the
0
substance ignited at about 600 •
For cloxacillin benzathine
To 60 mg" add 40 mL of methanol, shake to dissolve, add CHARACTERISTICS
25 mL of 1M sodium hydroxide and allow to stand for A black \powder.
30 minutes. Add 27.5 mL of 1M hydrochloric acid and Practically insoluble in water. It dissolves in mineral acids and
sufficient water to produce 100 mL, mix, transfer 20 mL of in solutions of the alkali hydroxides.
the solution to a stoppered fiask, add 30 mL of O.OlM iodine
VS close the fiask with a wet stopper and allow to stand for IDENTIFICATION
J
15 minutes protected from light. Titrate the excess of iodine A. Dissolve 50 mg, with warming, in 5 mL of hydrochloric
with 0.02M sodium thiosulfate VS, using starch mucilage, added acid and add 10 mL of water. To 2 mL of the solution add
towards the end of the titration, as indicator. Add a further 1 mL of 5M sodium hydroxide. A blue precipitate which
12 mg of the substance being examined to 10 mL of water, becomes pink on warming is produced. Reserve the
swirl to disperse, add 30 mL of O.OlM iodine VS and titrate remainder of the solution for use in test B.
Deltamethrin 55
Me H
O
;::-
;::-
~ I O
~
AJ
exhibits a well-defined maximum onIy at 265 nm. O H CN
TESTS
Light absorption 505.2 52918-63-5
Dissolve 40 mg in 10 mI of hot chloroform and, keeping the
solution warm, dilute slowly with 70 mI of absolute ethanol. Action and use
Cool and dilute to 100 mI with absolute ethanol. Immediately Insecticide (veterinary).
dilute 10 mI 10 100 mI with absolute ethanol. To 10 mI of the Preparation
solution add 10 mI of O.lM hydrochloric acid and dilute to Deltamethrin Pour-on
100 mI with absolute ethanol. The absorbance of the resulting
solution at the maximum at 265 nm is 0.38 to 0.42, DEFINITION
calculated with reference 10 the dried substance, Deltamethrin is (S)-(X-cyano-3-phenoxybenzyl-
Appendix II B. (lR,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyc1opropane
carboxylate. It contains not less than 97.0% and not more
than 101.0% of C22H19Br2N03.
56 Deltamethrin
where V titration volume obtained in the becisthemic 0.04 volume of propan-2-ol, 2 volumes of acetonitrile,
acid ch10ride test, 10 volumes of dichloromethane and 100 volumes of hexane.
PI weight of sample used in the becisthemic SYSTEM SUITABILITY
chloride test, The test is not valid unless, in the chromatogram obtained
Pz weight of sample used in this test. with solution (3), a peak due to (R)-deltamethrin appears
Each mI of 0.02M sodium hydroxide VS is equivalent to immediately before the principal peak, as indicated in the
5.959 mg of becisthemic acid, CSHlOBrzOz. reference chromatogram supplied with deltamethrin
Becisthemic anhydride impurity standard BPCRS.
To 1.0 g add 10 mI of O.OlM aniline in cyclohexane and 10 mI DETERMINATION OF CONTENT
of glacial acetic acid. Stopper the fiask and allow to stand at Calculate the content of C2zHI9BrzN03 using the dec1ared
room temperature for 1 hour. Titrate with O.OIM perchloric content of CZZHI9BrzN03 in deltamethrin BPCRS.
acid VS using crystal violet solution as indicatQt. Repeat the
procedure omitting the substance being examined. Correct IMPURITIES
the volume of titrant for any contribution due !to twice the The impurities limited by the requirements of this
becisthemic acid chloride content calculated. using the aboye monograph inc1ude:
formula. Each mI of O.OlM perchloric acid VS is equivalent to - Becisthemic acid,
5.779 mg of becisthemic anhydride, CI6HISBr403. - Beci~themic anhydride,
- Becisthemic acid chloride.
Related substances
Carry out the method for thin-Iayer chromatography,
Appendix nI A, using the following solutions in toluene.
(1) 2.0% w/v of the substance being examined.
(2) 0.5% w/v of the substance being examined.
(3) 0.020% w/v of the substance being examined.
(4) 0.010% w/v ofthe substance being examined.
Dembrexine Hydrochloride Monohydrate 57
20 - 25 50 --t 75 50 --t 25
Br
)J Br
Detection Spectrophotometer at 250 nm.
1njection 1O 1lL.
Relative retention : With reference to dembrexine
C13H17Br2N02,HCI,H20 433.6 52702-51-9
(retention tim'e = about 6 min): impurity A = about 2.3;
~E~ ___________________________________________
impurity B = l:\bout 1.3.
DEFINITION System suitability Reference solution (b):
trans-4- [(3,5-Dibromo-2-hydroxybenzyl)amino] cyc1ohexanol - resolution: minimum 2 between the peaks due to
hydrochIoride monohydrate. dembrexine and impurity E.
Content Limits:
98.0 per cent to 101.0 per cent (anhydrous substance). - impurities A, B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
CHARACTERS reference solution (a) (0.2 per cent);
Appearance - unspecijied impurities: for each impurity, not more than the
White or almost white, crystalline powder. area of the principal peak in the chromatogram obtained
Solubility with reference solution (a) (0.2 per cent);
Slightly soluble in water, freely soluble in methanol, slightly - total: not more than 2.5 times the area of the principal
soluble in anhydrous ethanol. peak in the chromatogram obtained with reference
IDENTIFICATION solution (a) (0.5 per cent);
- disregard limit: 0.5 times the area of the principal peak in
A. Infrared absorption spectrophotometry (2.2.24).
the chromatogram obtained with reference solution (a)
Comparison dembrexine hydrochloride monohydrate CRS. (0.1 per cent); disregard any peak due to the blank.
B. It gives reaction (a) of chlorides (2.3.1).
Water (2.5.12)
TESTS 3.5 per cent to 5.0 per cent, determined on 0.500 g.
Related substances Sulfated ash (2.4.14)
Liquid chromatography (2.2.29). Prepare the solutions Maximum 0.1 per cent, determined on 1.0 g.
immediately before use.
ASSAY
Test solution Dissolve 25.0 mg of the substance to be
Dissolve 0.350 g in 40 mL of methanol R. Add 40 mL of
examined in methanol R and dilute to 10.0 mL with the same
acetone R and 1 mL of 0.1 M hydrochloric acid. Carry out a
solvento
potentiometric titration (2.2.20) using 0.1 M sodium
Reference solution (a) Dilute 1.0 mL of the test solution to hydroxide. Read the volume added between the 2 points of
50.0 mL with methanol R. Dilute 1.0 mL of this solution to inflexion.
10. O mL with methanol R.
1 mL of 0.1 M sodium hydroxide is equivalent to 41.56 mg of
Reference solution (b) Dissolve 2.5 mg of tribromophenol R
C13HlSBr2CIN02'
(impurity E) in methanol R and dilute to 50.0 mL with the
same solvento To 1.0 mL of this solution add 1.0 mL of the IMPURITIES
test solution and dilute to 10.0 mL with methanol R. Specijied impurities A, B.
Blank solution methanol R. Other detectable impurities (the following substances would, if
Column: present at a sufficient level, be detected by one or other of
- size: 1 = 0.15 m, 0 = 4.0 mm; the tests in the monograph. They are limited by the general
- stationary phase: end-capped octadecylsilyl silica gel for acceptance criterion for other/unspecified impurities and/or
chromatography R (5 Ilm); by the general monograph Substances for pharmaceutical use
- temperature: 40 oc. (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Mobile phase:
Control of impurities in substances for pharmaceutical use):
- mobile phase A: dissolve 1.0 g of potassium dihydrogen
C, D, E.
phosphate R in 900 mL of water R, adjust to pH7.4 with
0.5 M potassium hydroxide and dilute to 1000 mL with
water R; mix 80 volumes of this solution with 20 volumes
of methanol R;
- mobile phase B: methanol R, acetonitrile R (20:80 V/V);
A. trans-4-[(3,5-dibromo-
2-hydroxybenzylidene)amino] cyc1ohexanol,
58 Detomidine Hydrochloride
H~~~-~
Dissolve 0.25 g in water R and dilute to 25 mL with the
same solvento
~ OH
Sr '# Sr
Related substances
Liquid chromatography (2.2.29).
Test solution Dissolve 25.0 mg of the substance to be
B. cis-4- [(3,5-dibromo-2-hydroxybenzyl)amino] cyc1ohexanol, examined in 20 mL of the mobile phase and dilute to
50.0 mL with the mobile phase.
R1 Reference solution (a) Dilute 0.20 mL of the test solution to
~OH 100.0 mL with the mobile phase.
R2 M R3
Reference solution (b) Dissolve 1 mg of detomidine
impurity B CRS in the mobile phase and dilute to 100 mL
with the mobile phase. Dilute 1 mL of this solution to
C. Rl = CHO, R2 = R3 = Br: 10 mL with reference solution (a).
3,5-dibromo-2-hydroxybenzaldehyde, Column:
D. Rl = CHO, R2 = R3 = H: 2-hydroxybenzaldehyde -- size: 1 = 0.15 m, 0 = 4.6 mm;
(salicylaldehyde), -- stationary phase: octylsilyl silica gel for chromatography R
E. Rl = R2 = R3 = Br: 2,4,6-tribromophenol. (5 /lm).
__________________________________________ ffiE~
Mobile phase acetonitrile R, 2.64 gIL solution of ammonium
phosphate R (35:65 V/V).
Flow rate 1 mUmin.
Detection Spectrophotometer at 220 nm.
Detomidine Hydrochloride Jnjection 20 /lL.
Run time 4 times the retention time of detomidine.
(Detomidine Hydrochloride for Veterinary Use)
Relative retention With reference to detomidine
Ph Eur monograph 1414)
(retention time = about 7 min): impurity A = about 0.4;
impurity B = about 2.0; impurity C = about 3.0.
System suitability Reference solution (b):
, Hel
-- resolution: minimum 5 between the peaks due to
detomidine and impurity B.
Limits:
222.7 90038-01-0 -- correction factor: multiply by 2.7 the area of any peak due
to impurity C and its diastereoisomer e1uting with a
Action and use relative retention time of about 3;
Alphaz-adrenoceptor agonist. -- impurity C: for the sum of the are as of the peaks due to
impurity C and its diastereoisomer, not more than
ffiE~ __________________________________________
2.5 times the area of the principal peak in the
DEFINITION chromatogram obtained with reference solution (a)
4-(2,3-Dimethylbenzyl)-IH-imidazole hydrochloride. (0.5 per cent);
-- any other impurity: for each impurity, not more than the
Content
area of the principal peak in the chromatogram obtained
98.5 per cent to 101.5 per cent (dried substance).
with reference solution (a) (0.2 per cent);
CHARACTERS --/ total: not more than 5 times the area of the principal peak
Appearance in the chromatogram obtained with reference solution (a)
White or almost white, hygroscopic, crystalline powder. (1 per cent);
Solubility -- disregard limit: 0.25 times the area of the principal peak in
Soluble in water, free1y soluble in ethanol (96 per cent), very the chromatogram obtained with reference solution (a)
slight1y soluble in methylene chloride, practically insoluble in (0.05 per cent).
acetone. Loss on drying (2.2.32)
mp Maximum 0.5 per cent, determined on 1.000 g by drying in
About 160 oC. oven at 105 oC.
IMPURITIES Solubility
Specijied impurities C. PracticalIy insoluble in water, sparingly soluble in
Other detectable impurities (the following substances would, if dimethylformamide, practicalIy insoluble in alcohol and
present at a sufficient level, be detected by one or other of methylene chIoride.
the tests in the monograph. They are limited by the general IDENTIFICATION
acceptance criterion for other/unspecified impurities and/or Infrared absorption spectrophotometry (2.2.24).
by the general monograph Substances for pharmaceutical use
Comparison Ph. Eur. reference spectrum of diclazun'l.
(2034). It is therefore not necessary to identify these /
impurities for demonstration of compliance. See also 5.10. TESTS
Control of impurities in substances for pharmaceutical use): A, B. Related substances
Liquid chromatography (2.2.29).
Test solution Dissolve 20.0 mg of the substance to be
W
NH
H3 C
1.# ,
I j and enantiomer
examined in"dimethylformamide R and dilute to 20.0 mL with
the same solvento
CH 3 H OH Reference solutil{n (a) Dissolve 5 mg of diclazurillor system
suitability CRS in dimethylformamide R and dilute to 5.0 mL
A. (RS)-(2,3-dimethylphenyl)(lH-imidazol-4-yl)methanol, with the same solvento
Reference solution (b) Dilute 1.0 mL of the test solution to
100.0 mL with dimethylformamide R. Dilute 5.0 mL ofthe
solution to 20.0 mL with dimethylformamide R.
and enantiomer Column:
-- size: 1 = 0.10 m, 0 = 4.6 mm,
-- stationary phase: base-deactivated octadecylsilyl silica gel for
chromatography R (3 ¡..tm),
-- temperature: 35 oc.
B. (RS)-(1-benzyl-1H-imidazol-5-yl)
Mobile phase:
(2, 3-dimethylphenyl) methanol,
-- mobile phase A: mix 10 volumes of a 6.3 giL solution of
ammonium formate R adjusted to pH 4.0 with anhydrous
formic acid R, 15 volumes of acetonitrile R and 75 volumes
of water R,
-- mobile phase B: mix 10 volumes of a 6.3 giL solution of
ammoniumformate R adjusted to pH 4.0 with anhydrous
formic acid R, 85 volumes of acetonitrile R and 5 volumes
C. 4- [(2,3-dimethylcyclohexyl)methyl] -lH-imidazole. of water R,
___________________________________________ PhEw Time Mobile phase A Mobile phase B
(mio) (per ceot VM (per ceot VM
0-20 100 ~ O O ~ 100
20 - 25 O 100
Diclazuril
(Diclazuril for Veterinary Use, Flow rate 1.0 mUmin.
Ph Eur monograph 1718) Detection Spectrophotometer at 230 nm.
Injection 5 ¡..tL.
System suitability Reference solution (a):
-- peak-to-valley ratio: minimum of 1.5, where Hp = height
and enantiomer aboye the baseline of the peak due to impurity D and
H v = height aboye the baseline of the lowest point of the
curve separating this peak from the peak due to diclazuril.
Limits:
-- correction factors: for the calculation of contents, multiply
407.6 101831-37-2 the peak areas of the following impurities by the
corresponding correction factor: impurity D = 1.9;
Action and use
impurity H = 1.4,
Antiprotozoal (veterinary); coccidiosis.
-- impurity D: not more than 0.4 times the area of the
~Ew ___________________________________________ principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent),
DEFINITION
-- any other impurity: not more than the area of the principal
(RS)-( 4-Chlorophenyl) [2,6-dichloro-4-(3,5-dioxo- peak in the chromatogram obtained with reference
4,5-dihydro-1 ,2,4-triazin-2 (3H) -yl)phenyl] acetonitrile. solution (b) (0.25 per cent),
Content ~ total: not more than 4 times the area of the principal peak
99.0 per cent to 101.0 per cent (dried substance). in the chromatogram obtained with reference solution (b)
CHARACTERS (1.0 per cent),
Appearance
White or light yellow powder.
60 Difioxacin Hydrochloride Trihydrate for Veterinary U se
H\ eN el
~~
RV el~N./'..,.NH andenantiomer
~0o
R'
elA)el~R
record new spectra using the residues.
B. It gives reaction (a) of chlorides (2.3.1).
C. Water (se e Tests).
E. R = NH2 : (RS)-( 4-amino-
2,6-dichlorophenyl) (4-chlorophenyl)acetonitrile,
H. R = H: (RS)-( 4-chlorophenyl)
(2, 6-dichlorophenyl) acetonitrile,
Difloxacin Hydrochloride Trihydrate for Veterinary Use 61
Dihydrostreptomycin Sulfate
Dihydrostreptomycin Sulphate
(Dihydrostreptomycin Sulfate for Veterinary Use,
Ph Eur monograph 0485)
c. 1-(4-chlorophenyl)-6-fiuoro-7-(4-methylpiperazin-l-yl)-
4-oxo-l ,4-dihydroquinoline-3-carboxylic acid,
5490-27-7
DEFINITION
Main compound
bis [N,N' , ,- [( lR,2R,3S,4R,5R,6S)-4- [[5-deoxy-2-0- [2-deoxy-
E. 7 -chIoro-l-(4-fiuorophenyl)-6-(4-methylpiperazin-l-yl)-
2-(methylamino )-C'i-L-glucopyranosyl] -3-C-(hydroxymethyl)-
4-oxo-l ,4-dihydroquinoline-3-carboxylic acid,
C'i-L-lyxofuranosyl]oxy]-2,5,6-trihydroxycyc1ohexane-
1,3-diyl] diguanidine] trisulfate.
Sulfate of a substance obtained by catalytic hydrogenation of
streptomycin or by any other means.
Semi-synthetic product derived from a fermentation producto
Stabilisers may be added.
Content:
-- sum of the percentage contents of dihydrostreptomycin sulfate
and streptomycin sulfate: 95.0 per cent to 102.0 per cent
(dried substance);
-- streptomycin sulfate: maximum 2.0 per cent (dried
F. 6-fiuoro-N, l-bis(4-fiuorophenyl)-7 -( 4-methylpiperazin- substance) .
l-yl)-4-oxo-l ,4-dihydroquinoline-3-carboxamide,
PRODUCTION
The method of manufacture is validated to demonstrate that
i,
the product, if tested, would comply with the following test.
Abnormal toxicity (2.6.9)
Inject into each mouse 1 mg dissolved in 0.5 mL of water for
injections R.
CHARA¿TERS
Appearance
White or almost white, hygroscopic powder.
Solubility
G. 7 -chloro-6-fiuoro-l-(4-fiuorophenyl)-4-oxo- Freely soluble in water, practically insoluble in acetone, in
1,4-dihydroquinoline-3-carboxylic acid. ethanol (96 per cent) and in methanol.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Dihydrostreptornycin Sulfate 63
NH
-- impurity C: not more than twice the area of the principal
peak in the chromatogram obtained with reference HO HN)lNH 2
solution (b) (2.0 per cent);
-- impurities A J B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
HO--ÜOH
reference solution (b) (1.0 per cent); HO HN NH 2
-- any other impurity: for each impurity, not more than the y
NH
area of the principal peak in the chromatogram obtained
with reference solution (b) (1.0 per cent);
-- total: not more than 5 times the area of the principal peak A. N J N"'-[(lR,2s,3S,4R,5r,6S)-2,4,5,6-
in the chromatogram obtained with reference solution (b) tetrahydroxycyc1ohexane-1 ,3-diyl] diguanidine (streptidine),
(5.0 per cent);
-- disregard limit: the area of the principal peak in the NH
chromatogram obtained with reference solution (c) HO HN)lNH 2
(0.1 per cent); disregard the peak due to streptomycin.
Heavy metals (2.4.8)
20 ppm.
HO--ÜOH
1.0 g complies with test C. Prepare the reference solution
using 2 mL of lead standard solution (lO ppm Pb) R.
HO ......... O
~~)
O HN
yNH NH 2
Sulfated ash
Dimetridazole Not more than 0.1 %, Appendix IX A.
ASSAY
Me
I Carry out Method I for non-aqueous titration,
OZN--(r Me Appendix VIII A, using 0.3 g and crystal violet solution as
indicator. Each mL of O.lM perchloric acid VS is equivalent to
14.11 mg of C sH 7 N 3 0 2 •
STORAGE
141.1 551-92-8
Dimetridazole should be protected from light.
Action and use
Antiprotozoal (veterinary).
DEFINITION
Dimetridazole is 1,2-dimethyl-5-nitroimidazole. It contains
Dimpylate\
not less than 98.0% and not more than 101.0% of
C sH 7N 30 2, ca1culated with reference to the anhydrous Me
1~ ~
substance.
CHARACTERISTIC
An almost white to brownish yellow powder which darkens Pr' N O-P-OEt
I
on exposure to light; odourless or almost odourless, OEt
Slightly soluble in water; freely soluble in chloroform; sparingly
soluble in ethanol (96%); slightly soluble in ether. 304.4 333-41-5
IDENTIFICATION
Action and use
A. The infrared absorption spectrum, Appendix n A, is
Insecticide.
concordant with the reference spectrum of dimetridazole
(RSV 16). DEFINITION o
B. The light absorption, Appendix n B, in the range 230 to Dimpylate is O,O-diethyl O-(2-isopropyl-6-methylpyrimidin-
350 nm, of a 0:002% w/v solution in methanol exhibits a 4-yl)phosphorothioate. It contains not less than 95.0% and
well-defined maximum on1y at 309 nm. The absorbance at the not more than 101.0% of C12H21N203PS, ca1culated with
maximum at 309 nm is about 1.3. reference to the anhydrous substance.
C. Dissolve 0.1 g in 20 mL of ether, add 10 mL of a 1% w/v CHARACTERISTICS
solution of picric acid in ether, induce crystallisation and allow A clear, yellowish brown, slight1y viscous liquido
to stand. A precipitate is produced which after washing with
Practically insoluble in water; miscible with ethanol (96%),
ether and drying at 105° has a melting point of about 160°,
with ether and with most organic solvents.
Appendix V A.
TESTS IDENTIFICATION
A. The infrared absorption spectrum, Appendix n A, is
Melting point
concordant with the reference spectrum of dimpylate (RSV 49).
138° to 141°, Appendix V A.
B. In the Assay, the chromatogram obtained with solution
2-Methyl-5-nitroimidazole
(1) shows a peak with the same retention time as the peak
Carry out the method for thin-layer chromatography,
due to dimpylate in the chromatogram obtained with
Appendix nI A, using the following solutions in chloroform.
solution (3).
Protect the solutions from light.
(1) 2.0% w/v of the substance being examined. TESTS
Toluene
(2) 0.010% w/v of 2-methyl-5-nitroimidazole BPCRS.
Not more than 1% v/v when determined by the test for
CHROMATOGRAPHIC CONDITIONS residual solvents, Appendix VIn L.
(a) Use as the coating silica gel F254. Related substances
(b) Use the mobile phase as described be1ow. Carry out the method for gas chromatography,
(c) Apply 5 ¡.tL of each solution. Appendix III B, using the following solutions. Solution (1)
(d) Develop the plate to 15 cm. contains 0.5% w/v of the substance being examined in
dichloromethane. Solution (2) contains 0.5% w/v of the
(e) After removal of the plate allow it to dry in air and
substance being examined and 0.025% w/v of diethyl
examine under ultraviolet light (254 nm).
phthalate (internal standard) in dichloromethane. Solution (3)
MOBILE PHASE contains 0.0020% w/v of the substance being examined and
1 volume of propan-2-o1 and 9 volumes of chloroform. 0.025% w/v of diethyl phthalate in dichloromethane. Solution
LIMITS
(4) contains 0.5 % w/v of dimpylate for chromatography BPCRS
and 0.025% w/v of dz'ethyl phthalate in dichloromethane.
Any spot in the chromatogram obtained with solution (1) Solution (5) contains 0.012% v/v of toluene in
corresponding to 2-methyl-5-nitroimidazole is not more
dichloromethane.
intense than the spot in the chromatogram obtained with
solution (2) (0.5%). The chromatographic procedure may be carried out using
(a) a fused silica capillary column (15 m x 0.32 mm) coated
Water with a 1 ¡.tm film of dimethyl silicone gum (SE-54 is suitable)
Not more than 1.0% w/w, Appendix IX C. Use 1.0 g. fitted with a precolumn (0.2 m x 0.53 mm) coated with a
66 Dimpylate
1 11m film of dimethylsilicone gum, the temperature The chromatographic procedure may be carried out using
programme described below with the inlet port at room (a) a fused silica capillary column (15 m x 0.53 mm) coated
temperature and the detector at 280 0 and (b) hydrogen as the with a 1.5 11m film of dimethyl silicone gum (SE-54 from
carrier gas at a flow rate of 40 mL per minute and nitrogen as J & W Scientific is suitable) at a temperature of 1100
the make up gas with a flow rate of 50 mL per minute. increasing linearly at arate of 60 per minute to 215 0 with the
inlet port at 250 0 and the detector at 250 0 and (b) helium as
the carrier gas at a flow rate of 20 mL per minute with
Time (Minutes) Temperature Comment
nitrogen as the make up gas with a flow rate of 10 mL per
0-1 35° isothermal minute.
The assay is not valid unless, in the chromatogram obtained
1 - 15.5 35°~180° linear in crease
with solution (3), the resolution factor between the peaks due
100/minute
to dimpylate and the internal standard is at least 2.
15.5 - 23.5 180° isothermal Calculate the content of C12H21N2Ü3PS from the
23.5 - 30.5 1800~250° linear increase chromatograms obtained and using the declared content of
10 0/minute C12H21N2Ü3PS in dimpylate BPCRS.
N~
Water
Not more than 0.1 % w/w, Appendix IX C. Use 2 g.
ASSAY pri)lN"lo
Carry out the method for gas chromatography, I
Et
Appendix III B, using solutions in 4-methylpentan-2-one
containing (1) 0.2% w/v of the substance being examined,
(2) 0.2% w/v of the substance being examined and F. 3-Ethyl-2-isopropyl-6-methyl-4-oxo~3,4-
0.15% w/v of diethyl phthalate (internal standard) and (3) dihydropyrimidine,
0.2% w/v of dimpylate BPCRS and 0.15% w/v of diethyl
phthalate.
Dinitolmide 67
G.4-Hydroxy-2-isopropyl-6-methylpyrimidine,
s11
o
11
EtO - P - O - P - OEt
I I
OEt OEt N. O-ethyl O,O-(2-isopropyl-6-methylpyrimidin-
4-yl)phosphorothiophate.
H. Tetraethyl thionopyrophosphate,
s11
S
11
Dinitolmide:
EtO - P - O - P - OEt
I I
OEt OEt
1. Tetraethyl dithionopyrophosphate,
Me
N~ 148-01-6
Prl
,)l"Á
N
'2
O-P-OEt
Action and use
I Antiprotozoal (veterinary).
OEt
DEFINITION
J. 0,0-DiethylO-(2-isopropyl-6-methylpyrimidin- Dinitolmide is 3,5-dinitro-o-toluamide. It contains not less
4-yl)phosphate, than 98.0% and not more than 100.5% of C sH 7N 3 0 s,
calculated with reference to the dried substance.
Me CHARACTERISTICS
1i ~
A cream to light tan powder.
Practically insoluble in water; soluble in acetone; slight1y
soluble in chloroform, in ethanol (96%) and in ether.
Prl N' S-P-OEt
I
OEt
IDENTIFICATION
A. The infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum of dinitolmide
K. 0,0-Diethyl S-(2-isopropyl-6-methylpyrimidin- (RSV 17).
4-yl)phosphorothiophate,
B. Heat 1 g with 20 mL of 9M sulfuric acid under a reflux
condenser for 1 hour, cool, add 50 mL of water and filter.
Me The melting point of the residue, after washing with water
Pr
1\ ~
N O-P-SEt
I
OEt
and drying at 105°, is about 205°, Appendix V A.
TESTS
Acid value
Not more than 5.0, Appendix X B, using 0.5 g and 50 mL
of ethanol (96%) as the solvento
L. O,S-Diethyl 0-(2-isopropyl-6-methylpyrimidin- Melting point
4-yl)phosphorothiophate, 177° to 181°, Appendix V A.
Related substances
Carry out the method for thin-layer chromatography,
Appendix III A, using a silica gel F 2S4 precoated plate
(Merck plates are suitable) and a mixture of 5 volumes of
glacial acetic acid, 10 volumes of methanol and 85 volumes of
chloroform as the mobile phase. Apply separately to the plate
10 IlL of each of three solutions in acetone containing (1)
2.5% w/v of the substance being examined, (2) 0.0125% w/v
ofthe substance being examined and (3) 0.0125% w/v of
o-toluic acid. After removal of the plate allow it to dry in air
and examine under ultraviolet light (254 nm). Spray with
M.Bis(2-isopropyl-6-methylpyrimidin-4-yl)· sulfide, titanium(IlI) chloride solution diluted 1 to 5 with water,
heat at 100° for 5 minutes and spray with alcoholic
dimethylaminobenzaldehyde solution. When examined under
68 Diprenorphine Hydrochloride
ultraviolet light the spot in the chromatogram obtained with exhibits a maximum on1y at 287 nm. The absorbance at the
solution (3) is more intense than any corresponding spot in maximum is about 0.70.
the chromatogram obtained with solution (1) (0.5%). C. The light absorption, Appendix n B, in the range 230 to
By each method of visualisation any secondary spot in the 350 nm of a 0.02% w/v solution in O.lM sodium hydroxide
chromatogram obtained with solution (1) is not more intense exhibits a maximum on1y at 301 nm. The absorbance at the
than the spot in the chromatogram obtained with maximum is about 1.1.
solution (2) (0.5%). D. Yields reaction A characteristic of chlorides, Appendix VI.
Loss on drying
When dried to constant weight at 105°, loses not more than
TESTS
1.0% of its weight. Use 1 g. Acidity
pH of a 2.0% w/v solution, 4.5 to 6.0, Appendix V L.
ASSAY
Specific optica1 rotation
Dissolve 0.15 g in acetone and dilute to 50 mL. To 10 mL of
In a solution prepared by dissolving 0.5 g in sufficient
this solution add 10 mL of glacial acetic acid and 15 mL of a
methanol to produce 25 mL, -97.0 to -107.0, calculated with
40% w/v solution of sodium acetate. Maintain a stream of
reference to the dried substance, Appendix V F.
carbon dioxide through the flask throughout the
determination. Add 25 mL of O.lM titanium(lIl) chloride VS Re1ated substances
and allow to stand for 5 minutes. Add 10 mL of hydrochloric Carry out the method for thin-layer chromatography,
acid, 10 mL of water, and 1 mL of a 10% w/v solution of Appendix In A, using silica gel G as the coating substance
potassium thiocyanate. Titrate with O.lM ammonium iron(lIl) and a mixture of 1 volume of water, 5 volumes of methanol,
sulfate VS until the solution becomes colourless and then 30 volumes of butan-2-one, 80 volumes of acetone and
orange. Repeat the operation without the substance being 100 volumes of cyclohexane as the mobile phase. Apply
examined. The difference between the titrations represents separately to the plate 20 ¡.tL of each of two solutions of the
the amount of titanium(m) chloride required to reduce the substance being examined in methanol containing (1)
dinitolmide. Each mL of O.lM titanium(lIl) chloride VS is 2.0% w/v and (2) 0.020% w/v. Add at each point of
equivalent to 1.876 mg of CgH7N 30 S. application 10 ¡.tL of a mixture of 4 volumes of methanol and
1 volume of 13.5M ammonia. After removal of the plate,
allow it to dry in air and spray with a 1% w/v solution of
iodine in methanol. Any secondary spot in the chromatogram
Diprenorphine Hydrochloride obtained with solution (1) is not more intense than the spot
in the chromatogram obtained with solution (2) (1 %).
Loss on drying
When dried to constant weight at 105°, loses not more than
2.0% of its weight. Use 1 g.
Sulfated ash
Not more than 0.1 %, Appendix IX A.
ASSAY
Carry out Method I for non-aqueous titration,
Appendix VIII A, using 0.5 g, adding 7 mL of mercury(Il)
acetate solution and using crystal violet solution as indicator.
Each mL ofO.1Mperchloric acid VS is equivalent to 46.21 mg
462.1 16808-86-9
C26H3SN04,HCl.
Action and use STORAGE
Opio id receptor antagonist. Diprenorphine Hydroch1oride should be protected from light.
Preparation
Diprenorphine Injection
DEFINITION
Diprenorphine Hydrochloride is N-cyclopropylmethyl- Enilconazole
7 ,8-dihydro-7 ct-(1-hydroxy-1-methylethyl)-6-0-methyl-
6ct,14ct-ethanonormorphine hydroch1oride. It contains not (Bnilconazole for Veterinary Use,
less than 98.5% and not more than 101.0% of Ph Bur monograph 1720)
C26H3sN04,HCI, calculated with reference tb the dried
substance. CI
CHARACTERISTICS
A white or almost white, crystalline powder.
CI~H ,
Sparingly soluble in water; slight1y soluble in ethanol (96%); H2C~O~ and enantiomer
H2e~o~
HU and enantiomer
mixture and dilute to 50.0 mL with the solvent mixture.
Dilute 4.0 mL of this solution to 10.0 mL with the solvent
mixture.
Plate TLC silica gel F254 plate R (2-10 Ilm).
N Mobile phase butanol R~ water R~ anhydrous acetic acid R~
() N
ethyl acetate R (15:15:20:50 V/V/V/V).
Application 10 1lL.
F. 1- [(2RS) - 2-(3,4-dichlorophenyl)-2-(prop-2-enyloxy)ethyl]- Development Over 3/4 of the plateo
1H-imidazole. Drying In air.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur Detection Examine in ultraviolet light at 254 nm.
Results:
- impurity A: any spot due to impurity A is not more
intense than the spot in the chromatogram obtained with
the reference solution (0.2 per cent).
Enrofloxacin Related substances
(Enrofioxacin for Veterinary Use, Liquid chromatography (2.2.29).
Ph Eur monograph 2229) Test solution Dissolve 50 mg of the substance to be
examined in the mobile phase and dilute to 50.0 mL with
the mobile phase.
Reference solution (a) Dissolve 10 mg of enrofioxacin for
system suitability CRS (containing impurities B and C) and
dilute to 10 mL with the mobile phase.
Reference solution (b) Dilute 1.0 mL of the test solution
to 50.0 mL with the mobile phase. Dilute 1.0 mL ofthis
solution to 10.0 mL with the mobile phase.
359.4 93106-60-6
Column:
Action and use - size: 1 = 0.15 m, 0 = 4.6 mm;
Fluoroquinolone antibacterial (veterinary). - stationary phase: base-deactivated end-capped octadecylsilyl
szlica gel for chromatography R (5 Ilm);
~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ - temperature: 40 oC.
DEFINITION Mobzle phase Mix 15 volumes of methanol R and 85 volumes
1-Cyc1opropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo- of a 2.9 gIL solution of phosphoric acid R, previously adjusted
1,4-dihydroquinoline-3-carboxylic acid. to pH 2.3 with triethylamine R.
Content Flow rate 1.5 mUmin.
98.5 per cent to 101.5 per cent (dried substance). Detection Spectrophotometer at 270 nm.
CHARACTERS Jnjection 10 1lL.
Appearance Run time 3 times the retention time of enrofloxacin.
Pale yellowish or light yellow, crystalline powqer. Jdentification of impurities Use the chromatogram supplied
Solubility with enrofioxacin for system suitabz1ity CRS and the
Practically insoluble in water, freely soluble in methylene chromatogram obtained with reference solution (a) to
chloride, slight1y soluble in methanol. identify,the peaks due to impurities B and C.
1,
Limits:
- impunty B: not more than 2.5 times the area of the
principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent);
- impunty C: not more than the area of the principal peak
in the chromatogram obtained with reference solution (b)
(0.2 per cent);
- unspecified impunties: for each impurity, not more tl}an the B. ciprofloxacin,
area of the principal peak in the chromatogram obtained
with reference solution (b) (0.20 per cent);
- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference sOÚltion (b)
(1.0 per cent);
- disregard limit: 0.5 times the area of the principal peak in
the chromatogram obtained with reference solution (b)
(0.1 per cent).
Heavy metals (Lt¡.~8) C. 1-cyclopropyl-7 -( 4-ethylpiperazin-l-yl)-4-oxo-
Maximum 20 ppm. 1,4-dihydroquinoline-3-carboxylic acid,
Dissolve 1.5 g in a mixture of 5 mL of 2 M acetic acid and
10 mL of water R. Filter. 12 mL of the filtrate after adding
2 mL of water R (instead of buffersolution) complies with
test E. Prepare the reference solution using 12 mL of lead
standard solution (2 ppm Pb) R.
Loss on drying (2.2.32)
Maximum 1.0 per cent, determined on 2.000 g by drying
under high vacuum at 120 oC for 6 h.
E. 6-chloro-1-cyclopropyl-7 -(4-ethylpiperazin-1-yl)-4-oxo-
Sulfated ash (2.4.14)
1,4-dihydroquinoline-3-carboxylic acid,
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.250 g in 100 mL of anhydrous acetic acid R and
titrate with 0.1 M perchlonc acid determining the end-point
potentiometrically (2.2.20).
1 mL of 0.1 M perchlonc acid is equivalent to 35.94 mg of
C19H22FN303·
STORAGE
Protected from light. F. 1-cyclopropyl-7 -(4-ethylpiperazin-1-yl)-6-fluoroquinolin-
4(1H)-one,
IMPURITIES
Specified impunties A, B, C.
Other detectable impunties (the following substances would, if
present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. G. 7-[ (2-aminoethyl)amino] -1-cyclopropyl-6-fluoro-4-oxo-
Control of impunties in substances for pharmaceutical use):
1,4-dihydroquinoline-3-carboxylic acid.
E, F, G.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
A. 7-chloro-l-cyclopropyl-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid,
72 Etamiphylline Camsilate
y
Sulfated ash
Not more than 0.1 %, Appendix IX A.
ASSAY
Carry out the method for liquid chromatography, NHAc
Appendix III D, using the following solutions in a mixture of
equal volumes of methanol and water. Solution (1) contains E. ethy14-acetamido-2-ethoxybenzoate.
0.002% w/v of the substance being examined. Solution (2)
contains 0.002% w/v of ethopabate BPCRS. Solution (3)
contains 0.002% w/v of ethopabate BPCRS and 0.010% w/v
of methyl 4-acetamido-2-hydroxybenzoate BPCRS. Etorphine Hydrochloride
The chromatographic conditions described under Related
substances may be used.
The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peaks due
to ethopabate and methyl 4-acetamido-2-hydroxybenzoate is ,HCI
at least 1.2.
Calculate the content of C 12 H 1SN0 4 in the substance being
examined using the dec1ared content of C 12H 1S N0 4 in
ethopabate BPCRS.
IMPURITIES
448.0 13764-49-3
COOH
Action and use
¡YOH
DEFINITION
A. 4-aminosalicylic acid, Etorphine Hydrochloride is (6R, 7R, 14R)-7 ,8-dihydro-
7-[(1R)-1-hydroxy-1-methylbutyl] -6-0-methyl-
6,14-ethenomorphine hydrochloride. It contains not less than
98.0% and not more than of 101.0% of CzsH33N04,HCl,
calculated with reference to the dried substance.
74 Febantel
CAUTION Etorphine Hydrochloride is extremely potent and solution (1) is not more intense than the spot in the
extraordinary care should be taken in any procedure in which it is chromatogram obtained with solution (2) (1 %).
used. Any spillage on the skin should be washed off at once. In the Loss on drying
case of accidental injection or absorption through broken skin or When dried to constant weight at 1050, loses not more than
mucous membraneJ a reversing agent should be injected 4.0% of its weight. Use 1 g.
immediately.
Sulfated ash
CHARACTERISTICS Not more than 0.1 %, Appendix IX A.
A white or almost white, microcrystalline powder.
ASSAY
Sparingly soluble in water and in ethanol (96%); very slight1y Carry out Method I for non-aqueous titration,
soluble in chloroform; practically insoluble in ether. Appendix vln A, using 0.5 g, adding 7 mL of mercury(lI)
IDENTIFICATION acetate solution and using crystal violet solution as indicator.
A. The light absorption, Appendix n B, in the range 230 to Each mL of O.lM perchloric acid VS is equivalent to 44.80 mg
350 nm of a 0.02% w/v solution in O.lM hydrochloric acid of C2sH33N04,HCl.
exhibits a maximum onIy at 289 nm. The absorbance at the STORAGE
maximum is about 0.68. Etorphine Hydrochloride should be protected from light.
B. The light absorption, Appendix n B, in the range 230 to
350 nm of a 0.02% w/v solution in O.lM sodium hydroxide
exhibits a maximum only at 302 nm. The absorbance at the
maximum is about 1.2.
C. Carry out the method for thin-layer chromatography, Febantel
Appendix In A, in subdued light using silica gel GF254 as the (Febantel for Veterinary UseJ Ph Eur monograph 2176)
coating substance and a mixture of 10 volumes of
diethylamine, 20 volumes of ethyl acetate and 70 volumes of
toluene as the mobile phase. Apply separately to each half of
the plate 1 IlL of each of two solutions in methanol containing
(1) 2.0% w/v of the substance being examined and (2)
1.8% w/v of diprenorphine BPCRS. Add at each point of
application 10 IlL of a mixture of 4 volumes of methanol and
1 volume of 13.5M ammonia. After removal of the plate,
allow it to dry in air and examine under ultraviolet light
(254 nm). Spray one half of the plate with a mixture of
446.5 58306-30-2
5 volumes of chloroplatinic acid solution, 35 volumes of dilute
potassium iodide solution and 60 volumes of acetone. Spray the Action and use
other half of the plate with a mixture of 1 volume of iron(III) Antihelminthic.
chloride solution R1 and 1 volume of dilute potassium
PhE~ ___________________________________________
hexacyanoferrate(III) solution. The principal spot in the
chromatogram obtained with solution (1) has an Rf value of DEFINITION
about 1.15 relative to that of the spot in the chromatogram Dimethyl N,N'-[[[2-[(methoxyacetyl)amino]-
obtained with solution (2), absorbs ultraviolet light and yields 4-(phenylsulfanyl)phenyl] imino] methylene] dicarbamate.
a reddish violet colour with the iodoplatinate spray reagent
and a blue colour with the iron-hexacyanoferrate spray Content
reagent. 97.5 per cent to 102.0 per cent (dried substance).
D. Yields reaction A characteristic of chlorides, Appendix VI. CHARACTERS
Appearance
TESTS
White or almost white, crystalline powder.
Acidity
pH of a 2.0% w/v solution, 4.0 to 5.5, Appendix V L. S 01 ubility
Practically insoluble in water, soluble in acetone, slight1y
Specific optica1 rotation
soluble in anhydrous ethanol.
In a solution prepared by dissolving 0.5 g in sufficient
methanol to produce 25 mL, -122 to -132, ca1culated with It shows polymorphism (5.9).
reference to the dried substance, Appendix V F. IDENTIFICATION
Related substances Infrared absorption spectrophotometry (2.2.24).
Carry out in subdued light the method for thin~layer Comparison febantel CRS.
chromatographYJ Appendix nI A, using silica gel G as the If the spectra obtained in the solid state show differences,
coating substance and a mixture of 10 volumes of dissolve the substance to be examined and the reference
diethylamine, 20 volumes of ethyl acetate and 70 volumes of substanccl separately in acetone R, evaporate to dryness and
toluene as the mobile phase. Apply separately to the plate record new spectra using the residues.
20 ¡..tL of each of two solutions of the substance being
examined in methanol containing (1) 2.0% w/v and (2) TESTS
0.020% w/v. Add at each point of application 10 IlL of a Related substances
mixture of 4 volumes of methanol and 1 volume of Liquid chromatography (2.2.29).
13.5M ammonia. After removal of the plate, allow it to dry in Solvent mixture acetonitrile RJ tetrahydrofuran R (50:50 V/V).
air and spray with a 1% w/v solution of iodine in methanol. Test solution (a) Dissolve 0.100 g of the substance to be
Any secondary spot in the chromatogram obtained with examined in the solvent mixture and dilute to 10.0 mL with
the solvent mixture.
Fenbendazole 75
100
MeÚO-~=s
10 - 40 O
lOMe
MeS ~
Flow rate 1 mUmin.
Deteetion Spectrophotometer at 280 nm. 278.3 55-38-9
Injeetion 10 ¡.tL. Action and use
Retention time fenbendazole = about 19 min. Insecticide.
System suitability Reference solution (d):
- resolution: minimum 1.5 between the peaks due to DEFINITION
fenbendazole and mebendazole. Fenthion is O,O-dimethyl 0-4-methylthio-
Limits: m-¡tolylphosphorothioate. It contains not Iess than 90.0% and
- impurity A: not more than 2.5 times the area of the not more than 100.5% of ClOHlSÜ3PS2'
corresponding peak in the chromatogram obtained with CHARACTERISTICS
reference solution (b) (0.5 per cent); A yellowish brown, oily substance.
- impurity B: not more than 2.5 times the area of the
corresponding peak in the chromatogram obtained with Irnmiscible with water; miscible with ehlorojorm and with
reference solution (c) (0.5 per cent); ethanol (96%).
- any other impurity: for each impurity, not more than twice IDENTIFICATION
the area of the principal peak in the chromatogram A. The infrared absorption speetrum, Appendix II A, is
obtained with reference solution (a) (0.5 per cent); concordant with the rejerenee speetrum of fenthion (RSV 21).
- total: not more than 4 times the area of the principal peak B. Carry out the method for thin-Iayer ehromatography,
in the chromatogram obtained with reference solution (a) Appendix In A, using siliea gel GF254 as the coating
(1 per cent); " , substance and a mixture of 1 volume of aeetone and
- disregard limit: 0.2 times the area of the principal peak in 4 volumes of hexane as the mobile phase. Apply separately to
the chromatogram obtained with reference solution (a) the pIate 2 ¡.tL of each of two solutions in ehloroform
(0.05 per cent). containing (1) 0.5% w/v of the substance being examined
Heavy metals (2.4.8) and (2t 0.5% w/v of fenthion BPCRS. After removal of the
Maximum 20 ppm. pIate, allow it to dry in air and examine under ultraviolet light
1.0 g complies with test C. Prepare the reference solution (254 nm). The spot in the chromatogram obtained with
using 2 mL of lead standard solution (lO ppm Pb) R. solution (1) corresponds to that in the chromatogram
Loss on drying (2.2.32) obtained with solution (2).
Maximum 1.0 per cent, determined on 1.000 g by drying in C. To 0.15 mL add 3 mL of propan-2-01 and 0.2 g of
an oven at 105 oC for 3 h. potassium hydroxide and heat on a water bath for 15 minutes.
Fluanisone 77
Add 5 mL of water, heat for a further 5 minutes, cool, dilute (RSV 22). Ifthe spectra are not concordant, dissolve 0.1 g of
to 50 mL with water and add 3 mL of iodindted potassium the substance being examined in 3 mI of dichloromethane and
iodide solution. A green colour is produced which gradually evaporate the solvent at room temperature, scratching the
fades. side of the container occasiona1ly with a glass rod and
Related substances prepare a new spectrum of the residue.
Carry out the method' for thin-layer chromatography, B. The light absorption, Appendix II B, in the range 230 to
Appendix III A, using a silica gel F 2S4 precoated plate 350 nm of a 0.002% w/v solution in a mixture of 9 volumes
(Merck silica gel 60 F 2S4 plates are suitable) and a mixture of of propan-2-ol and 1 volume of O.IM hydrochloric acid exhibits
1 volume of acetone and 4 volumes of hexane as the mobile a well-defined maximum onIy at 243 nm. The absorbance
phase. Apply separately to the plate 10 /1L of each of three at 243 nm is about 1.1.
solutions of the substance being examined in methanbl e. Heat 0.5 mI of chromic-sulphuric acid mixture in a small
containing (1) 2.0% w/v, (2) 0.12% w/vand (3) 0.040% w/v. test tube in a water bath for 5 minutes; the solution wets the
After removal of the plate, allow it to dry in air and examine side of the tube readily and there is no greasiness. Add 2 to
under ultraviolet light (254 nm). Any spot in the 3 mg of the slÍbstance being examined and again heat in a
chromatogram obtained with solution (1) with an Rf value of water bath for 5 minutes; the solution does not wet the side
about 0.36 relative to that of the principal spot is not more of the tube and does not pour easily from the tube.
intense than the spot in the chromatogram obtained with
TESTS
solution (2) (6%) and any other secondary spot is not more
intense than the spot in the chromatogram obtained with
Melting point
solution (3) (2%). 72° to 76°, Appendix V A.
Related substances
ASSAY
Carry out the method for thin-layer chromatography,
Carry out the method for gas chromatography,
Appendix III A, using the following solutions.
Appendix III B, using solutions in chloroform containing (1)
0.4% w/v offenthion BPCRS and 0.2% w/v of dibutyl (1) 2.0% w/v of the substance being examined.
phthalate (internal standard), (2) 0.4% w/v of the substance (2) 0.010% w/v of the substance being examined.
being examined and (3) 0.4% w/v of the substance being (3) 0.020% w/v of 4' -fluoro-4-chlorobutyrophenone BPCRS.
examined and 0.2% w/v of the internal standard. (4) 0.010% w/v of 1-(2-methoxyphenyl)piperazine BPCRS.
The chromatographic procedure may be carried out using CHROMATOGRAPHIC CONDITIONS
(a) a glass column (1.5 m x 4 mm) packed with acid-
(a) Use as the coating silica gel GF 2S4 precoated plate
washed, silanised diatomaceous support (80 to 100 mesh) coated
(Merck silica gel 60 plates are suitable).
with 3% w/w of phenyl methyl silicone fluid (OV 17 is
suitable) and maintained at 225°. (b) Use the mobile phase as described below.
Calculate the content of ClOHlS03PS2 using the declared (c) Apply 10 /11 of each solution.
content of ClOHlSÜ3PS2 in fenthion BPCRS. (d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and expose to iodine
vapour for 15 minutes.
MOBILE PHASE
Fluanisone 10 volumes of ethanol (96%) and 90 volumes of chloroform.
LIMITS
In the chromatogram obtained with solution (1):
any spots corresponding to 4'-fluoro-4-chlorobutyrophenone
and 1-(2-methoxyphenyl)piperazine are not more intense
than the spots in the chromatograms obtained with solutions
(3) and (4) respectively (1 % and 0.5%, respectively);
356.4 1480-19-9 any other secondary spot in the chromatogram obtained with
solution (1) is not more intense than the spot in the
Action and use chromatogram obtained with solution (2) (0.5%).
Dopamine receptor antagonist; neuroleptic. Loss on drying
When dried to constant weight at 40° at a pressure not
DEFINITION exceeding 0.7 kPa, loses not more than 0.5 % of its weight.
Fluanisone is 4' -fluoro-4- [4-(2-methoxyphenyl)piperazin- Use 1 g.
1-yl]-butyrophenone. It contains not less than 98.0% and not
more than 101.0% of C21H2SFN202, calculated with
Sulphated ash
reference to the dried substance. Not more than 0.1 %, Appendix IX A.
CHARACTERISTICS ASSAY
White or almost white to buff-coloured crystals or powder; Carry out Method I for non-aqueous titration,
odourless or almost odourless. It exhibits polymorphism. Appendix VIII A, using 0.15 g and crystal violet solution as
indicator. Each mI of 0.1 M perchloric acid VS is equivalent to
Practically insoluble in water; freely soluble in chloroform, in
17.82 mg of C21H2sFN202'
ethanol (96%), in ether and in dilute solutions of organic
acids. STORAGE
Fluanisone should be protected from light.
IDENTIFICATION
A. The infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum of fluanisone
78 Flunixin Meglumine
Column:
Flunixin Meglumine -- size: l = 0.125 m, 0 = 4.0 mm,
(Flunixin Meglumine for Veterinary Use, -- stationary phase: octadeeylsilyl siliea gel for ehromatography R
Ph Eur monograph 1696) (5 11m).
Mobile phase Mix 300 volumes of water R and 700 volumes
of aeetonitrile R, and add 0.25 volumes of phosphorie acid R.
P:\
HO H
~' CH 3
Flow rate 1.0 mUmin.
H'- " --OH Deteetion Spectrophotometer at 254 nm.
HO '- H
HO H
Jnjeetion 10 1lL.
HO Run time 5 times the retention time of fiunixin.
Relative retention With reference to fiunixin
491.5 42461-84-7 (retention time = about 3.1 min): impurity A = about 0.4;
impurity C = about 0.6; impurity B = about 0.7;
Action and use impurity D = about 4.2.
Cyclo-oxygenase inhibitor; analgesic; anti-infiammatory. System suitability Reference solution (a):
PhE~ ___________________________________________ -- resolution: minimum 3.5 between the peaks due to
impurity B and fiunixin.
DEFINITION Limits:
2-[ [2-Methyl-3-(trifiuoromethyl)phenyl] amino] pyridine- -- eorrection factor: for the calculation of content, multiply the
3-carboxylic acid, 1-deoxy-1-(methylamino)-D-glucitol. peak area of impurity C by 1.9,
Content -- impurity A: not more than the area of the corresponding
99.0 per cent to 101.0 per cent (dried substance). peak in the chromatogram obtained with reference
solution (b) (0.2 per cent),
CHARACTERS
-- impurity B: not more than the area of the corresponding
Appearance peak in the chromatogram obtained with reference
White or almost white, crystalline powder. solution (b) (0.2 per cent),
Solubility -- impurities C, D: for each impurity, not more than the area
Freely soluble in water and in methanol, practically insoluble of the peak due to fiunixin in the chromatogram obtained
in acetone. with reference solution (b) (0.2 per cent),
IDENTIFICATION -- any other impurity: for each impurity, not more than the
A. Specific optical rotation (2.2.7): - 9.0 to - 12.0 (dried area of the peak due to fiunixin in the chromatogram
substance), determined on solution S (see Tests). obtained with reference solution (b) (0.2 per cent),
-- total: not more than 2.5 times the area of the peak due to
B. Infrared absorption spectrophotometry (2.2.24).
fiunixin in the chromatogram obtained with reference
Comparison jlunixin meglumine CRS. solution (b) (0.5 per cent),
TESTS -- disregard limit: 0.25 times the area of the peak due to
Solution S fiunixin in the chromatogram obtained with reference
Dissolve 2.50 g in earbon dioxide-free water R and dilute to solution (b) (0.05 per cent).
50.0 mL with the same solvento Loss on drying (2.2.32)
Appearance of solution Maximum 0.5 per cent, determined on 1.000 g by drying in
Solution S is clear (2.2.1) and not more intensely coloured an oven at 105 oC for 4 h.
than reference solution Y7 (2.2.2, Method Jl). Sulfated ash (2.4.14)
pH (2.2.3) Maximum 0.1 per cent, determined on 1.0 g.
7.0 to 9.0 for solution S. ASSAY
Related substances Dissolve 0.175 g in 50 mL of anhydrous aeetie acid R. Titrate
Liquid chromatography (2.2.29). with 0.1 M perehlorie acid, determining the end-point
Test solution Dissolve 50.0 mg of the substance to be potentiometrically (2.2.20).
examined in the mobile phase and dilute to 10.0 mL with 1 mL of 0.1 M perehlorie acid is equivalent to 24.57 mg of
the mobile phase. C21H28F3N3Ü7'
ASSAY
The potency of equine serum gonadotrophin is estimated by
comparing under given conditions its effect of increasing the
mass of the ovaries of immature female rats with the same
effect of the International Standard of equine serum
B. 2-methyl-3-(trifiuoromethyl)aniline, gonadotrophin or of a reference preparation calibrated in
International Vnits.
The International Vnit is the activity contained in a stated
amount of the International Standard, which consists of a
mixture of a freeze-dried extract of equine serum
gonadotrophin from the serum of pregnant mares with
lactose. The equivalence in International Vnits of the
International Standard is stated by the World Health
Organisation. "
V se immature female rats of the same strain, 21 10 28 days
old, differing inage by not more than 3 days and having
D. ethyl 2-[[2-methyl- masses such that the difference between the heaviest and the
3-(trifiuoromethyl)phenyl] amino] pyridine-3-carboxylate. lightest rat is not more than 10 g. Assign the rats at random
________________________ ~ _________________ PhE~
to 6 equal groups of not fewer than 5 animals. If sets of 6
litter mates are available, assign one litter mate from each set
to each group and mark according to litter.
Choose 3 doses of the reference preparation and 3 doses of
the preparation to be examined such that the smallest dose is
Serum Gonadotrophin sufficient to produce a positive response in sorne of the rats
(Bquine Serum Gonadotrophin for Veterinary Use, and the largest dose does not produce a maximal response in
Ph Bur monograph 0719) aH the rats. Vse doses in geometric progression: as an initial
approximation total doses of 8 ID, 12 IV and 18 IV may be
Action and use tried, although the dose will depend on the sensitivity of the
Equine serum gonadotrophin. animals used and may vary widely.
Preparation Dissolve separately the total quantities of the preparation to
Serum Gonadotrophin Injection be examined and of the reference preparation corresponding
to the doses to be used in sufficient of a sterile 9 giL solution
PhE~ ___________________________________________
of sodium chloride R containing 1 mg/mL of bovine albumin R
DEFINITION such that each single dose is administered in a volume of
Equine serum gonadotrophin for veterinary use is a dry about 0.2 mL. Store the solutions at 5 ± 3 oc.
preparation of a glycoprotein fraction obtained from the Inject subcutaneously into each rat the dose aHocated to its
serum or plasma of pregnant mares. It has follic1e-stimulating group. Repeat the injections 18 h, 21 h, 24 h, 42 h and 48 h
and luteinising activities. The potency is not less than after the first injection. N ot less than 40 h and not more than
1000 IV of gonadotrophin activity per milligram, calculated 72 h after the last injection, euthanise the rats and remove
with reference to the anhydrous substance. the ovaries. Remove any extraneous fiuidand tissue and
weigh the 2 ovaries lmmediately. Calculate the results by the
PRODUCTION
usual statistical methods, using the combined mass of the
Equine serum gonadotrophin may be prepared by
2 ovaries of each animal as the response.
precipitation with alcohol (70 per cent VIV) and further
purificationbya suitable form of chromatography. It is The estimated potency is not les s than 80 per cent and not
prepared inconditions designed to minimise microbial more than 125 per cent of the stated potency.
contamination. The confidence limits (P = 0.95) of the estimated potency
are not less than 64 per cent and not more than 156 per cent
CHARACTERS of the stated potency.
Appearance
White or pale grey, amorphous powder. STORAGE
In an airtight container, protected from light, at a
Solubility temperature not exceeding 8 oC. If the substance is sterile,
Soluble in water. store in a sterile, airtight, tamper-proof container.
IDENTIFICATION LABELLING
When administered as prescribed in the assay it causes an The label states the potency in International Vnits per
increase in the mas s of the ovaries of immature female rats. milligram.
TESTS ___________________________________________ PhE~
Water (2.5.12)
Maximum 10.0 per cent, determined on 80 mg.
Bacterial endotoxins (2.6.14, method C)
Less than 0.035 IV per IV of equine serum gonadotrophin,
if intended for use in the manufacture of parenteral
preparations without a further appropriate procedure for the
removal of bacterial endotoxins.
80 Levamisole
Mobile phase:
Levamisole -- mobile phase A: dissolve 0.5 g of ammonium dihydrogen
(Levamisole for Veterinary UseJ phosphate R in 90 mL of water R; adjust to pH 6.5 with a
Ph Eur monograph 1728) 40 gIL solution of sodium hydroxide R and dilute to
100 mL with water R,
-- mobile phase B: acetonitrile R.
Time Mobile phase A Mobile phase B
(min) (per cent VM (percent VM
0-8 90 ~ 30 10 ~ 70
8 - 10 30 70
204.3 14769-73-4
IMPURlTIES
Reference solution (b) Dilute 1.0 mL of the test solution to
100.0 mL with methanol R. Dilute 5.0 mL ofthe solution to
25.0 mL with methanol R.
Column: and enantiomer
-- size: 1 = 0.10 m, el = 4.6 mm,
-- stationary phase: base-deactivated octadecylsilyl silica gelfor
chromatography R (3 ~m).
A. 3-[(2RS)-2-amino-2-phenylethyl]thiazolidin-2-one,
Lufenuron 81
Levomepromazine
H Me Anhydrous Lufenuron
~NMe2 (Lufenuron (Anhydrous) for Veterinary Use,
Ph Eur monograph 2177)
(X
~ I
NX)oMe
S
~ I
and enantiomer and enantiomer
328.5 60-99-1
el
HO~
I~ o o F
# ~)l~)6
el
F
~ I
B. 1-(2,5-dichloro-4-hydroxyphenyl)-
H. 1,3-bis[2,5-dichloro-4-(1,1,2,3,3,3-
3-(2,6-difluorobenzoyl)urea, hexafluoropropoxy)phenyl] urea.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
and enantiomer
Marbofloxacin
(Marbofioxacin for Veterinary Use~
H F
F3e>Y0~~ o
11 )6I
O F
and enantiomer
F F # N~N ~
H H
el F ~
362.4 115550-35-1
D. 1-[2-chloro-4-[(2RS)-1,1,2,3,3,3-
hexafluoropropoxy]phenyl]-3-(2,6-difluorobenzoyl)urea, Action and use
Fluroquinolone antibacterial.
H,{X_o~el
F3e~
F F
~
I
#
el
N
H
o
)l
N
H
n
o
F
~
~
el
I
and enantiomer
~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
DEFINITION
9-Fluoro-3-methyl-1 0-(4-methylpiperazin-1-yl)-7 -oxo-
2,3-dihydro-7H-pyrido[3,2,1-V] [4,1,2]benzoxadiazine-
6-carboxylic acid.
E. 1-(2-chloro-6-fluorobenzoyl)-3-[2,5-dichloro- Content
4- [(2RS)-1, 1,2,3,3,3-hexafluoropropoxy]phenyl] urea, 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
H F el Appearance
~~ ~
Solubility
I Slightly soluble in water, sparingly soluble or slightly soluble
el F ~ in methylene chloride, very slightly soluble in
ethanol (96 per cent).
F. 1-[2,5-dichloro-4-[(2RS)-1,1,2,3,3,3-
IDENTIFICATION
hexafluoropropoxy] phenyl] -3-(2-fluorobenzoyl)urea,
Infrared absorption spectrophotometry (2.2.24).
Comparison marbofioxacin CRS.
TESTS
Absorbance (2.2.25)
Maximum 0.20, determined at 450 nm.
Dissolve 0.400 g in borate buffer solution pH 10.4 R and dilute
to 10.0 mL with the same buffer solution.
G. 2,5-dichloro-4-[[ [(2, 6-difluorophenyl) carbonyl] Related substances
carbamoyl] amino]phenyl phenyl carbonate, Liquid chromatography (2.2.29). Cany out the test protected
from light.
Solvent mixture methanol R~ water R (23:77 V/V).
Test solution To 0.100 g ofthe substance to be examined
add 80 mL of the solvent mixture, sonicate until dissolution
and dilute to 100.0 mL with the solvent mixture.
Reference solution (a) Dilute 5.0 mL of the test solution to
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 50.0 mL with the solvent mixture.
Reference solution (b) Dissolve 10 mg of marbofioxacin for
peak identification CRS (containing impurities A, B, C, D and
84 Marbofloxacin
E) in the solvent mixture and dilute to 10 mL with the 1 mL of 0.1 M perchloric acid is equivalent to 36.24 mg of
solvent mixture. C17H19FN404·
Column: STORAGE
- size: l = 0.15 m, 0 = 4.6 mm; Protected from light.
- stationary phase: end-capped polar-embedded octadecylsilyl
amorphous organosilica polymer R (3.5 ¡.tm); IMPURITIES
- temperature: 40 oC. Specified impurities AJ B J C DJ E.
J
Mobile phase Mix 230 volumes of methanol R and 5 volumes Other detectable impurities (the following substances would, if
of glacial acetic acid R with 770 volumes of a 2.70 gIL present at a sufficient leve1, be detected by one or other of
solution of sodium dihydrogen phosphate R containing 3.50 giL the tests in the monograph. They are limited by the general
of sodium octanesuljonate R and previously adjusted to pH 2.5 acceptance criterion for other/unspecified impurities and/or
with phosphoric acid R. by the general monograph Substances for pharmaceutical use
Flow rate 1.2 mUmin. (2034). Ir is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Detection Spectrophotometer at 315 nm. Control of impurities in substances for pharmaceutical use): F.
Injection 10 ¡.tL.
Run time 2.5 times the retention time of marbofioxacin.
Identification of impurities U se the chromatogram supplied
with marbofloxacin for peak identification CRS and the
chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, B, C, D and E.
Relative retention With reference to marbofioxacin
(retention time = about 33 min): impurity B = about 0.5;
impurity A = about 0.7; impurity C = about 0.9; A. 6,7 -difiuoro-8-hydroxy-1-(methylamino )-4-oxo-
impurity D = about 1.3; impurity E = about 1.5. 1,4-dihydroquinoline-3-carboxylic acid,
System suitability Reference solution (b):
- resolution: minimum 1.5 between the peaks due to
impurity C and marbofioxacin, and minimum 4.0
between the peaks due to marbofioxacin and impurity D.
Limits:
- correction factor: for the calculation of content, multiply the
peak area of impurity E by 1.5;
- impurities CJ DJ E: for each impurity, not more than twice
B. 9,1 O-difiuoro-3-methyl-7-oxo-2,3-dihydro-
the area of the principal peak in the chromatogram
7H-pyrido [3,2, 1-vJ [4,1,2] benzoxadiazine-6-carboxylic acid,
obtained with reference solution (a) (0.2 per cent);
- impurities A J B: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.1 per cent);
- unspecified impurities: for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent);
- total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent); C. R = F: 6,8-difiuoro-1-(methylamino)-
- disregard limit: the area of the principal peak in the 7-(4-methylpiperazin-1-yl)-4-oxo-1 ,4-dihydroquinoline-
chromatogram obtained with reference 3-carboxylic acid,
solution (a) (0.1 per cent). D. R = OH: 6-fiuoro-8-hydroxy-1-(methylamino)-
Heavy metals (2.4.8) 7-(4-methylpiperazin-1-yl)-4-oxo-1 ,4-dihydroquinoline-
Maximum 20 ppm. 3-carboxylic acid,
Dissolve 0.5 g in dilute acetic acid R and dilute to 30 mL with E. R = O-C 2H s : 8-ethoxy-6-fiuoro-1-(methylamino)-
the same solvento Adding 2 mL of water R instead of 2 mL 7-(4-methylpiperazin-1-yl)-4-oxo-1 ,4-dihydroquinoline-
of buffer solution pH 3.5 R, the filtrate complies with test E. 3-carboxylic acid,
Prepare the reference solution using 5 mL of le,ad standard
solution (2 ppm Pb) R. '
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying at
105 oC for 4 h.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g in a platinum
crucible. F. 4-[6-carboxy-9-fiuoro-3-methyl-7-oxo-2,3-dihydro-
ASSAY 7H-pyrido [3,2, 1-vJ [4, 1,2]benzoxadiazin.-l O-yl]-
Dissolve 0.300 g in 80 mL of glacial acetic acid R. Titrate 1-methylpiperazine l-oxide.
with 0.1 M perchloric acidJ determining the end-point __________________________________________ ~E~
potentiometrically (2.2.20).
Morantel Tartrate 85-
_
octadecylsilyl silica gel for chromatography (10 Ilm) (Spherisorb
eOOH el ODS 1 is suitable), (b) a mixture of 1 volume of glacial acetic
~~Me
& JJ
acid, 25 volumes of water and 75 volumes of methanol as the
mobile phase with a flow rate of 2 mL per minute and (c) a
~I el
detection wavelength of 254 nm.
The test is not valid unless the column efficiency is at least
4500 theoretical plates per metre, determined using the peak
296.2 644-62-2 due to the internal standard in the chromatogram obtained
with solution (1).
Action and use In the chromatogram obtained with solution (3) the area of
Cyclo-oxygenase inhibitor; analgesic; anti-inflarnmatory. the peak irnmediately preceding the peak due to
meclofenamic aicid is not greater than one-seventh of the area
DEFINITION of the peak due to the internal standard (0.05% of
Meclofenamic Acid is N-(2,6-dichloro-m-tolyl)anthranilic 2,6-dichloro-3-ni:ethylaniline). The area of any other
acid. It contains not--Iess than 98.5% and not more than secondary peak is not greater than the area of the peak due to
100.5% of CI4HllClzNOz, calculated with reference tó the the internal standard (0.35%).
dried substance.
Loss on drying
CHARACTERISTICS When dried at 1050 to constant weight, loses not more than
A white or almost white, crystalline powder. 0.5% of its weight. Use 1 g.
Practically insoluble in water; soluble in dimethyiformamide Sulfated ash
and in 1M sodium hydroxide; sparingly soluble in ether; slight1y Not more than 0.1 %, Appendix IX A.
soluble in chloroform and in ethanol (96%).
ASSAY
IDENTIFICATION Dissolve 0.6 g in 100 mL ofwarm absolute ethanol previously
A. The infrared absorption spectrum, Appendix II A, is neutralised to phenol red solution and titrate with -O.IM sodium
concordant with the reference spectrum of meclofenamic acid hydroxide VS using phenol red solution as indicatór. Each mL
(RSV 26). of O.IM sodium hydroxide VS is equivalent to 29.62 mg of
B. The light absorption, Appendix II B, in the range 230 to CI4HIIClzNOz.
350 nm of a 0.002% w/v solution in O.lM sodium hydroxide
exhibits two maxima, at 279 nm and 317 nm. The
absorbances at the maxima are about 0.45 and about 0.33,
respective1y. Morantel Tartrate
C. Dissolve about 25 mg in 15 mL of chloroform.
The solution exhibits a strong blue fluorescence when (Morantel Hydrogen T arta te for Veterinary Use,
examined under ultraviolet light. Ph Eur monograph 1546)
D. Dissolve about 1 mg in 2 mL of sulfuric acid and add
0.05 mL of 0.02M potassium dichromate. An intense purple
colour is produced, which rapidly fades to purplish brown.
TESTS
Clarity and co1our of solution
A 5.0% w/v solution in 1M sodium hydroxide is not more
opalescent than reference suspension 11, Appendix IV A, and is 370.4 26155-31-7
not more intense1y coloured than reference solution BYs, Action and use
Appendix IV B, Method II. Antihe1minthic.
Light absorption
~E~ __________________________________________
Absorbance of a 0.002% w/v solution in O.OlM methanolic
hydrochloric acid at the maximum at 279 nm, not less than DEFINITION
0.400 and not more than 0.445, and at the maximum at 1-Methyl-2- [(E)- 2-(3-methylthiophen-2-yl) ethenyl]-
335 nm, not les s than 0.440 and not greater than 0.490, 1,4,5,6-tetrahydropyrimidine hydrogen tartrate.
Appendix II B.
Content
Heavy meta1s 98.5 per cent to 101.5 per cent (dried substance).
1 g complies with limit test C for heavy metals, Appendix VII.
Use 2 mL of lead standard solution (lO ppm Pb) to prepare the CHARACTERS
standard (20 ppm). Appearance
White or pale yellow, crystalline powder.
Related substances
Carry out the method for liquid chromatography, Solubility
Appendix III D, using the following solutions in absolute Very soluble in water and in ethanol (96 per cent), practically
ethanol. Solution (1) contains 0.0035% w/v of ethyl insoluble in ethyl acetate.
meclofenamate BPCRS (internal standard). Solution (2) IDENTIFICATION
contains 1.0% w/v solution of the substance being examined. First identification B.
Solution (3) contains 1.0% w/v of the substance being
Second identification A, C, D.
examined and 0.0035% w/v of the internal standard.
A. Melting point (2.2.14): 167 oC to 172 oC.
86 Morantel Tartrate
G. (23E,25S)-50-desmethyl-28-deoxy-25-[(1E)-1,3- L. (23Z)-moxidectin.
dimethylbut-l-enyl] -23-(methoxyimino )milbemycin B, __________ ______________________________
~ ~E~
Nandrolone Laurate
Me~
o
H. 2,5-didehydro-5-deoxymoxidectin, 456.7 26490-31-3
DEFINITION
Nandrolone Laurate is 3-oxo-estr-4-en-17B-yllaurate.
It contains not less than 97.0% and not more than 103.0%
of C30H4S03, calculated with reference to the dried
substance.
CHARACTERISTICS
A white to creamy white, crystalline powder.
1. (23S)-23-des(methoxyimino)- Practically insoluble in water; freely soluble in chloroform, in
23- [(methylsulfanyl)methoxy] moxidectin, ethanol (96%), in ether, in fixed oils and in esters of fatty
acids.
IDENTIFICATION
A. The infrared absorption spectntm, Appendix II A, is
concordant with the reference spectntm of nandrolone laurate
(RSV 30).
B. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions in chloroform.
(1) 0.5% w/v of the substance being examined.
(2) 0.5% w/v of nandrolone laurate BPCRS.
(3) Mix equal volumes of solutions (1) and (2).
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F254 precoated plate the
surface of which has been modified by chemically-bonded
J.R = CHz-S-CH3, R' = H: octadecylsilyl groups (Whatman KC 18F plates are suitable).
7-0- [(methylsulfanyl)methyl] moxidectin,
(b) U se the mobile phase as described below.
K. R = H, R' = CO-C 6H 4 -pNO z:
5-0-(4-nitrobenzoyl)moxidectin, (c) Apply 5 IlL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air until the odour of
solvent is no longer detectable and heat at 100° for
90 Nitroxinil
IMPURITIES
Specified impurities A, D.
Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities andlor
by the general monograph Substances for pharmaceutical use
(2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): B, C, E. l-cyc1opropyl-5-[ (3R,5S)-3,5-dimethylpiperazin-l-yl]-
E, F, G. 6,7 ,8-trifiuoro-4-oxo-l ,4-dihydroquinoline-3-carboxylic acid,
F. l-cyc1opropyl-5,6, 7,8-tetrafiuoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid.
____________________________________ ~----~E~
Oxfendazole
(Oxfendazole for Veterinary Use,
Ph Eur monograph 1458)
H-~NWN
Action and use
Antihe1minthic.
HaC I I
F ~ R1 Preparation
R2 O Oxfendazole Oral Suspension
~E~ __________________________________________
C. Rl = C0 2H, R2 = H : l-cyc1opropyl-
7-[ (3R,5S)3,5-dimethylpiperazin-l-yl]-6,8-difiuoro-4-oxo- DEFINITION
1,4-dihydroquinoline-3-carboxylic acid, Methyl [5-(phenylsulfinyl)-IH-benzimidazol-2-yl]carbamate.
G. Rl = H, R2 = F: l-cyc1opropyl-7-[(3R,5S)-3,5- Content
dimethylpiperazin-l-yl] -5,6 ,8-trifiuoroquinolin-4( 1H)-one, 97.5 per cent to 100.5 per cent (dried substance).
CHARACTERS
Appearance
White or almost white powder.
Solubility
Practically insoluble in water, slight1y soluble in
ethanol (96 per cent) and in methylene ch1oride.
It shows ~olymorphism (5.9).
IDENTIFICATION
D. l-cyc1opropyl-7-[(3R,5S)-3,5-dimethylpiperazin-l-yl]- Infrared absorption spectrophotometry (2.2.24).
6,8-difiuoro-5-hydroxy-4-oxo-l,4-dihydroquinoline- Comparison oxfendazole CRS.
3-carboxylic acid,
If the spectra obtained in the solid state 'show differences,
dissolve the substance to be examined and the reference
substance separately in ethanol (96 per cent) R, evaporate to
dryness and record new spectra using the residues.
Oxyc1ozanide 93
CHARACTERISTICS ASSAY
A pale cream or cream coloured powder. Dissolve 0.25 g in 75 mL of anhydrous pyridine and pass a
Very slight1y soluble in water; freely soluble in acetone; soluble current of nitrogen through the solution for 5 minutes. Carry
in ethanol (96%); slight1y soluble in chloroform. out Method 11 for non-aqueous titration, Appendix VIII A,
maintaining a eurrent of nitrogen through the solution
IDENTIFICATION throughout the titration, using O.IM tetrabutylammonium
A. The infrared absorption spectrum, Appendix 11 A, is hydroxide VS as titrant and determining the end point
concordant with the reference spectrum of oxyc1ozanide potentiometrically. Eaeh mL of O.IM tetrabutylammonium
(RSV 33). hydroxide VS is equivalent to 20.07 mg of C 13 H 6 ClsN0 3 .
B. The light absorption, Appendix 11 B, in the range 250 to
350 nm of a 0.003% w/v solution in acidijied methanol
exhibits a maximum on1y at 300 nm. The absorbance at the
maximum is about 0.76, Appendix 11 B.
C. Melting point, about 208 0 , Appendix V A. Piperonyl Butoxide
TESTS
Ionisable chlorine
Dissolve 2 g in 100 mL of methanol, add 10 mL of
I.5M nitric acid and titrate with O.IM silver nitrate VS
determining the end point potentiometrically. Not more than
1.4 mL is required (0.25%).
Related substances
338.4 51-03-6
Carry out the method for liquid chromatography,
Appendix 111 D, using the following solutions. Action and use
(1) 0.1 % w/v of the substance being examined prepared by Insecticide.
dissolving it in a suitable volume of methanol and slowly
Preparation
diluting with water containing 0.1 % v/v orthophosphoric acid to
Compound Pyrethrum Spray
give a solution containing about the same ratio of methanol
to water as the mobile phase. DEFINITION
(2) Dilute 1 volume of solution (1) to 100 volumes with the Piperonyl Butoxide is 5-[2-(2-butoxyethoxy)ethoxymethyl]-
mobile phase. 6-propyl-l,3-benzodioxole. It contains not less than 85.0% of
CHROMATOGRAPHIC CONDITIONS C1 9 H300S'
(a) Use a stainless steel column (20 cm x 5 mm) paeked CHARACTERISTICS
with octadecylsilyl silica gel for chromatography (5 flm) (Hypersil A yellow or pale brown, oily liquido
ODS is suitable). Very slight1y soluble in water; miscible with chloroform, with
(b) Use isocratic elution and the mobile phase described ethanol (96%) with ether and with petroleum oils.
below.
IDENTIFICATION
(e) Use a flow rate of 2 mL per minute. A. The light absorption, Appendix 11 B, in the range 220 to
(d) Use an ambient column temperature. 350 nm of a 0.008% w/v solution in absolute ethanol exhibits
(e) Use a detection wavelength of 300 nm. well.;..defined maxima at 238 nm and 290 nm.
(f) Inject 20 flL of each solution. The absorbances at the maxima are about 1.2 and about 1.0,
respectively.
MOBILE PHASE
B. Dissolve 0.1 mg in 0.1 mL of acetonitrile, add 10 mg of
A mixture of methanol and water containing 0.1 % v/v of gallic acid and mix. Add 3 mL of sulfuric acid to form a lower
orthophosphoric acid (a mixture of 38 volumes of water and layer, allow.to stand for 1 minute and mix. A green colour is
62 volumes of methanol is usually suitable). produced.
LIMITS C. To 0.1 mL of a 0.05% w/v solution in ethanol (96%) add
In the chromatogram obtained with solution (1): 5 mL of tannic acid reagent, shake vigorously for 1 minute
the area of any secondary peak with a retention time less than and heat in a water bath for 5 minutes. A blue colour is
that of the principal peak is not greater than one third of the produced.
area of the principal peak in the chromatogram: obtained with TESTS
solution (2) (0.3%); Refractive index
the area of any secondary peak with a retention tiipe greater 1.494 to 1.504, Appendix V E.
than that of-rhe principal peak is not greater 'than the area of
Weight per rnL
the principal peak in the ehromatogram obtained with
1.050 to 1\:.065 g, Appendix V G.
solution (2) (1 %).
Sulfated ash
Loss on drying
Not more than 0.2%, Appendix IX A.
When dried to constant weight at 60 0 at a pressure not
exceeding 0.7 kPa, loses not more than 1.0% of its weight. ASSAY
Use 1 g. Carry out the method for gas chromatogrq,phy,
Sulfated ash Appendix 111 B, using the following solutions in chloroform.
Not more than 0.2%, Appendix IX A. (1) 0.25% w/v of piperonyl butoxide CRS and 0.2% w/v of
tetraphenylethylene (internal standard).
(2) 0.25% w/v of the substance being examined.
Pyrethrum Flower 95
Acid-insoluble ash Titrate with O.OlM potassium iodate VS, running almost all
Not more than 1.0%, Appendix XI K. the required volume of titrant into the fiask in one portion.
Continue the titration, shaking the fiask vigorously for
ASSAY
30 seconds after each addition of the titrant, until the
Transfer 12.5 g in No. 1000 powder to an apparatus for the
chloroform is colourless. Repeat the operation without the
continuous extraction of drugs, Appendix XI F, and extract with
extract; the difference between the titrations represents the
aromatic-free petroleum spirit (boiling range~ 40° to 60°) for
amount of potassium iodate required. Each mL of
7 hours. Evaporate the extract to about 40 mL and allow to
O.OlM potassium iodate VS is equivalent to 5.7 mg of
stand ovemight at 0° to 5°. Add 20 mL of O.5M ethanolic
pyrethrin I.
potassium hydroxide and boil under a refiux condenser for
45 minutes. Transfer the solution to a beaker and wash the For pyrethrin 11
fiask with sufficient hot water, adding the washings to the Transfer the combined aqueous liquids reserved in the Assay
beaker, to produce a total volume of 200 mL. Boil until the for pyrethrin 1 to a beaker, cover with a watch glass and
volume is reduced to 150 mL, cool rapidly and transfer the evaporate to 50 mL within 35 to 45 minutes. Cool, washing
solution to a stoppered fiask, washing the beaker with three the underside of the watch glass with not more than 5 mL of
20 mL quantities of water and transferring any gummy water and adding the washings to the beaker. Filter through
residue to the fiask. Add 1 g of diatomaceous earth (Filtercel absorbent cOUon into a separating funnel, washing with
is suitable) and 10 mL of barium chloride solution, swirl gently successive quantities of 10, 7.5, 7.5, 5 and 5 mL of water.
and add sufficient water to produce 250 mL. Stopper the Saturate the aqueous liquid with sodium chloride, add 10 mL
fiask, shake vigorously until the separating liquid is clear and of hydrochloric acid and 50 mL of ether, shake for 1 minute,
filter the suspension through a filter paper (Whatman No. allow to separate, and remove the lower layer. Repeat the
1 is suitable). extraction successively with 50, 25 and 25 mL of ether. Wash
the combined ether extracts with three 10 mL quantities of a
For pyrethrin 1
saturated solution of sodium chloride and transfer the ether
Transfer 200 mL of the filtrate to a separating funnel, rinsing
layer to a fiask with the aid of 10 mL of ether. Remove the
the measuring vessel with two 5 mL quantities of water, and
bulk of the ether by distillation and remove the remainder
add 0.05 mL of phenolphthalein solution R1. Neutralise the
with a gentle current of air and dry the residue at 100° for
solution by the drop wise addition of hydrochloric acid and
10 minutes, removing any residual acid fumes with a gentle
add 1 mL of hydrochloric acid in excess. Add 5 mL of a
current of airo Add 2 mL of ethanol (96%) previously
saturated solution of sodium chloride and 50 mL of aromatic-
neutralised to phenolphthalein solution R1 and 0.05 mL of
free petroleum spirit (boiling range~ 40° to 60°), shake vigorously
phenolphthalein solution R1, swirl to dissolve the residue, add
for 1 minute, allow to separate, remove and retain the lower
20 mL of carbon dioxide-free water and titrate rapidly with
layer. Filter the petroleum spirit extract through absorbent
0.02M sodium hydroxide VS until the colour changes to
couon into a second separating funnel containing 10 mL of
brownish pink and persists for 30 seconds, keeping the fiask
water. Retum the aqueous layer to the first separating funnel
stoppered between additions of alkali. Repeat the operation
and repeat the extraction with 50 mL and then with 25 mL
using the aqueous liquid reserved for the repeat operation in
of aromatic-free petroleum spirit (boiling range~ 40° to 60°),
the Assay for pyrethrin I. The difference between the
reserving the aqueous layer for the assay of pyrethrin II, and
titrations represents the volume of 0.02M sodium hydroxide VS
filtering the petroleum spirit extracts through the same
required. Each mL of 0.02M sodium hydroxide VS is
absorbent cOUon into the second separating funnel. Shake
equivalent to 3.74 mg of pyrethrin II.
the combined petroleum spirit extracts and water for about
30 seconds and allow to separate; remove the lower layer and
add it to the aqueous liquid reserved for the assay of
pyrethrin II. Wash the combined petroleum spirit extracts
with a further 10 mL of water, adding the washings to the Ronidazole
reserved aqueous liquido
To the petroleum spirit extracts add 5 mL of O.lM sodium
Me O
hydroxide, shake vigorously for 1 minute, allow to separate
and remove the clear lower layer, washing the stem of the
separating funnel with 1 mL of water. Repeat the extraction
02N~ro)lNH2
by shaking for about 30 seconds with two quantities of
2.5 mL and 1.5 mL of O.lM sodium hydroxide and add the
extracts to the alkaline extracto Add to the fiask 10 mL of 200.2 7681-76-7
mercury(Il) sulfate solution, stopper, swirl and allow to stand in
the dark at 25° ± O.5°'Ior exactly 60 minutesafter the Action and use
addition of the mercury(n) sulfate solution. Add 20 mL of Antiprotozoal.
acetone and 3 mL of a saturated solution of sodzum chloride,
heat to boiling on a water bath, allow the precipitate to settle DEFINITION
and decant the supernatant liquid through a filter paper Ronidaz9le is (1-methyl-5-nitroimidazol-2-yl)methyl
(Whatman No. 1 is suitable), retaining most of the carbamate. It contains not less than 98.5% and not more
precipitate in the fiask. Wash the precipitate with 10 mL of than 101.0% of C 6 H sN 4 0 4 , calculated with reference to the
acetone, again boil, allow to seule and decant through the anhydrous substance.
same filter papero Repeat the washing and decanting with CHARACTERISTICS
three 10 mL quantities of hot chloroform. Transfer the filter A white to yellowish brown powder; odourless or almost
paper to the fiask, add 50 mL of a cooled mixture of three odourless.
volumes of hydrochloric acid and two volumes of water, 1 mL Slightly soluble in water, in chloroform and in
of strong iodine monochloride reagent and 6 mL of chloroform.
ethanol (96%); very slightly soluble in ether.
Selamectin 97
IDENTIFICATION hexopyranosyl)oxy]-20b-hydroxy-20-(hydroxyimino)-
A. The infrared absorption spectrum, Appendix n A, is 5' ,6,8, 19-tetramethyl-
concordant with the reference spectrum of ronidazole 3',4',5',6,6',7,10,11,14,15,17a,20,20a,20b-
(RSV 36). tetradecahydrospiro [2H, 17 H-11, 15-methanofuro [4,3,2-
B. The light absorption, Appendix n B, in the range 230 to pq] [2,6]benzodioxacyclooctadecine-13,2'-pyran]-17-one
350 nm of a 0.002% w/v solution in O.lM methanolic ((5Z,25S)-25-cyc1ohexyl-4' -O-de(2 ,6-dideoxy-3-0-methyl-
hydrochloric acid exhibits a maximum onIy at 270 nm. The Ci-L-arabino-hexopyranosyl)-5-demethoxy-
absorbance at the maximum is about 0.64. 25-de (1-methylpropyl)-22,23-dihydro-
5-(hydroxyimino)avermectin Ala).
C. Melting point, about 167°, Appendix V A.
Semi-synthetic product derived from a fermentation producto
TESTS
Content
Colour of solution
96.0 per cent 10 102.0 per cent (anhydrous substance).
A 0.5% w/v solution in methanol is not more intense1y
coloured than reference solution Y6, Appendix IV B, Method n. CHARACTERS
( l-Methyl-5-nitroimidazol-2-y1)methanol Appearance
Carry out the method for thin-layer chromatography, White or almost white, hygroscopic powder.
Appendix nI A, using sz1ica gel GF254 as~the coating Solubility
substance and a mixture of 5 volumes of glacial acetic acid, Practically insoluble in water, free1y soluble in isopropyl
5 volumes of methanol and 80 volumes of toluene as the alcohol, soluble in acetone and in methylene chloride,
mobile phase. Apply separately to the plate 20 ¡lL of each of sparingly soluble in methanol.
two solutions in acetone containing (1) 1.0% w/v of the
IDENTIFICATION
substance being examined and (2) 0.0050% w/v of {l-methyl-
Infrared absorption spectrophotometry (2.2.24).
5-nitroimidazol-2-yl)methanol BPCRS. After removal of the
plate, allow it to dry in air and examine under ultraviolet light Comparison selamectin CRS.
(254 nm). Any spot in the chromatogram obtained with TESTS
solution (1) corresponding to (1-methyl-5-nitroimidazol- Related substances
2-yl)methanol is not more intense than the spot in the Liquid chromatography (2.2.29).
chromatogram obtained with solution (2) (0.5%). Solvent mixture water R, acetonitrzte R (40:60 V/V).
Water Test solution Dissolve 25 mg of the substance to be
Not more than 0.5% w/w, Appendix IX C. Use 5 g. examined in the solvent mixture and dilute to 50 mL with
Sulfated ash the solvent mixture.
Not more than 0.1 %, Appendix IX A. Reference solution (a) Dilute 1.0 mL of the test solution to
ASSAY 100.0 mL with the solvent mixture.
Carry out Method I for non-aqueous titration, Reference solution (b) Dissolve 2.5 mg of selamectin for system
Appendix VIn A, using 0.3 g and determining the end point suitability CRS (containing impurities A, B, C and D) in the
potentiometrically. Each mL of O.lM perchloric acid VS is solvent mixture and dilute to 5 mL with the solvent mixture.
equivalent to 20.02 mg C 6H sN 4 0 4 . Column:
STORAGE - size: 1 = 0.15 m, O = 3.9 mm;
Ronidazole should be protected from light.
- stationary phase: end-capped octadecylsilyl silica gel for
chromatography R (4 ¡lm);
- temperature: 30 oC.
Mobile phase:
- mobile phase A: water R;
Selamectin - mobile phase B: acetonitrile R;
Time Mobile phase A Mobile phase B
(Selamectin for Veterinary Use,
(min) (per cent VJII) (per cent VIII)
Ph Eur monograph 2268)
0-28 40 60
28 - 45 40 ~ 20 60 ~ 80
Limits: IMPURITIES
- correction factor: for the ca1culation of content, multiply the Specified impurities A, B, C, D.
peak area of impurity D by 1.5;
- impurities A, B: for each impurity, not more than twice
the area of the principal peak in the chromatogram
obtained with reference solution (a) (2.0 per cent);
- impurities C, D: for each impurity, not more than
1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
(1.5 per cent);
- any other impurity: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (1.0 per cent);
- total: not more than 4 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(4.0 per cent);
A. (2aE,2'R,4E,4' S,5' S,6S,6'R,7S,8E,11R,15S,17aR,20Z,
- disregard limit: 0.2 times the area of the principal peak in
20aR,20bS)-6' -cyclohexyl-7 - [(2,6-dideoxy-3-0-methyl-
the chromatogram obtained with reference solution (a)
Cl-L-arabino-hexopyranosyl)oxy] -4' ,20b-dihydroxy-
(0.2 per cent).
20-(hydroxyimino)-5' ,6,8, 19-tetramethyl-
Heavy metals (2.4.8) 3',4',5',6,6',7,10,11,14,15,17a,20,20a,20b-
Maximum 20 ppm. tetradecahydrospiro[2H,17H-11,15-methanofuro[4,3,2-
Dissolve 2.0 g in ethanol (96 per cent) R and dilute to pq] [2,6]benzodioxacyclooctadecine-13,2'-pyran]-17-one
20.0 mL with the same solvento 12 mL of the solution ((5Z,21R,23S,25R)-25-cyclohexyl-4' -O-de(2,6-dideoxy-
complies with test B. Prepare the reference solution using 3-0-methyl-Cl-L-arabino-hexopyranosyl)-5-demethoxy-
lead standard solution (2 ppm Pb) obtained by diluting lead 25-de (l-methylpropyl)-22,23-dihydro-23-hydroxy-
standard solution (lOO ppm Pb) R with ethanol (96 per cent) R. 5-(hydroxyimino)avermectin AlJ,
Filter the solution through a membrane filter (nominal pore
size 0.45 ¡.tm). Compare the spots on the filters obtained with
the different solutions. Any brownish-black colour in the spot
from the test solution is not more intense than that in the
spot from the reference solution.
Water (2.5.12, Method A)
Maximum 7.0 per cent, determined on 0.20 g.
Sulfated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29).
Test solution Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 250.0 mL with B. (2aE,2' S,4E,5' S,6S,6' R,7S,8E,llR, 15S, 17aR,20Z,20aR,
the mobile phase. 20bS)-6' -cyclohexyl-7 - [(2,6-dideoxy-3-0-methyl-Cl":L-arabino-
hexopyranosyl)oxy]-20b-hydroxy-20-(hydroxyimino)-
Reference solution Dissolve 50.0 mg of selamectin CRS in the
5' ,6,8, 19-tetramethyl-5',6,6', 7,10,11,14,15,17a,20,20a,20b-
mobile phase and dilute to 250.0 mL with the mobile phase.
dodecahydrospiro [2H, 17H-11, 15-methanofuro [4,3,2-
Column: pq] [2,6]benzodioxacyclooctadecine-13,2' -pyran] -17 -one
- size: l = 0.15 m, 0 = 3.9 mm; ((5Z,25R)-25-cyclohexyl-4' -O-de (2,6-dideoxy-3-0-methyl-
- stationary phase: end-capped octadecylsilyl silica gel for Cl-L-arabino-hexopyranosyl)-5-demethoxy-
chromatography R (4 ¡.tm);
25-de(1-methylpropyl)-5-(hydroxyimino)avermectin Ala),
- temperature: 30 oC.
Mobile phase water R, acetonitrile R (20:80 V/V).
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 243 nm.
Injection 20 ¡.tL.
Run time Twice the retention time of selamectin.
Retention time Selamectin = about 9 min:
Ca1culate the percentage content of C43H63NOll from the
declared content of selamectin CRS.
STORAGE
In an airtight container.
172.9 10102-18-8
DEFINITION
Sodium Selenite contains not less than 44.0% and not more
than 46.0% of Se, calculated with reference to the dried
substance. Compound R R' Molee. Formula
CHARACTERISTICS
White to slightly greyish pink, granular powder.
Freely soluble in water; practically insoluble in ethanol (96%)
and in ether. (4R)-dihydro-
spectinomycin
Dissolve 2 g in carbon dioxide-free water prepared from distilled
water and dilute to 100 mL with the same solvent (solution
S). ~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ___
IDENTIFICATION DEFINITION
A. Dissolve 50 mg in 5 mL of 7M hydrochloric acid, add 2 mL Mixture of (2R,4aR,5aR,6S,7S,8R,9S,9aR,10aS)-4a,7,9-
of O.lM sodium thiosulfate and heat to boiling. A reddish trihydroxy-2-methyl-6,8-bis (methylamino) decahydro-
orange precipitate is produced. 4H-pyrano[2,3-b] [1,4]benzodioxin-4-one sulfate tetrahydrate
B. 1 mL of solution S yields the reactions characteristic of (spectinomycin sulfate tetrahydrate) and
sodium salts, Appendix VI. (2R,4R,4aS,5aR,6S, 7S,8R, 9S, 9aR, 1OaS)-2-methyl-
6,8-bis (methylamino )decahydro-2H-pyrano _
TESTS [2,3-b] [1,4]benzodioxine-4,4a,7,9-tetrol sulfate tetrahydrate
Clarity of solution ((4R)-dihydrospectinomycin sulfate tetrahydrate).
Solution S is not more opalescent than reference suspension IL
It is produced by Streptomyces spectabilis or by any other
Appendix IV A.
means.
Alkalinity
To 50 mL of solution S add 0.2 mL of thymolphthalein
solution. Not more than 0.25 mL of 1M hydrochloric acid VS is
required to change the colour of the solution from dark blue
to very pale blue.
100 Spectinomycin Sulfate Tetrahydrate
OH Spiramycin
H3C /~x;x: OH (Ph Eur monograph 0293)
HO "OH
HN,
CH 3
and epimer at C·
B. (2S,3RS,5R)-3-hydroxy-5-rnethyl-2-[[(lr,2R,3S,4r,5R,6S)-
2,4,6-trihydroxy-3, S-bis (rnethylarnino) cyc1ohexyl]
oxy]tetrahydrofuran-3-carboxylic acid (actinospectinoic acid),
Compound R Molee Formula Mr
8025-81-8
C. Rl = CH3 , R2 = R4 = H, R3 = OH:
(2R,4S,4aS,5aR,6S, 7S,8R, 9S, 9aR, 10aS)-2-rnethyl- Action and use
6,8-bis(rnethylarnino )decahydro-2H-pyrano [2,3- Antibacterial.
b] [1,4]benzodioxine-4,4a, 7,9-tetrol
((4S)-dihydrospectinornycin),
102 Spiramycin
~E~ ___________________________________________
DEFINITION
Sulfadimidine contains not les s than 99.0 per cent and not
more than the equivalent of 101.0 per cent of 4-amino-
OHC N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide,
CH 3 H-- calculated with reference 10 the dried substance.
h oo , CHARACTERS
V
:3C(~/CH3) ~~ ,
White or almost white powder or crystals, very slight1y
HO ~~ , soluble in water, soluble in acetone, slight1y soluble in
CH 3 OH H
OH alcohol. It dissolves in solutions of alkali hydroxides and in
dilute mineral acids.
CH 3 It melts at about 197 oC, with decomposition.
IDENTIFICATION
D. (4R,5S,6S, 7R,9R,10R,11E,13E,16R)-6-[[3,6-dideoxy- First identification A~ B.
4-0-(2,6-dideoxy-3-C-methyl-rt-L-ribo-hexopyranosyl)-
Second identification B~
C, D.
3-(dimethylamino)-~-D-glucopyranosyl]oxy]-10-[(2,6-
dideoxy-3-C-methyl-rt-L-ribo-hexopyranosyl) oxy] -4-hydroxy- A. Examine by infrared absorption spectrophotometry
5-methoxy-9, 16-dimethyl-7 -(2-oxoethyl)oxacyclohexadeca- (2.2.24), comparing with the spectrum obtained with
1l,13-dien-2-one (spiramycin V), sulfadimidine CRS. Examine the substances prepared as discs.
B. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
obtained with test solution (a) corresponds in position and
size 10 the principal spot in the chromatogram obtained with
reference solution (a).
C. Place 3 g in a dry tube. Immerse the lower part of the
tube, inclined at 45°, in a silicone oil bath and heat to about
270 oC. The substance to be examined decomposes and a
white or yellowish-white. sublimate is formed which, after
recrystallisation from toluene R and drying at 100 oC, melts
(2.2.14) at 150 oC to 154 oC.
D. Dissolve about 5 mg in 10 mL of 1 M hydrochloric acid.
Dilute 1 mL of the solution to 10 mL with water R.
The solution, without further acidification, gives the reaction
ofprimary aromatic amines (2.3.1).
TESTS
Appearance of solution
Dissolve 0.5 g in a mixture of 5 mL of dilute sodium hydroxide
solution R and 5 mL of water R. The solution is not more
in~ensely coloured than reference solution Ys, BYs or GY s
(2.2.2~ Method JI).
Acidity
To 1.25 g, finely powdered, add 25 mL of carbon dioxide-free
F. spiramycin dimer. water R. Heat at about 70 oC for 5 min. Cool in iced water
___________________________________________ ~E~
for about 15 min and filter. To 20 mL of the filtrate add
0.1 mL of bromothymol blue solution R1. Not more than
0.2 mL of 0.1 M sodium hydroxide is required to change the
colour of the indicator.
Related substances
Sulfadimidine Examine by thin-Iayer chromatography (2.2.27), using silica
(Ph Eur monograph 0295) gel GF254 Ras the coating substance.
Test solution (a) Dissolve 20 mg of the substance to be
examined in 3 mL of a mixture of 2 volumes of concentrated
ammonia R and 48 volumes of methanol R and dilute to
5.0 mL with the same mixture of solvents.
Test solution (b) Dissolve 0.10 g of the substance to be
examined in 0.5 mL of concentrated ammonia R and dilute to
5.0 mL with methanol R. If the solution is not clear, heat
gent1y until dissolution is complete.
278.3 57-68-1
Reference solution (a) Dissolve 20 mg of sulfadimidine CRS in
Action and use 3 mL of a mixture of 2 volumes of coneentrated ammonia R
Sulfonamide antibacterial. and 48 volumes of methanol R and dilute to 5.0 mL with the
same mixture of solvents.
Preparation
Sulfadimidine Injection
Sulfamerazine 105
Heavy metals (2.4.8) to the principal spot in the chromatogram obtained with
1.0 g complies with limit test C for heavy metals (20 ppm). reference solution (a).
Prepare the standard using 2 mL of lead standard solution C. Dissolve 0.5 g in 1 mL of a 40 per cent V/V solution of
(10 ppm Pb) R. sulfuric acid R, heating gentIy. Continue heating until a
Loss on drying (2.2.32) crystalline precipitate appears (about 2 min). Cool and add
Not more than 0.5 per cent, determined on 1.000 g by 10 mL of dilute sodium hydroxide solution R. Cool again, add
drying in an oven at 105 oC. 25 mL of ether R and shake the solution for 5 mino Separate
Sulfated ash (2.4.14) the ether layer, dry over anhydrous sodium sulfate R and filter.
Not more than 0.1 per cent, determined on 1.0 g. Evaporate the ether by heating in a water-bath. An oily
residue is obtained which becomes crystalline on cooling;
ASSAY if necessary, scratch the wall of the container with a glass
Dissolve 0.2500 g in a mixture of 20 mL of dilute hydrochloric rod. The residue melts (2.2.14) at 102 oC to 106 oC.
acid R and 50 mL of water R. Cool the solution in iced D. Dissolve about 5 mg in 10 mL of 1 M hydrochloric acid.
water. Carry out the determination of primary aromatic Dilute 1 mL of the solution to 10 mL with water R.
amino- nitrogen (2.5.8), determining the end-point The solution, without further acidification, gives the reaction
e1ectrometrically. of primary aromatic amines (2.3.1).
1 rnL of 0.1 M sodium nitrite is equivalent to 26.43 mg of
TESTS
CllH12N402S,
Appearance of solution
STORAGE Dissolve 1.0 g in a mixture of 10 mL of 1 M sodium
Store protected from light. hydroxide and 15 mL of water R. The solution is clear (2.2.1)
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur and not more intensely coloured than reference solution Y 4
or BY4 (2.2.2, Method JI).
Acidity
To 1.25 g, finely powdered, add 25 mL of carbon dioxide-free
water R. Heat at 70 oC for 5 mino Cool in iced water for
Sulfametoxypyridazine about 15 min and filter. To 20 mL ofthe filtrate add 0.1 mL
(Sulfamethoxypyridazine for Veterinary Use, of bromothymol blue solution Rl. Not more than 0.5 mL of
Ph Eur monograph 0638) 0.1 M sodium hydroxide is required to change the colour of
the indicator.
Related substances
Examine by thin layer chromatography (2.2.27), using
TLC silica gel GF254 plate R.
Test solution (a) Dissolve 0.10 g of the substance to be
examined in acetone R and dilute to 5 mL with the same
solvento
280.3 80-35-3
Test solution (b) Dilute 1 mL of test solution (a) to 10 mL
Action and use with acetone R.
Sulfonamide antibacterial. Reference solution (a) Dissolve 20 mg of
~E~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ___ sulfamethoxypyridazine CRS in acetone R and dilute to 10 mL
with the same solvento
DEFINITION Reference solution (b) Dilute 2.5 mL of test solution (b) to
Sulfamethoxypyridazine for veterinary use contains not less 50 mL with acetone R.
than 99.0 per cent and not more than the equivalent of Apply separately to the plate 5 ¡.tL of each solution. Deve10p
101.0 per cent of 4-amino-N-(6-methoxypyridazin- over a path of 15 cm using a mixture of 1 volume of dilute
3-yl)benzenesulfonamide, calculated with reference to the ammonia Rl, 9 volumes of water R, 30 volumes of 2-
dried substance. propanol R and 50 volumes of ethyl acetate R. Dry the plate at
CHARACTERS 100-105 oC and examine in ultraviolet light at 254 nm.
A white or slightIy yellowish, crystalline powder, colouring Any spot in the chromatogram obtained with test solution
slowly on exposure to light, practically insoluble in water, (a), apart from the principal spot, is not more intense than
sparingly soluble in acetone, slightIy soluble in alcohol, very the spot in the chromatogram obtained with reference
slightIy soluble in methylene chloride. It dissolves in solutions solution (b) (0.5 per cent).
of alkali hydroxides and in dilute mineral acids. Heavy metals (2.4.8)
It melts at about 180 oC, with decomposition. 1.0 g complies with limit test D for heavy metals (20 ppm).
Prepare the standard using 2 mL of lead standard solution
IDENTIFICATION
(10 ppm fb) R.
First identification A, B.
Loss on drying (2.2.32)
Second identification B, C, D.
Not more than 0.5 per cent, determined on 1.000 g by
A. Examine by infrared absorption spectrophotometry drying in an oven at 105 oC.
(2.2.24), comparing with the spectrum obtained with
sulfamethoxypyridazine CRS. Examine the substances prepared Sulfated ash (2.4.14)
as discs. Not more than 0.1 per cent, determined on 1.0 g.
B. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position and size
Sulfanilamide 107
(e) After removal of the plate, allow it to dry in air until the
Sulfaquinoxaline solvent has evaporated and examine under ultraviolet light
(254 nm).
MOBILE PHASE
20 volumes of IBM ammonia, 40 volumes of methanol and
60 volumes of chloroform.
LIMITS
In the chromatogram obtained with solution (1):
any spot corresponding to NI ,N2 -diquinoxalin-
59-40-5
2-ylsulphanilamide is not more intense than the spot in the
Action and use chromatogram obtained with solution (2) (3%);
Sulfonamide antibacterial. any other secondary spot is not more intense than the spot in
the chromatogram obtained with solution (3) (1 %).
DEFINITION Loss on drying
Sulfaquinoxaline is N 1-quinoxalin-2-ylsulphanilamide. When dried to constant weight at 105°, loses not more than
It contains not less than 98.0% and not more-than 101.0% 1.0% of its weight. Use 1 g.
of C14H12N402S, calculated with reference to the dried
Sulphated ash
substance.
Not more than 0.1 %, Appendix IX A. Ignite at 600° and use
CHARACTERISTICS 1.5 g.
A yellow powder.
ASSAY
Practically insoluble in water; very slight1y soluble in ethanol Dissolve 0.65 g in 10 mI of a mixture of equal volumes of
(96%); practically insoluble in ether. It dissolves in aqueous 1M sodium hydroxide and water. Add 20 mI of glycerol, 20 mI
solutions of alkalis. of 9M sulphuric acid and 5 g of potassium bromide, cool in ice
IDENTIFICATION and titrate slowly with O.lM sodium nitrite VS, stirring
A. The infrared absorption spectrum, Appendix n A, is constantly and determining the end point electrometrically.
concordant with the reference spectrum of sulfaquinoxaline Each mI of O.lM sodium nitrite VS is equivalent to 30.03 mg
(RSV 41). of C14H12N402S.
B. The light absorption, Appendix n B, in the range 230 to STORAGE
350 nm of a 0.001 % w/v solution in O.OlM sodium hydroxide Sulfaquinoxaline should be protected from light.
exhibits a maximum only at 252 nm. The absorbance at
252 nm is about 1.1.
C. Yields the reaction characteristic of primary aromatic
amines, Appendix VI, dissolving 4 mg in 2 mI of warm Sulfathiazole Sodium
2M hydrochloric acid. An orange-red precipitate is produced.
TESTS
Acidity
To 2 g add 100 mI of water, heat at 70° for 5 minutes, cool
to 20° and filter. 50 mI of the filtrate requires for titration to
pH 7.0 not more than 0.2 mI of O.lM sodium hydroxide VS,
Appendix V L.
Heavy metals C9HsN3Na02Sb1 V2H 20 304.3 144-74-1 (anhydrous)
Dissolve the residue obtained in the test for Sulphated ash in C9HsN3Na02Sb5H20 367.4 6791-71-5
1 mI of 2M hydrochloric acid and dilute to 14 mI with water.
12 mI of the solution complies with limit test A for heavy Action and use
metals, Appendix VII. Use lead standard solution (2 ppm Pb) to Sulfonamide antibacterial.
prepare the standard (20 ppm).
DEFINITION
Related substances Sulfathiazole Sodium is the hydrated sodium salt of
Carry out ~e method for thin-layer chromatography, N 1-thiazol-2-yl sulfanilamide, either the sesquihydrate or the
Appendix nI A, using the following solutions. pentahydrate. It contains not les s than 99.0% and not more
(1) Dissolve 0.40 g of the substance being examined in 4 mI than 101.0% of C9HsN3Na02S2, calculated with reference to
of 1M sodium hydroxide and add sufficient methanol to produce the dried substance.
100 mI.
CHARACTERISTICS
(2) 0.012% w/v ofNI,N2 -diquinoxalin-2-
A white or yellowish white, crystalline powder or granules.
ylsulphanilamide BPCRS in methanol.
Freely soluble in water; soluble in ethanol (96%).
(3) 0.0040% w/v of sulphanilamide in methanol.
IDENI}FICATION
CHROMATOGRAPHIC CONDITIONS
A. The infrared absorption spectrum of the dried substance,
(a) Use a silica gel F 254 precoated plate (Merck silica gel 60
Appendix n A, is concordant with the reference spectrum of
F 254 plates are suitable). sulfathiazole sodium (RSV 42). -
(b) Use the mobile phase as described below.
(e) Apply 5 111 of each solution.
(d) Develop the plate to 15 cm.
Testosterone Phenylpropionate 109
R2
D. (3aR,4R,6S,8R,9R,9aR, 10R)-6-ethenylhydroxy-
4,6,9,1 0-tetramethyl-5-oxodecahydro-3a, 9-propano-
3aH-cyc1opentacyc1oocten-8-yl
[[2-(diethylamino)ethyl] sulfanyl] acetate, N. (2E)-4-[2-[[(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-
5-hydroxy-4, 6, 9,1 O-tetramethyl-l-oxodecahydro-
3a, 9-propano-3aH-cyc1opentacyc1oocten-8-yl] oxy]-
2-oxoethoxy]-4-oxobut-2-enoic acid (pleuromutilin
22-fumarate),
112 Tiamulin Hydrogen Fumarate
Related substances
Liquid chromatography (2.2.29).
Ammonium carbonate buffer solution pH 10. O Dissolve 10.0 g
OH of ammonium carbonate R in water R, add 22 mL of perchloric
acid solution R and dilute to 1000.0 mL with water R. Adjust
to pH 10.0 with concentrated ammonia R1.
Solvent mixture ammonium carbonate buffer solution
Q. (3 aS,4R, 5S, 6S, 8R,9R, 10R)-6-ethenyl-2,5-dihydroxy-
pH 10.0, acetonitrile R1 (50:50 V/V).
4,6,9,1 0-tetramethyl-2,3,4, 5, 6,7, 8,9-octahydro-3a,9-prop ano-
3aH-cyclopentacycloocten-8-yl Test solution Dissolve 0.200 g of the substance to be
[[2-( diethylamino )ethyl] sulfanyl] acetate (3,4-didehydro- examined in the solvent mixture and dilute to 50.0 mL with
2-hydroxytiamulin), the solvent mixture.
Reference solution (a) Dissolve 0.200 g of tiamulin hydrogen
~ ~:rCH'
fumarate CRS in the solvent mixture and dilute to 50.0 mL
with the solvent mixture.
~"'~CH,
I Reference solution (b) Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture.
Reference solution (c) Dissolve 40.0 mg of fumaric acid R in
R. N-benzyl-N,N-dibutylbutan-1-aminium. the solvent mixture and dilute to 50.0 mL with the solvent
___________________________________________ PhE~
mixture.
Reference solution (d) Dissolve 4 mg of tiamulin for peak
identification CRS (tiamulin hydrogen fumarate containing
impurities B, C, D, F, H and 1) in the solvent mixture and
Tiamulin Hydrogen Fumarate dilute to 1 mL with the solvent mixture.
(Tiamulin Hydrogen Fumarate for Veterinary use, Column:
Ph Eur monograph 1659) - size: 1 = 0.15 m, 0 = 4.6 mm,
- stationary phase: end-capped octadecylsilyl silica gel for
(CH 3 chromatography R (5 ¡.tm),
- temperature: 30 oC.
H3C~N~S~O
Mobile phase acetonitrile R1, ammonium carbonate buffer
O solution pH 10. O, methanol R1 (21 :30:49 V/V/V).
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 212 nm.
lny"ection 20 ¡.tL.
610 55297-96-6 Run time 3 times the retention time of tiamulin.
- total: not more than 3 times the area of the principal peak M. R1 = R2 = CO-CH3: (3aS,4R,5S,6S,8R,9R,9aR,10R)-
in the chromatogram obtained with reference solution (b) 6-ethenyl-4,6,9, 10-tetramethyl-1-oxodecahydro-
(3.0 per cent), 3a, 9-propano-3 aH-cyc1opentacyc100cten-5,8-diyl diacetate
- disregard limit: 0.1 times the area of the principal peak in (mutilin 11,14-diacetate),
the chromatogram obtained with reference solution (b) P. R1 = CO-CHz-O-SOz-C6Hs, R2 = H:
(0.1 per cent); disr,egard any peak present in reference (3aS,4R,5S,6S,8R, 9R, 9aR, 10R)-6-ethenyl-5-hydroxy-
solution (c). 4,6,9,10-tetramethyl-1-oxodecahydro-3a, 9-propano-
Loss on drying (2.2.32) 3aH-cyc1opentacyc100cten-8-yl [(phenylsulfonyl) oxy] acetate,
Maximum 0.5 per cent, determined on 1.000 g by drying in T. R1 = CO-CHz-[S-CHz-CH2-hN(C2Hs)2, R2 = H:
an oven at 105 oC. (3aS,4R,5S,6S,8R, 9R, 9aR, 10R)-6-ethenyl-5-hydroxy-
ASSAY 4,6,9,1 0-tetramethyl-1-oxodecahydro-3a, 9-propano-
Liquid chromatography (2.2.29) as described in the test for 3aH-cyc1opentacyc100cten-8-yl
re1ated substances with the following modification. [[2-[[2-( diethylamino)ethyl] sulfanyl] ethyl] sulfanyl] acetate,
Injection Test solution and reference solution (a).
Calculate the percentage content of C32HsINOgS from the
dec1ared contentof-tiamulin hydrogen fumarate CRS.
STORAGE
Protected from light. B. R = CHz-C 6Hs: 2-(benzylsulfanyl)-
IMPURITIES N,N-diethylethanamine,
Specijied impurities A, B, C, D, E, F, G, H, 1, J, K, L, M, C. R = S-CHz-CHz-N(C2Hs)2: 2,2'-(disulfane-
S, T. 1,2-diyl) bis (N,N-diethylethanamine),
Other detectable impurities (the following substances would, if O. R = H: 2-(diethylamino)ethanethiol,
present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or
by the general monograph SubstCmces for pharmaceutical use
(2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): N, O,
P, Q, R.
D. (3aR,4R,6S,8R,9R,9aR,10R)-6-ethenylhydroxy-
4,6,9,10-tetramethyl-5-oxodecahydro-3a,9-propano-
3aH-cyc1opentacycloocten-8-yl
[[2-(diethylamino)ethyl] sulfanyl] acetate,
R2
A. R1 = R2 = H: (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-
5,8-dihydroxy-4, 6,9,10-tetramethyloctahydro-3a, 9-propano-
3aH-cyc1opentacyc100cten-1 (4H)-one (mutilin),
G. R1 = CO-CH 20H, R2 = H:
(3aS,4R,5S,6S,8R, 9R, 9aR, 10R)-6-ethenyl-5-hydroxy- E. (3aS,4R,6S,8R, 9R, 9aR, 10R)-6-ethenyl-
4,6,9,10-tetramethyl-1-oxodecahydro-3a, 9-propano-3 aH- 4,6,9,10-tetramethyl-1,5-dioxodecahydro-3a,9-propano-
cyc1opentacyc100cten-8-yl hydroxyacetate (pleuromutilin), 3aH-cyclopentacyc100cten-8-yl
J. R1 = CO-CH3, R2 = H: (3aS,4R,5S,6S,8R,9R,9aR,lOR)- [[2-( diethylamino )ethyl] sulfanyl] acetate (ll-oxotiamulin),
6-ethenyl-5-hydroxy-4,6, 9,1 0-tetramethyl-1-oxodecahydro- F. impurity of unknown structure with a re1ative retention of
3a, 9-propano-3aH-cyc1opentacycloocten-8-yl acetate (mutilin about 0.8,
14-acetate) ,
K. R1 = H, R2 = CO-CH3:
(3aS,4R,5S,6S,8R, 9R, 9aR, 10R)-6-ethenyl-8-hydroxy-
4,6,9,1 0-tetramethyl-1-oxodecahydro-3a, 9-propano-3aH-
cyc1opentacycloocten-5-yl acetate (mutilin 11-acetate),
L. R1 = CO-CHz-O-SOZ-C6H4-pCH3, R2 = H:
(3aS,4R,5S,6S,8R, 9R, 9aR, 10R)-6-ethenyl-5-hydroxy-
4,6,9,10-tetramethyl-1-oxodecahydro-3a, 9-propano-
3aH-cyc1opentacyc100cten-8-yl
[[ (4-methylphenyl)sulfonyl] oxy] acetate (pleuromutilin
H. (2E)-4-[ (2RS)-2-[ (3aS,4R,5S,6R,8R, 9R,9aR, 10R)-8-[[[[2-
22-tosylate) ,
(diethylamino )ethyl] sulfanyl] acetyl] oxy] -5-hydroxy-4,6, 9,10-
tetramethyl-1-oxodecahydro-3a, 9-propano-3 aH-
cyclopentacyc100cten-6-yl] -2-hydroxyethoxy] -4-oxobut-
2-enoic acid (19,20-dihydroxytiamulin 20-fumarate),
114 Tylosin
Tylosin
(Tylosinfor Veterinary Use, Ph Eur monograph 1273)
H~O~
O
I
osyl t--J
H3C~¿:'CH~3~ ,' ~~' O~,'~'H ~
1. (2E)-4-[[(3aS,4R,5S,6S,8R,9R,9aR,10R)-8-[[[[2-
(diethylamino)ethyl] sulfanyl] acetyl] oxy] -6-ethenyl-
1, 5-dihydroxy-4, 6, 9,1 O-tetramethyldecahydro-3a, 9-propano-
3 aH-cyc1opentacyc1oocten-2-yl] oxy] -4-oxobut-2-enoic acid
(2,3-dihydroxytiamulin 2-fumarate), O HO " O --H
I H
R1 OH H C HO
3 R2 OCH 3
Name Mol. Formula R1 R2 R3
tylosin A C46H77N017 osyl OCH 3 CHO
tylosin B C39H6SN014 H OCH 3 CHO
tylosin C C4sH7SN017 osyl OH CHO
tylosin D C46H79N017 osyl OCH 3 CH 20H
N. (2E)-4-[2-[[(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-
5-hydroxy-4,6, 9,1 0-tetramethyl-1-oxodecahydro- Action and use
3 a, 9-propano-3aH-cyc1opentacyc1oocten-8-yl] oxy]- Macrolide antibacterial.
2-oxoethoxy]-4-oxobut-2-enoic acid (pleuromutilin Preparation
22-fumarate), Tylosin Injection
~E~ __________________________________________
DEFINITION
Mixture of macrolide antibiotics produced by a strain of
OH Streptomyces fradiae or by any other means. The main
component of the mixture is
(4R,5S,6S, 7R,9R, 11E, l3E, 15R,16R)-15-[[(6-deoxy-2,3-di-O-
methyl-~-D-allopyranosyl)oxy]methyl]-6-[[3,6-dideoxy-4-0-
(2,6-dideoxy-3-C-methyl-cx-L-ribo-hexopyranosyl)-
Q. (3aS,4R,5S,6S,8R,9R, 10R)-6-ethenyl-2,5-dihydroxy- 3-(dimethylamino )-~-D-glucopyranosyl] oxy] -16-ethyl-
4,6,9,10-tetramethyl-2,3,4,5,6, 7,8,9-octahydro-3a,9-propano- 4-hydroxy-5, 9, 13-trimethyl-7-(2-oxoethyl) oxacyc1ohexadeca-
3aH-cyc1opentacyc1oocten-8-yl 1l,13-diene-2,10-dione (tylosin A, M r 916). Tylosin B
[[2-( diethylamino )ethyl] sulfanyl] acetate (3,4-didehydro- (desmycosin, M r 772), tylosin C (macrocin, M r 902) and
2-hydroxyriamulin), tylosin D (relomycin, M r 918) may also be presento They
coritribute to the potency of the substance to be examined.
Potency
Minimum 900 IU/mg (dried substance).
CHARACTERS
Appearance
Almost white or slight1y yellow powder.
R. N-benzyl-N,N-dibutylbutan-1-aminium, Solubility
Slight1y soluble in water, freely soluble in anhydrous ethanol
and in methylene chloride. It dissolves in dilute solutions of
mineral acids.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
and epimer at C* Comparison tylosin CRS.
B. Examine the chromatograms obtained in the test for
composition.
Results The principal peak in the chromatogram obtained
S. (lRS,3aR,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-1-ethyl-
with the test solution is similar in retention time and size to
1,5-dihydroxy-4,6,9,1 O, 12, 12-hexamethyldecahydro-
the principal peak in the chromatogram obtained with
3a, 9-propano-3aH-cyc1opentacyc1oocten-8-yl
reference solution (a).
[[2-(diethylamino)ethyl]sulfanyl]acetate.
__________________________________________ ~E~
C. Dissolve about 30 mg in a mixture of 0.15 mL of water R,
2.5 mL of acetic anhydride R and 7.5 mL of pyridine R. Allow
to stand for about 10 mino No green colour develops.
Tylosin Phosphate 115·
H~:'~q~OH
of tylosin D CRS in the solvent mixture and dilute to 10 mL
with the solvent mixture.
Column: CH 3 OH
OH
- size: 1 = 0.20 m, 0 = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
CH 3
(5 Ilm);
- temperature: 35 oC.
Mobile phase Mix 40 volumes of acetonitn7e R and A. desmycinosyltylosin,
60 volumes of a 200 gIL solution of sodium perchlorate R
previously adjusted to pH 2.5 using 1 M hydrochloric acid.
Flow rate 1.0 mUmin.
Detection Spectrophotometer at 290 nm.
Injection 20 1lL.
Retention time Tylosin A = about 12 mino
Identijication of peaks Use the chromatogram supplied with
tylosin phosphate for peak identijication CRS and the
chromatogram obtained with reference solution (a) to
identify the peaks due to tylosins A, B, C and D.
System suitability Reference solution (b):
- resolution: minimum 2.0 between the peaks due to tylosins
B. tylosin A aldol.
A and D.
___________________________________________ PhEw
Limits:
- tylosin A: minimum 80.0 per cent;
- sum of tylosins A, B, C and D: minimum 95.0 per cent.
Tyramine
Maximum 0.35 per cent and maximum 0.15 per cent, if Tylosin Phosphate
intended for use in the manufacture of parenteral
preparations. (Tylosin Phosphate Bulk Solution for Veterinary Use,
In a 25.0 mL volumetric flask, dissolve 50.0 mg in 5.0 mL of Ph Eur monograph 1661)
a 3.4 gIL solution of phosphoric acid R. Add 1.0 mL of
pyridine R and 2.0 mL of a saturated solution of ninhydrin R
(about 40 giL). Close the flask with a piece of aluminium foil
and heat in a water-bath at 85 oC for 30 mino Cool the
solution rapidly and dilute to 25.0 mL with water R. Mix and
measure immediately the absorbance (2.2.25) of the solution
at 570 nm using a blank solution as the compensation liquido
The absorbance is not greater than that of a standard
prepared at the same time and in the same manner using
5.0 mL of a 35 mglL solution of tyramine R in a 3.4 gIL
solution of phosphoric acid R. If intended for use in the
manufacture of parenteral preparations, the absorbance is not
Tylosin R1 R2 R3 Mol. Formula Mr
greater than that of a standard prepared at the same time and
A osyl OCH 3 CHO C46 H77 N0 17 916
in the same manner using 5.0 mL of a 15 mgIL solution of
B H OCH 3 CHO C39H6SN014 772
tyramine R in a 3.4 gIL solution of phosphoric acid R.
C osyl OH CHO C4sH7SN017 902
D osyl OCH 3 CH 20H C46H79N017 918
116 Tylosin Phosphate
Action and use Referenee solution (b) Dissolve 2 mg of tylosin CRS and 2 mg
Macrolide antibacterial. of tylosin D CRS in a mixture of equal volumes of
PhE~ ___________________________________________
aeetonitrile R and water R and dilute to 10 mL with the same
mixture of solvents.
DEFINITION Referenee solution (e) Dilute 1.0 mL of reference solution (a)
Solution of the dihydrogen phosphate of a mixture of to 100.0 mL with a mixture of equal volumes of aeetonitrz7e R
macrolide antibiotics produced by a strain of Streptomyees and water R. Dilute 1.0 mL of this solution to 10.0 mL with
fradiae or by any other means. a mixture of equal volumes of aeetonitrile R and water R.
The main component is the phosphate of Column:
(4R,5S,6S, 7R,9R, 11E, 13E,15R, 16R)-15-[[(6-deoxy-2,3-di-O- - size: l = 0.20 m, 0 = 4.6 mm;
methyl-~-D-allopyranosyl) oxy] methyl] -6- [[3, 6-dideoxy-4-0- - stationary phase: oetadeeylsilyl siliea gel for ehromatography R
(2, 6-dideoxy-3-C-methyl-et-L-ribo-hexopyranosyl)- (5 ¡.tm);
3-(dimethylamino )-~-D-glucopyranosyl] oxy]-16-ethyl- - temperature: 35 oc.
4-hydroxy-5,9,13-trimethyl-7-(2-oxoethyl)oxacyclohexadeca- Mobile phase Mix 40 volumes of aeetonitrz'le R and
11, 13-diene-2,1 O-dione (tylosin A phosphate). 60 volumes of a 200 giL solution of sodium perehlorate R
The phosphates of tylosin B (desmycosin phosphate), tylosin previously adjusted to pH 2.5 using a 36.5 giL solution of
C (macrocin phosphate) and tylosin D (relomycin hydroehlorie aeid R.
phosphate) may also be presento The solution also contains Flow rate 1.0 mUmin.
sodium dihydrogen phosphate.
Deteetion Spectrophotometer at 290 nm.
Potency
Injeetion 20 ¡.tL.
Minimum 800 IV per milligram of dry residue. Tylosins
A, B, C and D contribute to the potency. Run time 1.8 times the retention time of tylosin A.
Identifieation of tylosins V se the chromatogram supplied with
CHARACTERS
tylosin phosphate for peak identijieation CRS and the
Appearance
chromatogram obtained with reference solution (a) to
Yellow or brownish-yellow, viscous liquido
identify the peaks due to tylosins A, B, C and D.
Solubility Relative retention With reference to tylosin A (retention
Miscible with water. time = about 12 min): impurity A = about 0.35; tylosin
IDENTIFICATION C = about 0.5; tylosin B = about 0.6; tylosin D = about
A. Vltraviolet and visible absorption spectrophotometry 0.85; impurity B = about 0.9.
(2.2.25). System suitability Reference solution (b):
Test solution Dilute an amount of the preparation to be - resolution: minimum 2.0 between the peaks due to tylosin
examined equivalent to 400 000 IV of tylosin phosphate to D and tylosin A.
100.0 mL with water R. Dilute 1.0 mL ofthis solution to Limits:
100.0 mL with water R. - tylosin A: minimum 80.0 per cent;
Speetral range 230-350 nm. - sum of the eontents of tylosin A, tylosin B, tylosin C and
Absorption maximum At 290 nm. tylosin D: minimum 95.0 per cent;
- disregard limit: area of the principal peak inthe
Absorbanee at the absorption maximum Minimum 0.70. chromatogram obtained with reference solution (c).
B. Examine the chromatograms obtained in the test for
Tyramine
composition.
In a 25.0 mL volumetric fiask, dissolve an amount of the
Results The principal peak in the chromatogram obtained preparation to be examined equivalent to 50 000 IV of
with the test solution is similar in retention time and size to tylosin phosphate in 5.0 mL of a 3.4 giL solution of
the principal peak in the chromatogram obtained with phosphorie aeid R. Add 1.0 mL of pyridine R and 2.0 mL of a
reference solution (a). saturated solution of ninhydrin R (about 40 gIL). Close the
C. Dilute an amount of the preparation to be examined fiask with aluminium foil and heat in a water-bath at 85 oC
equivalent to 400 000 IV of tylosin phosphate in 10 mL of for 20-30 mino Cool the solution rapidly and dilute to
water R. The solution gives reaction (a) of phosphates 25.0 mL with water R. Mix and measure immediately the
(2.3.1). absorbance (2.2.25) ofthe solution at 570 nm using a blank
TESTS solution as the compensation liquido
pH (2.2.3) The absorbance is not greater than that of a standard
5.5 to 6.5. prepared at the same time and in the same manner using
Dilute 1.0 g in 10 mL of earbon dioxide-free water R. 5.0 mL of a 35 mglL solution of tyramine R in a 3.4 giL
solution of phosphorie aeid R.
Composition
Liquid chromatography (2.2.29): use the nonnalisation Phosphate
procedure. Prepare the solutions immediately before use. 8.5 per c~nt to 10.0 per cent of P0 4, calculated with
reference to the dry residue (see Assay).
Test solution Dilute an amount of the preparation to be
examined equivalent to 50 000 IV of tylosin phosphate to Test solution Dissolve an amount of the preparation to be
200 mL with a mixture of equal volumes of aeetonitrile R and examined equivalent to 200 000 IV of tylosin phosphate in
water R. 50 mL of water R. Add 5.0 mL of dilute sulfurie acid R and
dilute to 100.0 mL with water R. To 2.0 mL ofthis solution
Referenee solution (a) Dissolve 2 mg of tylosin phosphate for
add successively, mixing after each addition, 10.0 mL of
peak identifieation CRS (containing tylosins A, B, C and D) in
water R, 5.0 mL of ammonium molybdate reagent R2, 1.0 mL
a mixture of equal volumes of aeetonitrile R and water R and
of hydroquinone solution R and 1.0 mL of a 200 gIL solution
dilute to 10 mL with the same mixture of solvents.
Tylosin Tartrate 11 7
~
STORAGE
R3
H3
Protected from light, at a temperature of 2 oC to 8 oC. CH 3 /~O
--H O H
LABELLING
--H
The label states the concentration of the solution in O
Intemational Units per milligram of preparation.
H 3C
IMPURITIES CH 3
O
o
R2 OCH 3 2
OHC
CH 3 H--
. h oo ,
Ho~:lL(dOH
CH3 OH
OH
CH3
Action and use
Macrolide antibacterial.
A. desmycinosyltylosin A,
~E~ ___________________________________________
DEFINITION
Tartrate of a mixture of macrolide antibiotics produced by a
strain of Streptomyees fradiae or by any other means.
The main component of the mixture is
(4R,5S,6S, 7R,9R,11E, 13E, 15R, 16R)-15-[[(6-deoxy-2,3-di-0-
methyl-B-D-allopyranosyl) oxy] methyl] -6- [[3, 6-dideoxy-
4-0-(2 ,6-dideoxy-3-C-methyl-ex-L-ribo-hexopyranosyl)-
3-(dimethylamino)-B-D-glucopyranosyl] oxy] -16-ethyl-
4-hydroxy-5, 9, 13-trimethyl-7-(2-oxoethyl)oxacyc1ohexadeca-
1l,13-diene-2,10-dione (tylosin A, tartrate M r 1982).
Tylosin B (desmycosin, tartrate M r 1694), tylosin C
(macrocin, tartrate M r 1954) and tylosin D (relomycin,
118 Tylosin Tartrate
CHARACTERS 14 - 20 95 5
Appearance
White or yellowish, amorphous powder, hygroscopic. Flow rate 1.5 mUmin.
Deteetion Spectrophotometer at 200 nm.
Solubility
Freely soluble in water and in anhydrous ethanol, practically Injeetion 5 flL.
insoluble in tert-butyl methyl ether. Identifieation of impurities Use the chromatogram supplied
with valnemulin for peak identifieation CRS and the
IDENTIFICATION
chromatogram obtained with reference solution (c) to identify
A. Infrared absorption spectrophotometry (2.2.24).
the peaks due to impurities A, B and e.
Comparison valnemulin hydroehloride CRS.
Relative retention With reference to valnemulin
B. It gives reaction (a) of chlorides (2.3.1). (retention time = about 7 min): impurity D = about 0.2;
TESTS impurity A = about 0.7; impurity B = about 0.85;
pH (2.2.3) impurity E = about 0.9; impurity e = about 1.1.
3.0 to 6.0. System suitability Reference solution (b):
Dissolve 2.0 g in earbon dioxide-free water R and dilute to - resolution: minimum 1.5 between the peaks due to
20 mL with the same solvento impurity E and valnemulin.
Specific optical rotation (2.2.7) Limits:
+ 15.5 to + 18.0 (anhydrous substance). - eorreetion faetors: for the calculation of content multiply
the peak areas of the following impurities by the
Dissolve 0.250 g in water R and dilute to 25.0 mL with the
corresponding correction factor: impurity B = 3.2;
same solvento
impurity E = 4.2;
Related substances - impurity A: not more than 0.5 times the area of the
Liquid chromatography (2.2.29). principal peak in the chromatogram obtained with
Phosphate buffer solution pH 2.5 Dissolve 8.0 g of disodium reference solution (a) (0.5 per cent);
hydrogen phosphate R and 3.0 g of potassium dihydrogen - impurity B: not more than twice the area of the principal
phosphate R in water for ehromatography R and dilute to peak in the chromatogram obtained with reference
1000.0 mL with the same solvento Adjust to pH 2.5 with solution (a) (2.0 per cent);
phosphorie acid R. - impurity C: not more than the area of the principal peak
Solvent mixture Mix equal volumes of aeetonitrile R1 and in the chromatogram obtained with reference solution (a)
water for ehromatography R. (1.0 per cent);
- any other impurity: for each impurity, not more than
Test solution Dissolve 0.100 g of the substance to be
0.2 times the area of the principal peak in the
examined in the solvent mixture and dilute to 10. O mL with
chromatogram obtained with reference solution (a)
the solvent mixture.
(0.2 per cent);
Referenee solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with the solvent mixture.
120 Vedaprofen
- total: not more than 3 times the area of the principal peak B. R =H, X = S: (3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-
in the chromatogram obtained with reference solution (a) 5-hydroxy-4,6, 9,1 O-tetramethyl-l-oxodecahydro-
(3.0 per cent); 3a, 9-propano-3aH-cyc1openta [8] annulen-8-yl [(2-amino-
- disregard limit: 0.1 times the area of the principal peak in 1, l-dimethylethyl] sulfanyl] aceta te (dimethyl cysteaminyl
the chromatogram obtained with reference solution (a) pleuromulin) ,
(0.1 per cent); disregard the peak due to the chloride ion. C. R = D-Val-D-Val, X = S:
Water (2.5.12) (3aS,4R,5S,6S,8R, 9R, 9aR, 1OR) 6-ethenyl-5-hydroxy-
Maximum 4.0 per cent, determined on 0.500 g. 4,6,9,1 O-tetramethyl-l-oxodecahydro-3a, 9-propano-
3aH-cyc1openta[8]annulen-8-yl [[2-[[(2R)-2-[[(2R)-2-amino-
ASSAY
3-methylbutanoyl] amino] -3-methylbutanoyl] amino]-
Liquid chromatography (2.2.29).
1, l-dimethylethyl] sulfanyl] acetate (valyl-valneumulin),
Test solution Dissolve 40.0 mg of the substance to be
examined in a mixture of equal volumes of acetonitrile Rl and
water R and dilute to 50.0 mL with the same mixture of
solvents.
Reference solution Dissolve 50.0 mg of valnemulin hydrogen
tartrate CRS in a mixture of equal volumes of acetonitrzle Rl
D. (2R)-2-amino-3-methylbutanoic acid (D-valine),
and water R and dilute to 50.0 mL with the same mixture of
solvents.
Column:
- size: 1 = 0.125 m, (2) = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R
(3 flm);
- temperature: 45 oc.
Mobile phase Mix 43 volumes of acetonitrile Rl and
57 volumes of a solution containing 0.94 giL of disodium
E. (3aS,4R,5S,6S,8R, 9R, 9aR, 1OR) 6-ethenyl-5-hydroxy-
hydrogen phosphate R and 8.7 giL of potassium dihydrogen
4,6,9,1 O-tetramethyl-l-oxodecahydro-3a, 9-propano-3 aH-
phosphate R previously adjusted to pH 2.5 with phosphoric
cyc1openta[8] annulen-8-yl 2-hydroxyacetate (pleuromulin).
acid R.
- - - -_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ PhEur
Flow rate 1.2 mUmin.
Detection Spectrophotometer at 210 nm.
Injection 5 flL.
Run time 3 times the retention time of valnemulin Vedaprofen ****
(retention time = about 2.4 min). ** *
Calculate the percentage content of C31Hs3ClN20SS, using (Vedaprofen for Veterinary Use) *****
the dec1ared content of valnemulin hydrogen tartrate CRS and Ph Eur monograph 2248)
by multiplying by 0.841.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specijied impurities A) B) C.
Other detectable impurities (the following substances would, if
present at a sufficient leve!, be detected by one or other of
282.4 71109-09-6
the tests in the monograph. They are lin:ited by the general
acceptance criterion for other/unspecified impurities and/or Action and use
by the general monograph Substances for pharmaceutical use
Cyc1o-oxygenase inhibitor; analgesic; anti-inflarnmatory.
(2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. ~Ew _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
Control of impurities in substances for pharmaceutical use): D) E.
DEFINITION
(2RS)-2-( 4-Cyc1ohexyl-l-naphthyl)propanoic acid.
Content
98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white powder.
Solubility
A. R = D-Val, X = SO: (3aS,4R,5S,6S,8R,9R,9aR,10R)-6- Practicálly insoluble in water, free!y soluble in acetone,
ethenyl-5-hydroxy-4,6, 9,1 O-tetramethyl-l-oxodecahydro-3a, 9- soluble in methanol. It dissolves in dilute solutions of alkali
propano-3aH-cyc1openta[8]annulen-8-yl [[2-[[(2R)-2-amino- hydroxides.
3-methylbutanoyl] amino] -1, l-dimethylethyl] sulfinyl] acetate IDENTIFICATION
(valnemulin sulfoxide), Infrared absorption spectrophotometry (2.2.24).
Comparison vedaprofen CRS.
Xylazine Hydrochloride 121
Content Column:
98.0 per cent to 102.0 per cent (dried substance). - size: 1 = 0.15 m, 0 = 3.9 mm;
- stationary phase: end-capped octylsi1yl silica gel for
CHARACTERS
chromatography with polar incorporated groups R (5 ¡.tm);
Appearance
- temperature: 40 oC.
White or almost white, crystalline powder, hygroscopic.
Mobile phase:
Solubility - mobile phase A: mix 30 volumes of methanol R and
Freely soluble in water, very soluble in methanol, freely 70 volumes of a 2.72 gIL solution of potassium dihydrogen
soluble in methylene chloride. phosphate R adjusted to pH 7.2 with dilute sodium
IDENTIFICATION hydroxide solution Rj
A. Infrared absorption spectrophotometry (2.2.24). - mobile phase B: methanol R) acetonitrile R (30:70 V/V);
Comparison xylazine hydrochloride CRS. Time Mobile phase A Mobile phase B
B. It gives reaction (b) of chlorides (2.3.1). (min) (percent VM (per cent VIII)
0-15 89~ 28 11 ~ 72
TESTS
Solution S 15 - 21 28 72
ASSAY
Dissolve 0.200 g in 25 mL of ethanol (96 per cent) R.
Add 25 mL of water R. Titrate with 0.1 M sodium hydroxide~
determining the end-point potentiometricalIy (2.2.20).
1 mL of 0.1 M sodium hydroxide is equivalent to 25.68 mg
of C 1zH 17 CIN zS.
STORAGE
In an airtight container, protected from light.
IMPURITIES
Specijied impurities A~ B~ C~ D~ E.
A. 2,6-dimethylaniline (2,6-xylidine),
B. N~N'-bis(2,6-dimethylphenyl)thiourea,
C. 2,6-dimethylphenyl isothiocyanate,
D. N-(2,6-dimethylphenyl)-N'-(3-hydroxypropyl)thiourea,
Fortnulated Preparations
DIP CONCENTRATES
DEFINITION
Dip concentrates are preparations containing one or more
active substances, usually in the form of wettable powders,
pastes, solutions or suspensions, which are used to prepare
diluted solutions, suspensions or emulsions of active
substances. The diluted preparations are applied by complete
immersion of the animal.
POUR-ON PREPARATIONS
DEFINITION
Pour-on preparations contain one or more active substances
for the prevention and treatment of ectoparasitic andlor
endoparasitic infestations of animals. They are applied in
volumes which are usually greater than 5 mL by pouring
along the animal's dorsal midline.
128 General Monographs
TEAT SPRAYS
DEFINITION Eye Preparations of the British
Teat sprays contain one or more disinfectant active
substances, usually in the form of solutions which are sprayed Pharmacopoeia (Veterinary)
onto the teats of an animal pre- and post-milking, as In addition to the requirements of the European Pharmacopoeia,
appropriate, to reduce the population of pathogenic micro- the statements applicable to Eye Preparations of the British
organisms on the surfaces. Teat sprays may be Pharmacopoeia apply to those eye ointments that are the subject of
supplied/presented as ready-to-use preparations or they may an individual monograph in the British Pharmacopoeia
be prepared by dilution of teat spray concentrates. Pre- and (Veterinary) .
post-milking sprays often differ in formulation. Teat sprays
usually contain emollients to promote skin hydration, to
soften theskin and allow healing of lesions that would
otherwise harbour bacteria. ***
GRANULES
** **
UDDER-WASHES (Ph Eur monograph 0499) *****
DEFINITION Unless/otherwise justified and authorised, Granules comply with
U dder-washes contain one or more disinfectant active the appropriate requirements of the European Pharmacopoeia.
substances, usually in the form of solutions which are sprayed These requirements are reproduced in the British Pharmacopoeia.
onto the udder and teats of an animal to remove mud and
faecal contamination before the application of teat dips or
sprays. Udder-washes are usually prepared by the dilution
General Monographs 129
on this aspect are provided in the text on Microbiological conform to the definition given in the monograph on Tablets
quality of pharmaceutical preparations (5.1.4), see Table (0478).
5.1.4.-1. - Cutaneous use. A suitable applicator may be used for application into the
Sterile intrauterine preparations for veterinary use are uterus.
prepared using materials and methods designed to ensure
TESTS
sterility and to avoid the introduction of contaminants and
Disintegration
the growth of microorganisms; recommendations on this
Unless intended for prolonged local action, they comply with
aspect are provided in the text on Methods of preparation of
the test for disintegration of suppositories and pessaries
sterile products (5.1.1).
(2.9.2). Examine the state of the tablets after 30 min, unless
During development, it must be demonstrated that the otherwise justified and authorised.
nominal content can be withdrawn from the container of
liquid and semi-solid intrauterine preparations for veterinary
use presented in single-dose containers. INTRAUTERINE CAPSULES
TESTS DEFINITION
Uniformity of dosage units Intrauterine capsules are solid, single-dos e preparations. They
Single-dose intrauterine preparations for veterinary use are generally similar to soft capsules, differing only in their
comply with the test for uniformity of dosage units (2.9.40) shape and size. Intrauterine capsules have various shapes,
or, where justified and authorised, with the tests for usually ovoid. They are smooth and have a uniform external
uniformity of content and/or uniformity of mass shown appearance.
below. Herbal drugs and herbal drug preparations present in A suitable applicator may be used for application into the
the dosage form are not subject to the provisions ofthis uterus.
paragraph. TESTS
Uniformity of content (2.9.6) Disintegration
Unless otherwise prescribed or justified and authorised, solid Unless intended for prolonged local action, they comply with
single-dose preparations with a content of active substance the test for disintegration of suppositories and pessaries
less than 2 mg or less than 2 per cent of the total mass (2.9.2). Examine the state of the capsules after 30 min,
comply with test A (intrauterine tablets) or test B unless otherwise justified and authorised.
/
(intrauterine capsules) for uniformity of content of single-
dose preparations. If the preparation has more than 1 active
substance, the requirement applies only to those substances INTRAUTERINE SOLUTIONS,
which correspond 10 the aboye conditions. SUSPENSIONS AND EMULSIONS
Uniformity of mass (2.9.5) CONCENTRATESFORINTRAUTERINE
Solid single-dose intrauterine preparations for veterinary use SOLUTIONS
comply with the test for uniformity of mass of single-dose DEFINITION
preparations. If the test for uniformity of content is Intrauterine solutions, suspensions and emulsions are liquid
prescribed or justified and authorised for all the active preparations. Concentrates for intrauterine solutions are
substances, the test for uniformity of mass is not required. intended for administration after dilution.
Dissolution They may contain excipients, for example to adjust the
A suitable test may be carried out to demonstrate the viscosity of the preparation, to adjust or stabilise the pH, to
appropriate release of the active substance(s) from solid increase the solubility of the active substance(s) or to stabilise
single-dose intrauterine preparations for veterinary use, for the preparation. The excipients do not adversely affect the
example one of the tests described in Dissolution test for solid intended medical action, or, at the concentrations used,
dosage forms (2.9.3). cause undue local irritation.
When a dissblution test is prescribed, a disintegration test Intrauterine emulsions may show evidence of phase
may not be required. separation, but are readily redispersed on shaking.
Sterility (2.6.1) Intrauterine suspensions may show a sediment that is readily
Sterile intrauterine preparations for veterinary use comply dispersed on shaking to give a suspension which remains
with the test for sterility. Applicators supplied with the sufficient1y stable to enable a homogeneous preparation to
preparation also comply with the test for sterility. Remove be de1ivered.
the applicator with aseptic precautions from its package and They may be supplied in single-dos e containers. The
transfer it to a tube of culture medium so that it is container is adapted to deliver the preparation to the uterus
completely immersed. Incubate and interpret the results as or it may be accompanied by a suitable applicator.
described in the test for sterility.
PRODUCTION
LABELLING In the manufacture of intrauterine suspensions, measures are
The labe1 states: taken to ensure a suitable and controlled particle size with
- the na me of any added antimicrobial preservative; regard to the intended use.
- where applicable, that the preparation is sterile.
T ABLETS FOR INTRAUTERINE
INTRAUTERINE TABLETS SOLUTIONS AND SUSPENSIONS
DEFINITION DEFINITION
Intrauterine tablets are solid preparations each containing a Tablets intended for the preparation of intrauterine solutions
single dose of 1 or more active substances. They generally and suspensions are single-dose preparations which are
dissolved or dispersed in water at the time of administration.
132 General Monographs
- powders for injections or infusions; Multidase preparatians Multidose aqueous injections contain
- gel s for injections; a suitable antimicrobial preservative at an appropriate
-;- implants. concentration except when the preparation itself has adequate
PRODUCTION antimicrobial properties. When a preparation for parenteral
During the development of a parenteral preparation, the administration is presented in a multidose container, the
formulation for which contains an antimicrobial preservative, precautions to be taken for its administration and more
the effectiveness of the chosen preservative shall be particularly for its storage between successive withdrawals are
demonstrated to the satisfaction of the competent authority. given.
A suitable test method together with criteria for judging the Antimicrabial preservatives Aqueous preparations which are
preservative properties of the formulation are providecl under prepared using aseptic precautions and which cannot be
Efficacy af antimicrabial preservatian (5.1.3). terminally sterilised may contain a suitable antimicrobial
Parenteral preparations are prepared using materials and preservative in an appropriate concentration.
methods designed to ensure sterility and to avoid the N o antimicrobial preservative is added when:
introduction of contaminants and the growth of micro- - the volume to be injected in a single dose exceeds 15 mL,
organisms. Recommendations on this aspect are provided in unless otherwise justified;
the text on MethadsJJj preparatían af sterile praducts (5.1.1). - the preparation is intended for administration by routes
Water used in the manufacture of parenteral preparations where, for medical reasons, an antimicrobial preservative
complies with the requirements of water for injections in bulk is not acceptable, such as intracistemally, epidurally,
stated in the monograph on Water far injectíans (0169). intrathecally or by any route giving access to the
cerebrospinal fluid, or intra- or retro-ocularly.
TESTS
Such preparations are presented in single-dose containers.
Particulate contamination: sub-visible partic1es (2.9.19)
For preparations for human use, solutions for infusion or PRODUCTION
solutions for injection comply with the test. In the manufacture of injections containing dispersed
In the case of preparations for subcutaneous or intramuscular particles, measures are taken to ensure a suitable and
injection, higher limits may be appropriate. controlled particle size with regard to the intended use.
Radiopharmaceutical preparations are exempt from these Single-dase preparatíans The volume of the injection in a
requirements. Preparations for which the label states that the single-dose container is sufficient to permit the withdrawal
product is to be used with a final filter are exempt from these and administration of the nominal dose using a normal
requirements, providing it has been demonstrated that the technique (2.9.17). "-
filter delivers a solution that complies with the test. TESTS
F or preparations for veterinary use, when supplied in Uniformity of dosage units
containers with a nominal content of more than 100 mL and Single-dos e suspensions for injection comply with the test for
when the content is equivalent to adose of more than uniformity of dosage units (2.9.40) or, where justified and
1.4 mL per kilogram of body mass, solutions for infusion or authorised, with the test for uniformity of content shown
solutions for injection comply with the test for particulate below. Herbal drugs and herbal drug preparations present in
contamination: sub-visible particles. the dosage form are not subject to the provisions of this
Sterility (2.6.1) paragraph.
Parenteral preparations comply with the test for sterility. Uniformity of content (2.9.6)
STORAGE Unless otherwise prescribed or justified and authorised,
In a sterile, airtight, tamper-proof container. single-dose suspensions for injection with a content of active
substance les s than 2 mg or les s than 2 per cent of the total
LABELLING mass comply with test A for uniformity of content of single-
The label states: dos e preparations. If the preparation contains more than one
- the name and concentration of any added antimicrobial active substance, the requirement applies only to those
preservative; substances that correspond to the aboye conditions.
- where applicable, that the solution is to be used in
Bacteria! endotoxins - pyrogens
conjunction with a final filter;
A test for bacterial endotoxins (2.6.14) is carried out or,
- where applicable, that the preparation is free from
where justified and authorised, the test for pyrogens (2.6.8).
bacterial endotoxins or that it is apyrogenic.
Recommendations on the limits for bacterial endotoxins are
given in chapter 2.6.14.
INJECTIONS Preparatians far human use The preparation complies with a
DEFINITION test for bacterial endotoxins (2.6.14) or with the test for
Injections are sterile solutions, emulsions or suspensions. pyrogens (2.6.8).
They are prepared by dissolving, emulsi:fying or suspending Preparatians far veterinary use When the volume to be
the active substance(s) and any added excipients in water, in injected in a single dos e is 15 mL or more and is equivalent
a suitable non-aqueous liquid, that may be non-sterile where to adose of 0.2 mL or more per kilogram of body mass, the
justified, or in a mixture of these vehicles. preparation complies with a test for bacterial endotoxins
Solutions for injection, examined under suitable conditions of (2.6.14) or with the test for pyrogens (2.6.8).
visibility, are clear and practically free from particles. Any preparatian Where the label states that the preparation
Emulsions for injection do not show any evidence of phase is free from bacterial endotoxins or apyrogenic, respectively,
separation. Suspensions for injection may show a sediment the preparation complies with a test for bacterial endotoxins
which is readily dispersed on shaking to give a suspension (2.6.14) or with the test for pyrogens (2.6.8), respectively.
which remains sufficiently stable to enable the correct dose to
be withdrawn.
134 General Monographs
INFUSIONS TESTS
Uniformity of dosage units
DEFINITION
Powders for injections or infusions comply with the test for
Infusions are sterile, aqueous solutions or emulsions with unifonnity of dosage units (2.9.40) or, where justified and
water as the continuous phase. They are usually made authorised, with the tests for uniformity of content andlor
isotonic with respect to blood. They are principally intended unifonnity of mass shown below. Herbal drugs and herbal
for administration in large volume. Infusions do not contain drug preparations present in the dosage form are not subject
any added antimicrobial preservative. to the provisions of this paragraph.
Solutions for infusion, examined under suitable conditions of
Uniformity of content (2.9.6)
visibility are c1ear and practically free from partic1es.
Unless otherwise prescribed or justified and authorised,
Emulsions for infusion do not show any evidence of phase powders for injections or infusions with a content of active
separation. substance less than 2 mg or less than 2 per cent of the total
PRODUCTION mass, or with a unit mass equal to or less than 40 mg comply
In the manufacture of infusions containing dispersed with test A for uniformity of content of single-dose
particles, mea sures are taken to ensure a suitable and preparations. If the preparation contains more than one
controlled partic1e size with regard to the intended use. active substance, the requirement applies only to those
The volume of the infusion in the container is sufficient to substances that correspond to the above conditions.
permit the withdrawal and administration of the nominal Uniformity ofmass (2.9.5)
dos e using a nonnal technique (2.9.17). Powders for injections or infusions comply with the test for
unifonnity of mass of single-dose preparations. If the test for
TESTS
unifonnity of content is prescribed for all the active
Bacterial endotoxins - pyrogens
substances, the test for unifonnity of mass is no! required.
They comply with a test for bacterial endotoxins (2.6.14) or,
where justified and authorised, with the test for pyrogens Bacterial endotoxins - pyrogens
(2.6.8). For the latter test inject 10 mL per kilogram of body They comply with the requirements prescribed for injections
mas s into each rabbit, unless otherwise justified and or for infusions, after dissolution or suspension in a suitable
authorised. volume of liquido
LABELLING
CONCENTRATES FOR INJECTIONS OR The label states the instructions for the preparation of
INFUSIONS injections and infusions.
DEFINITION
Concentrates for injections or infusions are sterile solutions GELS FOR INJECTIONS
intended for injection or infusion after dilution. They are DEFINITION
diluted to a prescribed volume with a prescribed liquid Gels for injections are sterile gels with a viscosity suitable to
before administration. After dilution, they comply with the guarantee a modified release of the active substance(s) at the
requirements for injections or for infusions. sÍte of injection.
TESTS
Bacterial endotoxins - pyrogens IMPLANTS
They comply with the requirements prescribed for injections
or for infusions, after dilution to a suitable volume. DEFINITION
Implants are sterile, solid preparations of a size and shape
POWDERS FOR INJECTIONS OR suitable for parenteral implantation and release of the active
substance(s) over an extended period of time. Each dose is
INFUSIONS provided in a sterile container.
DEFINITION ___________________________________________ PhE~
TESTS
Loss on drying (2.2.32)
Unless otherwise justified and authorised, for premixes
occurring in granulated or powdered form, maximum
15.0 per cent, determined on 3.000 g by drying in an oven at
105 oC for 2 h.
LABELLING
The labe1 states:
-- the category of animal for which the premix is intended;
-- the instructions for the preparation of the medicated
feeding stuffs from the premix and the basic feed;
-- where applicable, the time that must elapse between the
cessation of feeding of the medicated feeding stuff and
collection of the material intended for human
consumption.
__________________________________________ ~E~
TABLETS ***
** **
(Ph Bur monograph 0478) *****
Unless otherwise justijied and authorised, T ablets comply with the
appropriate requirements oi the Buropean Pharmacopoeia. These
requirements are reproduced in the British Pharmacopoeia.
STORAGE
FORMULATED PREPARATIONS: Acepromazine Injection should be protected from Iight.
SPECIFIC MONOGRAPHS LABELLING
The strength is stated as the equivaIent amount of
acepromazine in a suitabIe dose-volume.
Acepromazine Injection
Action and use
Dopamine receptor antagonist; neuroleptic. Acepromazine Tablets
DEFINITION Action and use
Acepromazine Injection is a steriIe solution of Acepromazine Dopamine receptor antagonist; neuroleptic.
MaIeate in Water for Injections. The pH of the solution is
DEFINITION
adjusted to about 5 by the addition of Sodium Hydroxide.
Acepromazine TabIets contain Acepromazine MaIeate.
The injection complieswith the requirements stated under
The tablets comply with the requirements stated under T ablets and
Parenteral Preparations and with the following requirements.
with the following requirements.
Content of acepromazine, C19H22N20S
Content of acepromazine, C19H22N20S
92.5 to 107.5% ofthe stated amount.
92.5 to 107.5% ofthe stated amount.
IDENTIFICATION
IDENTIFICATION
A. To a volume containing the equivaIent of 20 mg of
A. To a quantity of the powdered tabIets containing the
acepromazine add 2 mL of water and 3 mL of 2M sodium
equivaIent of 20 mg of acepromazine add 2 mL of water
hydroxide, extract with two 5 mL quantities of cyclohexane
and 3 mL of 2M sodium hydroxide. Extract with two 5 mL
and evaporate to dryness under reduced pressure. The
quantities of cyclohexane and evaporate to dryness under
infrared absorption spectrum of the residue, Appendix II A, is
reduced pressure. The infrared absorption spectrum of the
concordant with the reference spectrum of acepromazine
residue, Appendix II A, is concordant with the reference
(RSV 01).
spectrum of acepromazine (RSV 01).
E. Complies with the test for identijication of phenothiazines,
E. Complies with the test for identijication of phenothiazines,
Appendix III A, appIying to the pIate 1 ¡.tL of each of the
Appendix III A, appIying to the pIate 1 ¡.tL of each of the
following solutions. For solution (1) extract a volume
following solutions. For solution (1) extract a quantity of the
containing the equivaIent of 20 mg of acepromazine with two
powdered tabIets containing the equivaIent of 20 mg of
5 mL quantities of chloroform and use the combined extracts.
acepromazine with two 5 mL quantities of chloroform. .
Solution (2) is a solution of acepromazine maleate BPCRS in
Solution (2) is a solution of acepromazine maleate BPCRS m
chloroform containing the equivaIent of 0.2% w/v of
chloroform containing the equivaIent of 0.2% w/v of
acepromazine.
acepromazine.
C. To 5 mg of the residue obtained in test A add 2 mL of
C. To a quantity of the powdered tabIets containing th~ .
sulfuric acid. A yellow colour is produced which becomes
equivaIent of 5 mg of acepromazine add 2 mL of sulfunc actd.
deep orange on warming for 2 minutes.
A yellow colour is produced which becomes deep orange on
D. To a volume of the injection containing the equivaIent of warming for 2 minutes.
25 mg of acepromazine add 2 mL of 5M sodium hydroxide
D. Dissolve as completely as possible a quantity of the
and shake with three 3 mL quantities of ether. Add 2 mL of
powdered tabIets containing the equivaIent of 25 mg of
bromine solution to the aqueous solution, warm in a water
acepromazine in a mixture of 3 mL of water and 2 m~ .of
bath for 10 minutes, heat to boiling and coo!. Add 0.25 mL
5M sodium hydroxide and shake with three 3 mL quantltles of
to a solution of 10 mg of resorcinol in 3 mL of suIfuric acid
ether. Add 2 mL of bromine solution to the aqueous solution,
and heat for 15 minutes in a water bath. A bIuish bIack
warm in a water bath for 10 minutes, heat to boiling and
colour deveIops.
coo!. Add 0.25 mL to a solution of 10 mg of resorcinol in
TESTS 3 mL of sulfuric acid and heat for 15 minutes on a water
Acidity bath. A bIuish bIack colour deveIops.
pH, 4.5 to 5.5, Appendix V L.
TESTS
ASSAY Related substances
To a volume containing the equivaIent of 40 mg of Comply with the test for related substances in phenothiazines,
acepromazine add 5 mL of 1M sodium hydroxide and extract Appendix III A, but using a mixture of 8 volumes of
with 50 mL quantities of chloroform until the chIoroform diethylamine, 17 volumes of butan-2-one and 75 volumes of
extract is colourless. Wash the extracts with the same 10 mL n-hexane as the mobile phase and using the following
of water and fiIter through a pIug of absorbent cotton solutions. For solution (1) shake a quantity of the powdered
previously moistened with chloroform. Evaporate the tabIets containing the equivaIent of 50 mg of acepromazine
combined extracts to dryness and dissolve the residue in with 10 mL of chloroform, fiIter, evaporate to dryness and
15 mL of acetic anhydride. Carry out Method I for non- dissolve the residue in 5 mL of methanol containing 0.5% v/v
aqueous titration, Appendix VIII A, using 0.02M perchloric acid of 13.5M ammonia. For solution (2) dilute 1 volume of
VS as titrant and crystal violet solution as indicator. Each mL solution (1) to 100 volumes with methanol containing
of 0.02M perchloric acid VS is equivaIent to 6.529 mg of 0.5% v/v of 13.5M ammonia.
C19H22N20S.
138 Albendazole Preparations
IDENTIFICATION
A. To a volume of oral suspension containing 25 mg of SYSTEM SUITABILlTY
Albendazole add 50 mL of O.lM sodium hydroxide and shake The test is not val id unless, in the chromatogram obtained
with the aid of ultrasound for 10 minutes. Dilute to 100 mL with solution (3), the resolution factor between the two
with the same solvent, filter through a 0.45-¡.un filter and principal peaks is at least 7.0.
dilute 1 volume ofthis solution to 10 volumes with the same
solvento The light absorption, Appendix II B, in the range LlMITS
240 to 340 nm of the resulting solution exhibits a maximum In the chromatogram obtained with solution (1):
at 308 nm, a minimum at 281 nm and a shoulder at the area of any secondary peak is not greater than the area of
269 nm. the principal peak in the chromatogram obtained with
B. To a volume of oral suspension containing 25 mg of solution (2) (1 %);
Albendazole add 50 mL of O.lM hydrochloric acid and shake the sum of the areas of any secondary peaks is not greater than
with the aid of ultrasound for 10 minutes. Dilute to 100 mL twice the area of the principal peak in the chromatogram
with the same solvent, filter through a 0.45-flm filter and obtained with solution (2) (2%).
dilute 1 volume of this solution to 10 volumes with the same Disregard any peak with an area less than 0.05 times the area
solvento The light absorption, Appendix II B, in the range of the principal peak in the chromatogram obtained with
240 to 340 nm of the resulting solution exhibits a maximum solution (2) (0.05%).
at 292 nm, a minimum at 273 nm and a shoulder at
261 nm. ASSAY
Carry out the method for liquid chromatography,
C. In the Assay, the chromatogram obtained with solution
Appendix III D, using the following solutions.
(1) shows a peak with the same retention time as the
principal peak in the chromatogram obtained with (1) Add 70 mL of 1% v/v solution of methanolic sulfuric acid
solution (2). to a quantity of the oral suspension containing 0.10 g of
Albendazole, stir for 15 minutes, mix with the aid of
TESTS ultrasound for 10 minutes and add sufficient 1% v/v solution
Acidity of methanolic sulfuric acid to produce 100 mL. Allow to stand
pH, 4.5 to 5.5, Appendix V L. and dilutb 5 volumes of the c1ear supematant to 25 volumes
Related substances with 1% v/v solution of methanolic sulfuric acid.
Carry out the method for liquid chromatography, (2) 0.020% w/v of albendazole BPCRS in 1% v/v solution of
Appendix III D, using the following solutions. metha~lic sulfuric acid.
(1) Dilute a quantity of the oral suspension with 1% v/v CHROMATOGRAPHIC CONDITIONS
solution of methanolic sulfuric acid to give a solution
The chromatographic conditions described under Related
containing 1.0% w/v of Albendazole and dilute 1 volume of
substances may be used.
the resulting solution to 2 volumes with the mobile phase.
Albendazole Preparations 139
DETERMINATION OF CONTENT (2) Dilute 1 volume of solution (1) to 100 volumes with the
Determine the weight per mL of the oral suspension, mobile phase.
Appendix V G, and calculate the content of C12HlSN302S, (3) Dissolve 25.0 mg of albendazole BPCRS and 25.0 mg of
weight in volume, using the dec1ared content of oxibendazole BPCRS in 5 mL of 1% v/v solution of methanolic
C12HlSN302S in albendazole BPCRS. sulfuric acid and dilute to 50 mL with the mobile phase.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with end-capped octadecylsilyl silica gel for chromatography
Albendazole Oral Suspension with (5 Ilm) (Waters Symmetry is suitable).
Minerals (b) U se gradient elution and the mobile phase described
below.
Action and use
(c) Use a flow rate of 0.7 mL per minute.
Benzimidazole antihelminthic.
(d) Use ambient column temperature.
DEFINITION (e) Use a detection wavelength of 292 nm.
Albendazole OraLSuspension with Minerals is a suspension (f) Inject 20 IlL of each solution.
of Albendazole in a suitable vehic1e containing cobalt and
MOBILE PHASE
selenium.
Mobile phase A 0.015M ammonium dihydrogen orthophosphate.
The oral suspension complies with the requirements stated under
Oral Liquids and with the following requirements. Mobile phase B methanol.
Content of albendazole, C12HlSN30ZS
95.0 to 105.0% of the stated amount. Time Mobile phase A% Mobile phase B% Comment
Content of Co (Minutes)
IDENTIFICATION (a) Use a stainless steel column (25 cm x 4.6 mm) packed
Extract a quantity containing the equivalent of 0.25 g of with octadecylsilyl silica gel for chromatography (5 ¡lm)
amoxicillin with three 20-rnL quantities of petroleum spirit (Hypersil 5 ODS is suitable).
(boiling range~ 120° to 160°) and discard the extracts. Wash (b) Use isocratic elution and the mobile phase described
the residue with ether and dry in a current of airo The residue below.
complies with the following tests. (c) Use a flow rate of 1 mL per minute.
A. The infrared absorption spectrum, Appendix II A, is (d) Use an ambient column temperature.
concordant with the reference spectrum of amoxicillin (e) Use a detection wavelength of 254 nm.
trihydrate (RSV 05).
(f) Inject 50 ¡lL of each solution.
B. Carry out the method for thin-layer chromatography,
MOBILE PHASE
Appendix III A, using the following solutions.
(1) Dissolve a quantity of the residue in sufficient sodium 8 volumes of mobile phase B and 92 volumes of mobile
hydrogen carbonate solution to produce a solution containing phase A.
the equivalent of 0.25% w/v of amoxicillin. Mobile phase A 1 volume of acetonitrzle and 99 volumes of a
(2) 0.25% w/v of amoxicillin trihydrate BPCRS in sodium 25% v/v solution of 0.2M potassium dihydrogen orthophosphate
hydrogen carbonate solution. adjusted to pH 5.0 with 2M sodium hydroxide.
(3) 0.25% w/v of each of amoxicillin trihydrate BPCRS and Mobile phase B 20 volumes of acetonitrile and 80 volumes of
ampicillin trihydrate BPCRS in sodium hydrogen carbonate a 25% v/v solution of 0.2M potassium dihydrogen orthophosphate
solution. adjusted to pH 5.0 with 2M sodium hydroxide.
(a) Use a TLC silica gel silanised plate (Merck silanised silica The Assay is not valid unless, in the chromatogram obtained
gel 60 F ZS4s (RP-18) plates are suitable). with solution (3), the resolution factor between the peaks due
to amoxicillin and cefadroxil is at least 2.0. If necessary,
(b) Use the mobile phase as described below.
adjust the composition of the mobile phase to achieve the
(c) Apply 1 ¡lL of each solution. required resolution.
(d) Develop the plate to 15 cm. DETERMINATION OF CONTENT
(e) After removal of the plate allow it to dry in air, expose to Calculate the content of C16H19N30SS in the injection from
iodine vapour until spots appear and examine in daylight. the chromatograms obtained and from the declared content
MOBILE PHASE of C16H19N30SS in amoxicillin trihydrate BPCRS.
10 volumes of acetone and 90 volumes of a 15.4% w/v LABELLING
solution of ammonium acetate adjusted to pH 5.0 with glacial The label states (1) the quantity of active ingredient in terms
acetic acid. of the equivalent amount of amoxicillin in a suitable dose-
SYSTEM SUITABILITY volume; (2) where appropriate, that the preparation is
The test is not valid unless the chromatogram obtained with Amoxicillin Oily Injection (Long Acting).
solution (3) shows two clearly separated spots.
142 Amoxicillin Preparations
ASSAY
Amoxicillin Veterinary Oral Powder Carry out the method for liquid chromatography,
Action and use Appendix III D, using the following solutions.
Penicillin antibacterial. (1) Add 80 mL of mobile phase A to a quantity of the
veterinary oral powder containing the equivalent of 60 mg of
DEFINITION amoxicillin and shake for 15 minutes. Mix with the aid of
Amoxicillin Veterinary Oral Powder is a mixture of ultrasound for 1 minute, add sufficient mobile phase A to
Amoxicillin Trihydrate, Lactose or other suitable diluent produce 100 mL, mix and filter (Whatman GF/C filter paper
and a stabilising agent. is suitable).
The veterinary oral powder complies with the requirements stated (2) 0.070% w/v of amoxicillin trihydrate BPCRS in mobile
under Veterinary Oral Powders and with the following phase A.
requirements. (3) 0.0004% w/v of cefadroxil BPCRS and 0.003% w/v of
Content of amoxicillin, C16H19N30SS amoxicillin trihydrate BPCRS in mobile phase A.
90.0 to 110.0% of the stated amount. CHROMATOGRAPHIC CONDITIONS
IDENTIFICATION (a) Use a stainless steel column (25 cm x 4.6 mm) packed
A. Carry out the method for thin-layer chromatography, with octadecylsilyl silica gel for chromatography (5 )lm)
Appendix III A, using the following solutions. (Hypersil 5 ODS is suitable).
(1) Dissolve a quantity of the veterinary oral powder (b) Use isocratic elution and the mobile phase described
containing the equivalent of 0.25 g of amoxicillin in sufficient below.
sodium hydrogen carbonate solution to produce 100 mL. (c) Use a flow rate of 1 mL per minute.
(2) 0.25% w/v of amoxicillin trihydrate BPCRS in sodium (d) Use an ambient column temperature.
hydrogen carbonate solution. (e) Use a detection wavelength of 254 nm.
(3) 0.25% w/v of each of amoxicillin trihydrate BPCRS and
(f) Inject 50 )lL of each solution.
ampicillin trihydrate BPCRS in sodium hydrogen carbonate
solution. MOBILE PHASE
CHROMATOGRAPHIC CONDITIONS
8 volumes of mobile phase B and 92 volumes of mobile
phase A.
(a) Use a TLC silica gel silanised plate (Merck silanised silica
gel 60 F ZS4s (RP-18) plates are suitable). Mobile phase A 1 volume of acetonitrile and 99 volumes of a
25% v/v solution of 0.2M potassium dihydrogen orthophosphate
(b) Use the mobile phase as described below.
adjusted to pH 5.0 with 2M sodium hydroxide.
(c) Apply 1 )lL of each solution.
Mobile phase B 20 volumes of acetonitrile and 80 volumes of
(d) Develop the plate to 15 cm. a 25% v/v solution of 0.2M potassium dihydrogen orthophosphate
(e) After removal of the plate allow it to dry in air, expose it adjusted to pH 5.0 with 2M sodium hydroxide.
to iodine vapour until spots appear and examine in daylight. SYSTEM SUITABILITY
MOBILE PHASE The Assay is not valid unless, in the chromatogram obtained
10 volumes of acetone and 90 volumes of a 15.4% w/v with solution (3), the resolutionfactor betweenthe peaks due
solution of ammonium acetate adjusted to pH 5.0 with glacial to amoxicillin and cefadroxil is at least 2.0. If necessary,
acetic acid. adjust the composition of the mobile phase to achieve the
SYSTEM SUITABILITY required resolution.
The test is not valid unless the chromatogram obtained with DETERMINATION OF CONTENT
solution (3) shows two clearly separated spots. C:¡llculate the content of C16H19N30SS in the veterinary oral
CONFIRMATION powder from the chromatograms obtained and from the
declared content of C16H19N30SS in amoxicillin
The principal spot in the chromatogram obtained with
trihydrate BPCRS.
solution (1) is similar in position, colour and size to that in
the chromatogram obtained with solution (2). LABELLING
B. Shake a quantity of the veterinary oral powder containing The quantity of active ingredient is stated in terms of the
the equivalent of 0.5 g of amoxicillin with 5 mL of water for equivalent amount of amoxicillin.
5 minutes, filter, wash the residue first with absolute ethanol
and then with ether and dry at a pressure not exceeding
0.7 kPa for 1 hour. Suspend 10 mg ofthe residue in 1 mL of
water and add 2 mL of a mixture of 2 mL of cupri-tartaric
solution Rl and 6 mL of water. A magenta colour is produced
Amoxicillin Tablets
immediatelY· Action 3¡nd use
C. Dissolve 0.1 mL of aniline in a mixture of 1 mL of Penicillinl, antibacterial.
hydrochloric acid and 3 mL of water. Cool the solution in ice
and add 1 mL of a freshly prepared 20% w/v solution of DEFINITION
sodium nitrite. Add the resulting mixture drop wise to a cold Amoxiéillin Tablets contain Amoxicillin Trihydrate.
solution of 0.1 g of the residue obtained in test B in 2 mL of The tablets comply with the requirements stated under T ablets
5M sodium hydroxide. The solution becomes deep cherry-red and with the following requirements.
and a copious dark brown precipitate is produced.
Content of amoxicillin, C16H19N30SS
90.0 to 110.0% of the stated amount.
Ampicillin Preparations 143
quantities of petroleum spirit (boiling rangeJ 120° to 160°). (bozting rangeJ 120° to 160°). Discard the extracts, wash the
Discard the extracts, wash the residue with 10 mL of ether residue with 10 mL of ether and dry the residue at 55°.
and dry in a current of airo Dissolve the residue in 50 mL of The residue produces an intense, persistent yellowish orange
phosphate buffer pH 7. O, shake well, filter and use the filtra te. colour when introduced into a non-Iuminous Bunsen burner
(2) 0.1 % w/v of anhydrous ampicülin BPCRS in phosphate flame on a platinum wire moistened with hydrochloric acid.
buffer pH 7. O. TESTS
CHROMATOGRAPHIC CONDITIONS Water
(a) Use a TLC silz"ca gel plate (Merck silica gel 60 plates Not more than 1.0% w/w, Appendix IX C. Use 1.5 g and a
are suitable). Impregnate the plate by spraying it with a mixture of 70 volumes of chloroform and 30 volumes of
0.1 % w/v solution of disodium edetate in a 5% w/v solution of anhydrous methanol as the solvento
sodium dihydrogen onhophosphate, allow the plate to dry in air ASSAY
and heat at 105° for 1 hour. Express, as far as possible, weigh and mix the contents of
(b) U se the mobile phase as described below. 10 containers. Extract a quantity of the mixed contents
(c) Apply 1 IlL of each solution. containing the equivalent of 50 mg of ampicillin with three
(d) Develop the plate to 15 cm. 15-mL quantities of petroleum spirit (boilz"ng rangeJ 120° to
160°) previously saturated with ampicillin sodium and
(e) After removal of the plate allow it to dry, heat at 105° for cloxacillin sodium. Discard the extract, wash the residue
lOto 15 minutes and spray with a mixture of 100 volumes of with ether previously saturated with ampicillin sodium and
starch mucilage, 6 volumes of glacial acetic acid and 2 volumes cloxacillin sodium, dry in a current of air, dissolve in water
of a 1% w/v solution of iodine in a 4% w/v solution of and dilute to 100 mL with water. Centrifuge and use the
potassium iodide. clear supernatant liquid (solution A).
MOBILE PHASE For ampicillin
5 volumes of butan-1-01, 10 volumes of a 0.1 % w/v solution Dilute 2 mL of solution A to 50 mL with buffered copper
of disodium edetate in a 5% w/v solution of sodium dihydrogen sulfate solution pH 5.2, transfer 10 mL to a stoppered test
onhophosphate, 30 volumes of glacial acetic acid and tube and heat in a water bath at 75° for 30 minutes. Rapidly
50 volumes of butyl acetate. cool to room temperature, dilute to 20 mL with buffered
CONFIRMATION copper sulfate solution pH 5.2 and measure the absorbance of
The principal spot in the chromatogram obtained with the resulting solution at the maximum at 320 nm,
solution (1) corresponds to that in the chromatogram Appendix n B, using in the reference cell a solution prepared
obtained with solution (2). by diluting 2 mL of solution A to 100 mL with buffered
copper sulfate solution pH 5.2. Calculate the content of
B. Carry out the method for thin-Iayer chromatography,
C16H19N304S in a container of average content from the
Appendix nI A, using the following solutions. absorbance obtained by carrying out the operation at the same
(1) Extract a quantity of the infusion containing the time using 2 mL of a solution prepared by dissolving 50 mg
equivalent of 0.130 g of cloxacillin with three 15-mL of anhydrous ampicillin BPCRS in 100 mL of water, diluting
quantities of petroleum spirit (boiling rangeJ 1200 to 160°). to 50 mL with buffered copper sulfate solution pH 5.2 and
Discard the extracts, wash the residue with 10 mL of ether beginning at the words 'transfer 10 mL. ..' and from the
and dry in a current of airo Dissolve the residue in 50 mL of declared content of C16H19N304S in anhydrous
phosphate buffer pH 7. O, shake well, filter and use the filtra te. ampicillin BPCRS.
(2) 0.28% w/v of cloxacillin sodium BPCRS in phosphate For cloxacillin
buffer pH 7. O. Dilute 2 mL of solution A to 100 mL with 1M hydrochloric
CHROMATOGRAPHIC CONDITIONS acid. Measure the absorbance of the resulting solution at the
(a) Use a TLC silica gel F254 silanised plate (Merck plates are maximum at 350 nm, Appendix n B, at 20° after exactly
suitable). 12 minutes using 1M hydrochloric acid in the reference cell.
Calculate the content of C19HlSClN30SS in a container of
(b) Use the mobile phase as described below.
average cbntent from the absorbance obtained by carrying out
(c) Apply 1 IlL of each solution. the operation at the same time using 2 mL of a solution
(d) Develop the plate to 15 cm. prepared by dissolving 0.14 g of cloxacillin sodium BPCRS in
(e) After removal of the plate allow it to dry, heat at 105° for 100 mL of water and from the declared content of
lOto 15 minutes and spray with a mixture of 100 volumes of C19H17CIN3NaOsS in cloxacillin sodium BPCRS. Each mg
starch mucilage, 6 volumes of glacial acetic acid and 2 volumes of C19H17CIN3NaOsS is equivalent to 0.9520 mg of
of a 1% w/v solution of iodine in a 4% w/v solution of C19HlSClN30SS.
potassium iodide. LABELLING
MOBILE PHASE The label states the quantity of Ampicillin Sodium in terms
1 volume üf formic acid, 30 volumes of acetone and of the equivalent amount of ampicillin and the quantity of
70 volumes of 0.05M potassium hydrogen phthalate that has Cloxacillin Sodium in terms of the equivalent amount of
been adjusted first to pH 6.0 with 5M sodium hydroxide and cloxacillin.
then to pH 9.0 with O.IM sodium hydroxide.
CONFIRMATION
The principal spot in the chromatogram obtained with
solution (1) corresponds to that in the chromatogram
obtained with solution (2).
C. Extract a quantity containing the equivalent of 50 mg of
ampicillin with three 15-mL quantities of petroleum spirit
Apramycin Preparations 145
CHROMATOGRAPHIC CONDITIONS Degas both mobile phases using helium. Equilibrate the
(a) Use a silica gel F 254 precoated plate (Merck silica column using a mixture containing 75% of mobile phase A
gel 60 F 254 plates are suitable). and 25% of mobile phase B. Mter each injection elute for
(b) Use the mobile phase as described below. 3 minutes using the same mixture and then carry out a linear
gradient elution for 6 minutes to 100% of mobile phase B.
(c) Apply 5 IlL of each solution.
Elute for a further 21 minutes using 100% of mobile
(d) Develop the plate to 10 cm. phase B, then step-wise re-equilibrate to a mixture of 75% of
(e) After removal of the plate, allow it to dry in air fOL mobile phase A and 25% of mobile phase B and elute for at
10 minutes, heat at 100° for 10 minutes, spray with sodium least 10 minutes.
hypochlorite solution whilst hot and cool for 5 minutes. Spray SYSTEM SUIT ABILITY
with absolute ethanolJ heat at 100° for 5 to 1O minute'~, or
The test is not valid unless, in the chromatogram obtained
until an area of the plate below the line of application gives
with solution (2), the resolution factor between compound A
at most a faint blue colour with one drop of a 1% w/v
and 3-hydroxyapramycin, identified as indicated in the
solution of potassium iodide containing 1% w/v soluble starchJ
reference chromatogram supplied with apramycin BPCRS, is
and spray with the potassium iodide-starch solution.
at least 0.8.
MOBILE PHASE
LIMITS
20 volumes of chloroformJ 40 volumes of 13.5M ammonia and
In the chromatogram obtained with solution (1):
60 volumes of methanolJ equilibrated for 1 hour before use.
the areas of any peaks corresponding to 3-hydroxyapramycin,
SYSTEM SUITABILITY
lividamine/2-deoxystreptamine (combined), compound A and
The test is not valid unless the chromatogram obtained with compound B (identified as indicated in the reference
solution (3) shows two clear1y separated principal spots. chromatogram supplied with apramycin BPCRS) are not
CONFIRMATION greater than 2.8, 2.0, 0.8 and 0.8 times respectively the area
The principal spot in the chromatogram obtained with of the principal peak in the chromatogram obtained with
solution (1) corresponds to that in the chromatogram solution (3) (7%, 5%, 2% and 2% respectively);
obtained with solution (2). the area of any other secondary peak is not greater than
B. In the test for Related substances, the retention time of 0.8 times the area of the principal peak in the chromatogram
the principal peak in the chromatogram obtained with obtained with solution (3) (2%);
solution (1) corresponds to that of the principal peak in the the sum of the areas of all the secondary peaks is not greater
chromatogram obtained with solution (2). than 6 times the area of the principal peak in the
C. Yields reaction A characteristic of sulfates, Appendix VI. chromatogram obtained with solution (3) (15%).
Disregard any peak with an area less than 0.04 times the area
TESTS
of the principal peak in the chromatogram obtained with
Related substances
solution (3) (0.1 %).
Carry out the method for liquid chromatography,
Appendix III D, using the following solutions. ASSAY
(1) Dissolve a quantity of the oral powder containing the Carry out the microbiological assay of antibiotics,
equivalent of 0.3 g of apramycin in water and dilute to Appendix XN A, Method B. The precision of the assay is
100 mL. such that the fiduciallimits of error are not les s than 95%
and not more than 105 % of the estimated potency.
(2) 0.30% w/v of apramycin BPCRS in water.
Calculate the content of apramycin in the veterinary oral
(3) Dilute 1 volume of solution (1) to 20 volumes with water.
powder taking each 1000 Units found to be equivalent to
CHROMATOGRAPHIC CONDITIONS 1 mg of apramycin. The upper fiducial limit of error is not
(a) Use a column (25 cm x 4 mm) packed with fast cation- les s than 97.0% and the lower fiduciallimit of error is not
exchange polymeric beads (13 11m) with sulfonic acid more than 110.0% ofthe stated contento
functional groups (Dionex Fast Cation-1 R is suitable) and a LABELLING
stainless steel post-column reaction coil (380 cm x 0.4 mm) The quantity of active ingredient is stated in terms of the
with internal baffies. U se in the reaction coil ninhydrin reagent
equivalent amount of apramycin.
1 at a flow rate approximately the same as that for the mobile
phase.
(b) U se gradient elution and the mobile phase described
below.
(c) Use a flow rate of 0.8 mL per minute. Apramycin Premix
(d) Use a column temperature of 130°. Maintain the post- Action and use
column reaction coil at the same temperature. Aminoglycoside antibacterial.
(e) Use a detection wavelength of 568 nm.
(f) Inject 20 IlL of each solution. DEFINITION
Apramycin Premix contains Apramycin Sulfate.
MOBILE PHASE
The premix complies with the requirements stated under Premixes
Mobz1e phase A A solution containing 1.961 % w/v of sodium
and with the following requirements.
citrate, 0.08% v/v of liquefied phenol and 0.5% v/v of
thiodiglycol, adjusted to pH 4.25 using hydrochloric acid. CHARACTERISTICS
Mobile phase B A solution containing 4.09% w/v of sodium Light brown granules.
chloride and 3.922% w/v of sodium citrate with 0.08% v/v of
liquefied phenol, adjusted to pH 7.4 with hydrochloric acid.
148 Aprarnycin Preparations
STORAGE
Calcium Borogluconate Injection should be protected from
Cefalonium Intramammary Infusion
light. It may be supplied in containers of glass type 111, (Dry COW)
Appendix XIX B.
Action and use
LABELLING Cephalosporin antibacterial.
The strength is stated as the amount of ca1cium in a suitable
dose-volume. The label also states the proportion of boric DEFINITION
acid presento Cefalonium Intramammary Infusion (Dry Cow) is a sterile
suspension of Cefalonium in a suitable non-aqueous vehic1e,
containing suitable suspending agents.
The intramammary infusion complies with the requirements stated
under Intramammary Infusions and with the following
Calcium Copperedetate Injection requirements.
Action and use Content of anhydrous cefalonium, C2oH18N40SS2
Used in the treatment of copper deficiency. 90.0 to 112.0% of the stated amount.
DEFINITION IDENTIFICATION
Ca1cium Copperedetate Injection is a sterile suspension of A. In the Assay, the retention time of the principal peak in
Ca1cium Copperedetate, with suitable stabilising and the chromatogram obtained with solution (1) corresponds to
dispersing agents, in an oil-in-water emulsiono that of the principal peak in the chromatogram obtained with
solution (2).
The injection complies with the requirements stated under
Parenteral Preparations and with the following requirements. B. To a quantity of the intramammary infusion containing
the equivalent of 20 mg of anhydrous cefalonium· add a few
Content of copper, Cu drops of sulfuric acid (80% v/v) containing 1% v/v of nitric
92.0 to 108.0% of the stated amount. acid and mix. A pale green colour is produced which
CHARACTERISTICS irnmediately changes to dark green.
Macroscopical A blue, opaque, viscous suspension. TESTS
Microscopical When diluted with glycerol and examined Related substances
microscopically, cubic crystals 10 to 30 ¡..tm in diameterare Carry out the method for liquid chromatography,
visible, but large plate-like crystals are absent. Appendix III D, using the following solutions prepared
IDENTIFICATION irnmediately before use and stored in a refrigerator between
Ignite 1 g and dissolve the residue by warming in 10 mL of a injections.
mixture of equal volumes of hydrochloric acid and water; filter (1) Disperse a quantity of the intramammary infusion
if necessary. The solution complies with the following tests. containing the equivalent of 0.10 g of anhydrous cefalonium
A. Neutralise 2 mL ofthe solution with 5M ammonia, and in 50 mL of petroleum spirit (boiling range, 60° to 80°), add
add 1 mL of 6M acetic acid and 2 mL of potassium iodide 100 mL of O.lM hydrochloric acid and shake vigorously by
solution. A white precipitate is produced and iodine is hand for 5 minutes and then mechanically for 30 minutes,
liberated, colouring the supernatant liquid brown. filter and use the lower layer.
B. To 5 mL ofthe solution add 25 mL ofa 10% v/v solution (2) Dilute 2 volumes of solution (1) to 100 volumes with
of mercaptoacetic acid and filter. Make the filtrate alkaline with 0.1 M hydrochloric acid.
5M ammonia and add 5 mL of a 2.5% w/v solution of (3) 0.0020% w/v of isonicotinamide in O.lM hydrochloric acid.
ammonium oxalate. A white precipitate is produced which is (4) 0.0050% w/v of each of cefalonium BPCRS and
soluble in hydrochloric acid but only sparingly soluble in isonicotinamide in 0.1 M hydrochloric acid.
6M acetic acid. CHROMATOGRAPHIC CONDITIONS
ASSAY (a) Use a stainless steel column (10 cm x 4.6 mm) packed
Evaporate a quantity containing the equivalent of 0.13 g of with partic1es of silica the surface of which has been modified
copper to dryness, ignite at 600° to 700°, cool and heat the with chemically-bonded hexylsilyl groups
residue with 5 mL of a mixture of equal volumes of (Spherisorb S5 C6 is suitable).
hydrochloric acid and water on a water bath for 15 minutes. (b) Use isocratic elution and the mobile phase described
Add 5 mL of water, filter and wash the residue with about below.
20 mL of water. Combine the filtrate and the washings, add
(c) Use a flow rate of 2 mL per minute.
10 mL of bromine water, boil to remove the bromine, cool
and add dilute sodium carbonate solution until a faint (d) Use an ambient column temperature.
permanent precipitate is produced. Add 3 g of potassium (e) Use a detection wavelength of 262 nm.
iodide and 5 mL of 6M acetic acid and titrate the liberated (±) Inject 10 ¡..tL of each solution.
iodine with O.lM sodium thiosulfate VS, using starch mucilage (g) For solution (1), allow the chromatography to proceed
as indicator, until only a faint blue colour remains; add 2 g for at least 3.5 times the retention time of the principal peak.
of potassium thiocyanate and continue the titration until the /
ASSAY (a) Use sz1ica gel Has the coating. Adjust the pH of a
Express, as far as possible, weigh and mix the contents of 10% w/v solution of disodium edetate to 8.0 with 10M sodium
10 containers. Carry out the method for liquid hydroxide and spray the solution evenly onto the plate (about
chromatography, Appendix nI D, using the following solutions 10 mL for a plate 100 mm x 200 mm). Allow the plate to
prepared immediately before use and stored in a refrigerator dry in a horizontal position for at least 1 hour. At the time of
between injections. use, dry the plate in an oven at 110 0 for 1 hour.
(1) Disperse a quantity of the mixed contents of the (b) Use the mobile phase as described below.
10 containers containing the equivalent of 75 mg of (c) Apply 1 IlL of each solution.
anhydrous cefalonium in 50 mL of petroleum spirit (boiling (d) Develop the plate to 15 cm.
0 0
rangeJ 60 to 80 add 100 mL of O.lM hydrochloric acid and
),
(e) After removal of the plate, allow it to dry in a current of
shake vigorously by hand for 5 minutes and then air and examine under ultraviolet light (365 nm).
mechanically for 30 minutes. Filter the lower layer and\ dilute
MOBILE PHASE
10 mL ofthe filtrate to 100 mL with O.lM hydrochloric acid.
(2) 0.0075% w/v of cefalonium BPCRS in O.lM hydrochloric
6 volumes of water, 35 volumes of methanol and 59 volumes
of dichloromethane.
acid.
SYSTEM SUIT ABILITY
CHROMATOGRAPHIC CONDITIONS
The test is not valid unless the chromatogram obtained with
The chromatographic conditions described under Related
solution (3) shows three clearly separated spots.
substances may be used.
CONFIRMATION
DETERMINATION OF CONTENT
The principal spot in the chromatogram obtained with
Calculate the content of C2oHlSN40SS2 in a container of
solution (1) corresponds to that in the chromatogram
average content using the declared content of C2oHlSN40SS2
obtained with solution (2).
in cefalonium BPCRS.
B. To a quantity of the oral powder containing 10 mg of
STORAGE Chlortetracycline Hydrochloride add 20 mL of warm ethanol
Cefalonium Intramammary Infusion (Dry Cow) should be (96%) J allow to stand for 20 minutes, filter and evaporate to
stored at a temperature not exceeding 30 0 • It should not be dryness on a water bath. A 0.1 % w/v solution of the residue
allowed to freeze. in phosphate buffer pH 7.6, when heated at 100 0 for 1 minute,
LABELLING exhibits a strong blue fluorescence in ultraviolet light.
The quantity of active ingredient is stated in terms of the Tetracycline hydrochloride and
equivalent amount of anhydrous cefalonium. 4-epichlortetracyc1ine hydrochloride
Not more than 8.0% and 6.0% respectively, determined as
described under the Assay. Inject separately solutions (1) and
(4).
MOBILE PHASE The test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor between the peak due to
20 volumes of dimethylfonnamide and 80 volumes of
hydrocortisone acetate (retention time about 25 minutes) and
O.lM oxalic acid the pH of which has been adjusted to 2.2
that of cloprostenol (retention time about 35 minutes) is at
with triethylamine.
least 6.
SYSTEM SUITABILITY
LIMITS
The assay is not valid unless, in the chromatogram obtained
In the chromatogram obtained with solution (1) the sum of
with solution (3), the resolution factor between the two
the areas of any secondary peaks is not more than 1.25 times
prinCipal peaks is at least 1.5.
the area of the principal peak in the chromatogram obtained
DETERMINATION OF CONTENT with solution (2) (2.5%).
Ca1culate the content of C22H23CIN208,HCI in the tablets
ASSAY
using the declared content of C22H23CIN208,HCI in
Carry out the method for liquid chromatography,
chlortetracycline hydrochloride BPCRS.
Appendix 111 D, using the following solutions.
(1) Dilute the injection, if necessary, in absolute ethanol to
contain the equivalent of 0.009% w/v of cloprostenol.
(2) 0.009% w/v of cloprostenol sodium BPCRS in absolute
Cloprostenol Injection ethanol.
(3) Dissolve 5 mg of hydrocortisone acetate BPCRS and
Action and use
2.5 mg of cloprostenol sodium BPCRS in absolute ethanol and
Prostaglandin (PGF2c:J analogue.
dilute to 10 mL with the mobile phase.
DEFINITION CHROMATOGRAPHIC CONDITIONS
Cloprostenol Injection is a sterile solution of Cloprostenol The chromatographic conditions described under Related
Sodium in Water for Injections. substances may be used.
The injection complies with the requirements stated under SYSTEM SUITABILITY
Parenteral Preparations and with the following requirements.
The test is not valid unless, in the chromatogram obtained
Content of cloprostenol, C22H29CI06 with solution (3), the resolution factor between the peak due to
90.0 to 110.0% of the stated amount. hydrocortisone acetate (retention time about 25 minutes) and
IDENTIFICATION that of cloprostenol (retention time about 35 minutes) is at
In the Assay, the chromatogram obtained with solution (1) least 6.
shows a peak with the same retention time as the peak due to DETERMINATION OF CONTENT
cloprostenol in the chromatogram obtained with solution (2). Calculate the content of C22H29CI06 in the injection using
Related substances the declared content of C22H29CI06 in cloprostenol
Carry out the method for liquid chromatography, sodium BPCRS.
Appendix 111 D, using the following solutions. STORAGE
(1) Dilute the injection, if necessary, in absolute ethanol to Cloprostenol Sodium Injection should be protected from
contain the equivalent of 0.009% w/v of cloprostenol. light.
(2) 0.00018% w/v of cloprostenol sodium BPCRS in absolute
LABELLING
ethanol.
The quantity of active ingredient is stated in terms of the
(3) Dissolve 5 mg of hydrocortisone acetate BPCRS and equivalent amount of cloprostenol in a suitable dose-volume.
2.5 mg of cloprostenol sodium BPCRS in absolute ethanol and
dilute to 10 mL with the mobile phase.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 5 mm) packed
with base-deactivated octadecylsilyl silica gel for chromatography
(Waters Symmetry ODS is suitable).
(b) Use isocratic elution and the mobile phase described
below.
(c) Use a flow rate of 1.8 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 220 nm.
154 Cloxacillin Preparations
DEFINITION DEFINITION
Cloxacillin Benzathine Intramammary Infusion (Dry Cow) is Cloxacillin Sodium Intramammary Infusion (Lactating Cow)
a sterile suspension of Cloxacillin Benzathine in a suitable is a sterile suspension of Cloxacillin Sodium in a suitable
non-aqueous vehic1e containing suitable suspending agents. non-aqueous vehic1e containing suitable suspending and
The intramammary infusion complies with the requirements stated dispersing agents.
under Intramammary Infusions and with the following The intramammary infusion complies with the requirements stated
requirements. under Intramammary Infusions and with the following
Content of cloxacillin, C19H18CIN30SS requirements.
90.0 to 110.0% of the stated amount. Content of cloxacillin, C19H18CIN30SS
90.0 to 110.0% ofthe stated amount.
IDENTIFICATION
Extract a quantity of the infusion containing the equivalent of IDENTIFICATION
75 mg of c10xacillin with three 15-rnL quantities of petroleum Extract a quantity containing the equivalent of 75 mg of
spirit (boiling range, 12rf to 160°), discard the extracts, wash c10xacillin with three 15-rnL quantities of petroleum spirit
the residue with ether and dry in a current of airo The residue (boiling range, 120° to 160°). Discard the extracts, wash the
obtained complies with the following tests. residue with ether and dry in a current of airo The residue
A. The infrared absorption spectrum, Appendix II A, is
obtained complies with the following tests.
concordant with the reference spectrum of c10xacillin A. The infrared absorption spectrum, Appendix II A, is
benzathine (RSV 12). concordant with the reference spectrum of c10xacillin sodium
B. Shake 50 mg with 1 mL of 1M sodium hydroxide for (RSV 13).
2 minutes, add 2 mL of ether, shake for 1 minute and allow B. Yields the reactions characteristic of sodium salts,
to separate. Evaporate 1 mL of the ether layer to dryness, Appendix VI.
dissolve the residue in 2 mL of glacial acetic acid and add Water
1 mL of dilute potassium dichromate solution. A golden yellow Not more than 1.0% w/w, Appendix IX C. Use 3 g and a
precipitate is produced. mixture of 70 volumes of chloroform and 30 volumes of
Water anhydrous methanol as the solvento
Not more than 2.0% w/w, Appendix IX C. Use 3 g and a ASSAY
mixture of 70 volumes of chloroform and 30 volumes of Express, as far as possible, weigh and mix the contents of
anhydrous methanol as solvento 10 containers. Carry out the method for liquid
ASSAY chromatography, Appendix III D, using the following
Express, as far as possible, weigh and mix the contents of solutions.
10 containers. Extract a quantity of the mixed contents (1) Extract a quantity of the mixed contents of the 10
containing the equivalent of 80 mg of c1oxacillin, with three containers containing the equivalent of 50 mg of c10xacillin
15 mL quantities of petroleum spirit (boiling range, 120° to with 15 mL of petroleum spirit (boiling range, 120° to 160°),
16rf) previously saturated with cloxacillin benzathine. Discard centrifuge and discard the supernatant liquido Repeat the
the extract, wash the residue with ether previously saturated extraction with a further two 15-mL quantities of petroleum
with cloxacillin benzathine, dry in a current of air, dissolve in spirit (boiling range, 120° to 160°). Shake the residue with
25 mL of methanol and dilute to 50 mL with water. Dilute 20 mL of ether, centrifuge and dry in a current of air until
2 mL to 100 mL with buffered copper sulfate solution pH 2. O, the solvents have evaporated. Dissolve the final residue in
transfer 10 mL to a stoppered test tube and heat in a water sufficient of the mobile phase to produce 50 mL and dilute
bath at 70° for 20 minutes. Rapidly cool to room 5 volumes of the resulting solution to 50 volumes with the
temperature, dilute to 20 mL with absolute ethanol and mobile phase.
measure the absorbance of the resulting solution at the (2) 0.011 % w/v of cloxacillin sodium BPCRSin the mobile
maximum at 338 nm, Appendix II B, using in the reference phase.
cell 10 mL of the unheated buffered solution of the (3) 0.01 % w/v of each of cloxacillin sodium BPCRS and
substance being examined, diluted to 20 mL with absolute fiucloxacillin sodium BPCRS in the mobile phase.
ethanol.
CHROMATOGRAPHIC CONDITIONS
Calculate the content of C19HlSC1N30SS in a container of
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
average content from the absorbance obtained by carrying out
the procedure simultaneously using 2 mL of a solution with octadecylsilyl silica gel for chromatography (5 /lm)
(Hypersil 5 ODS is suitable).
prepared by dissolving 105 mg of cloxacillin
benzathine BPCRS in 50 mL of a mixture of equal volumes (b) Use isocratic elution and the mobile phase described
of methanol and water and from the dec1ared content of below.
C19HlSCIN30SS in cloxacillin benzathine BPCRS. (c) Use a fiow rate of 1 mL per minute~
LABELLING (d) U se an ambient column temperature.
The quantity of active ingredient is stated in terms of the (e) Use a detection wavelength of 225 nm.
equivalent amount of c1oxacillin. (±) Inject 20 /lL of each solution.
Co-trimazine Preparations 155
using the declared content of C lO H lON 40 ZS in an Rf value of about 0.3 corresponds to the principal spot in
sulfadiazine BPCRS. the chromatogram obtained with solution (4).
When trimethoprim and sulfadiazine injection is prescribed ASSAY
or demanded, Co-trimazine Injection shall be dispensed or For trimethoprim
supplied. Extract the combined chloroform extracts from the Assay for
sulfadiazine with four 50-mL quantities of a 5% v/v solution
of 6M acetic acid. Wash the combined aqueous extracts with
5 mL of chlorojorm, discard the chloroform layer and dilute to
Co-trimazine Veterinary Oral Powder 250 mL with a 5% v/v solution of 6M acetic acid. Dilute
20 mL to 100 mL with water. Determine the absorbance of
Trimethoprim and Sulfadiazine Veterinary Oral Powder
the resulting solution at the maximum at 271 nm,
Appendix II B, and calculate the content of
Action and use
Dihydrofolate reductase inhibitor + sulfonamide antibacterial. CI4HISN403 taking 204 as the value of A(l %, 1 cm) at the
maximum at 271 nm.
DEFINITION For ·sulfadiazine
Co-trimazine Veterinary Oral Powder consists of Transfer a quantity of the powder containing 0.125 g of
Trimethoprim and Sulfadiazine in the proportion of one part Sulfadiazine to a separating funnel containing 20 mL of
to five parts mixed with suitable wetting, dispersing and O.lM sodium hydroxide and extract with four 50-mL quantities
suspending agents. of chlorojorm. Wash each ch1oroform extract with the same
The veterinary oral powder complies with the requirements stated two 10-mL quantities of 0.1M sodium hydroxide. Combine the
under Veterinary Oral Powders and with the jollowing aqueous washings and the aqueous layer from the separating
requirements . funnel and reserve the combined chloroform extracts for the
Assay for trimethoprim. Dilute the combined aqueous
Content of trimethoprim, C14H18N403
solutions to 250 mL with water, filter and dilute 10 mL of
92.5 to 107.5% ofthe stated amount.
the filtrate to 200 mL with water. To 2 mL of the resulting
Content of sulfadiazine, C lOH 1ON 40 2 S solution add 0.5 mL of 4M hydrochloric acid and 1 mL of a
92.5 to 107.5% ofthe stated amount. 0.1 % w/v solution of sodium nitrite and allow to stand for
IDENTIFICATION 2 minutes. Add 1 mL of a 0.5% w/v solution of ammonium
Carry out the method for thin-layer chromatography, sulfamate and allow to stand for 3 minutes. Add 1 mL of a
Appendix III A, using the following solutions. 0.1 % w/v solution of N-(1-naphthyl)-ethylenediamine
dihydrochloride, allow to stand for 10 minutes and dilute to
(1) Shake a quantity ofthe powder containing 0.2 g of
25 mL with water. Measure the absorbance of the resulting
Sulfadiazine with sufficient l.4M methanolic ammonia to
solution at the maximum at 538 nm, Appendix II B, using in
produce 100 mL, centrifuge and use the supematant liquido
the reference cell a solution prepared at the same time and in
(2) Shake a quantity of the powder containing 0.2 g of the same manner using 2 mL of water and beginning at the
Trimethoprim with sufficient l.4M methanolic ammonia to words 'add 0.5 mL of 4M hydrochloric acid.. .'. Calculate the
produce 100 mL, centrifuge and use the supematant liquido content of ClOHION40ZS from the absorbance obtained by
(3) 0.2% w/v solution of sulfadiazine BPCRS in repeating the operation with 2 mL of a 0.0025% w/v solution
1.4M methanolic ammonia. of sulfadiazine BPCRS in 0.0005M sodium hydroxide and
(4) 0.2% w/v solution of trimethoprim BPCRS in beginning at the words 'add 0.5 mL of 4M hydrochloric acid
1.4M methanolic ammonia.
(5) Mix equal volumes of solutions (3) and (5). When trimethoprim and sulfadiazine veterinary oral powder
CHROMATOGRAPHIC CONDITIONS
is prescribed or demanded, Co-trimazine Veterinary Oral
Powder shall be dispensed or supplied.
(a) Use as the coating silica gel GF254 •
(b) Use the mobile phase as described be1ow.
(c) Apply 5 ¡.tL of each solution.
(d) Develop the plate to 15 cm. Co-trimazine Tablets
(e) After removal ofthe plate, dry in a current of air, spray Trimethoprim and Sulfadiazine Tablets
with a 0.1 % w/v solution of 4-dimethylaminobenzaldehyde in a
Action and use
mixture of 1 mL of hydrochloric add and 100 mL of ethanol
Dihydrofolate reductase inhibitor + sulfonámide antibacterial.
(96%), allow to dry and spray with dz1ute potassium
iodobismuthate solution. DEFINITION
MOBILE PHASE Co-trimazine Tablets contain Trimethoprim and Sulfadiazine
5 volumes of water, 15 volumes of dimethylformamide and in the proportion one part to five parts.
75 volumes of ethyl acetate. The tablets Icomply with the requirements stated under T ablets and
SYSTEM SUITABILITY with the joÚowing requirements.
The test is not valid unless the chromatogram obtained with Content of trimethoprim, Cl~18N403
solution (5) shows two clear1y separated spots. 92.5 to 107.5% ofthe stated amount.
CONFIRMATION Content of sulfadiazine, C 1OH 1ON40 2 S
92.5 to 107.5% ofthe stated amount.
The spot in the chromatogram obtained with solution (1)
with an Rfvalue of about 0.7 corresponds to the principal IDENTIFICATION
spot in the chromatogram obtained with solution (3) and the Comply with the test described under Co-trimazine Oral
spot in the chromatogram obtained with solution (2) with Suspension using solutions prepared in the following manner
Deltamethrin Preparations 157
as solutions (1) and (2). For solution (1) shake a quantity of ASSAY
the finely powdered tablets containing 0.2 g of Sulfadiazine Heat a quantity containing 0.2 g of Decoquinate with 50 mL
with sufficient l.4M methanolic ammonia to produce 100 mL, of chloroform in an apparatus for the continuous extraction of
centrifuge and use the supernatant liquido For solution (2) drugs, Appendix XI F, for eight reflux cyc1es. Cool and add
shake a quantity of the finely powdered tablets containing sufficient chloroform to produce 100 mL. Dilute 5 mL to
0.2 g of Trimethoprim with sufficient l.4M methanolic 100 mL with absolute ethanol. To 5 mL add 10 mL of
ammonia to produce 100 mL, centrifuge and use the O.lM hydrochloric acid and dilute to 100 mL with absolute
supernatant liquido ethanol. Measure the absorbance of the resulting solution at
ASSAY the maximum at 265 nm, Appendix II B. Dissolve 50 mg of
decoquinate BPCRS in 10 mL of hot chloroform and, keeping
Weigh and finely powder 20 tablets.
the solution warm, add slowly 70 mL of absolute ethanol.
For trimethoprim Cool, dilute to 100 mL with absolute ethanol and immediately
Carry out the Assay for trimethoprim described under dilute 10 mL to 100 mL with absolute ethanol. To 10 mL of
Co-trimazine Veterinary Oral Powder. the resulting solution add 10 mL of O.lM hydrochloric acid
For sulfadiazine and dilute to 100 mL with absolute ethanol. Calculate the
Carry out the Assay for sulfadiazine described under content of C24H3SNOs in the premix from the absorbances
Co-trimazine Veterinary Oral Powder using a quantity of the obtained using the dec1ared content of C24H3SNOs in
powdered tablets containing 0.125 g of Sulfadiazine. Repeat decoquinate BPCRS.
the operation using 2 mL of a 0.0025% w/v solution of
sulfadiazine BPCRS in 0.0005M sodium hydroxide beginning at
the words 'add 0.5 mL of 4M hydrochloric acid ... '. Calculate
the content of C lO H lON 40 2S in the oral suspension from the Deltamethrin Pour-on
absorbances obtained using the dec1ared content of
C lOH lON 4 0 2 S in sulfadiazine BPCRS. Action and use
Insecticide (veterinary).
When trimethoprim and sulfadiazine tablets are prescribed or
demanded, Co-trimazine Tablets shall be dispensed or DEFINITION
supplied. Deltamethrin Pour-on is a pour-on solution. It contains
Deltamethrin in a suitable, oily vehic1e.
The pour-on complies with the requirements stated under
Decoquinate Premix Veterinary Liquid Preparations for Cutaneous Application and
with the following requirements.
Action and use Content of deltamethrin, C22H19Br2N03
Antiprotozoal (veterinary). 90.0 to 110.0% of the stated amount.
DEFINITION IDENTIFICATION
Decoquinate Premix contains Decoquinate. In the Assay, the chromatogram obtained with solution (1)
shows a peak with the same retention time as the peak in the
The premix complies with the requirements stated under Premixes
chromatogram obtained with solution (2).
and with the following requirements.
Content of decoquinate, C24H3SNOs ASSAY
95.0 to 105.0% of the statedamount. Carry out the method for liquid chromatography,
Appendix III D, using the following solutions.
IDENTIFICATION
(1) Mix a weighed quantity of the preparation being
Carry out the method for thin-layer chromatography,
examined containing 30 mg of Deltamethrin with sufficient
Appendix III A, using the following solutions. hexane to produce 100 mL. Dilute 1 volume 10 4 volumes
(1) Heat a quantity containing 0.1 g of Decoquinate with with hexane.
40 mL of chloroform for 20 minutes on a water bath under a (2) 0.0075% w/v of deltamethrin BPCRS in hexane.
reflux condenser, cool and filter.
(3) 0.0075% w/v of deltamethrin impurity standard BPCRS in
(2) 0.25% w/v of decoquinate BPCRS in chloroform.
hexane.
CHROMATOGRAPHIC CONDlTIONS
CHROMATOGRAPHIC CONDlTIONS
(a) Use as the coating silica gel GF254• (a) Use a stainless steel column (25 cm x 4.6 mm) packed
(b) Use the mobile phase as described below. with partic1es of silica the surface of which has been modified
(c) Apply 10 ¡..tL of each solution. with chemically-bonded nitro-phenyl groups (5 ¡..tm)
(d) Develop the plate 10 15 cm. (Nuc1eosil-N02 is suitable).
(e) After removal of the plate, dry in air and examine under (b) U se isocratic elution and the mobile phase described
ultraviolet light (254 nm). below.
MOBILE PHASE
(c) Use a flow rate of 2 mL per minute.
30 volumes of ethanol (96%) and 70 volumes of chloroform. (d) Use an ambient column temperature.
(e) Use a detection wavelength of 230 nm.
CONFIRMATION
(f) Inject 20 ¡..tL of each solution.
The principal spot in the chromatogram obtained with
solution (1) corresponds in position and colour to that in the MOBILE PHASE
chromatogram obtained with solution (2). hexane containing 0.25% v/v of propan-2-ol.
158 Diprenorphine Preparations
A. The infrared absorption spectrum, Appendix II A, is A. The infrared absorption spectrum, Appendix II A, is
eoneordant with the reference spectrum of etamiphylline eoneordant with the reference spectrum of etamiphylline
(RSV 19). (RSV 19).
B. Yields the reaetions eharaeteristie of xanthines, B. Yields the reaetions eharaeteristie of xanthines,
Appendix VI. Appendix VI.
TESTS Related substances
Acidity Carry out the method for thin-layer chromatography,
pH, 3.9 to 5.4, Appendix V L. Appendix III A, using the following solutions.
Related substances (1) Shake a quantity of the powdered tablets eontaining
Carry out the method for thin-layer chromatography, 0.2 g of Etamiphylline Camsilate with 5 mL of methanol and
Appendix III A, using the following solutions. eentrifuge.
(1) Dilute the injeetion with suffieient water to produce a (2) Dilute 1 volume of solution (1) to 500 volumes with
solution eontaining 3.5% w/v of Etamiphylline Camsilate. methanol.
(2) Dilute 1 volume of solution (1) to 500 volumes with CHROMATOGRAPHIC CONDITIONS
water. (a) Use as the eoating silica gel HF254 •
CHROMATOGRAPHIC CONDITIONS (b) Use the mobile phase as deseribed below.
(a) Use as the eoating silica gel HF254• (e) Apply 10 IlL of eaeh solution.
(b) U se the mobile phase as deseribed below. (d) Develop the plate to 15 cm.
(e) Apply 10 III of eaeh solution. (e) After removal of the plate, allow it to dry in air and
(d) Develop the plate to 15 cm. examine under ultraviolet light (254 nm).
(e) After removal of the plate, allow it to dry in air and MOBILE PHASE
examine under ultraviolet light (254 nm). 1 volume of 13.5M ammonia, 20 volumes of ethanol (96%)
MOBILE PHASE and 80 volumes of chloroform.
1 volume of 13.5M ammonia, 20 volumes of ethanol (96%) LIMITS
and 80 volumes of chloroform. Any secondary spot in the ehromatogram obtained with
LIMITS solution (1) is not more intense than the spot in the
ehromatogram obtained with solution (2) (0.2%).
Any secondary spot in the ehromatogram obtained with
solution (1) is not more intense than the spot in the ASSAY
ehromatogram obtained with solution (2) (0.2%). Weigh and powder 20 tablets. Dissolve a quantity of the
powder eontaining 0.5 g of Etamiphylline Camsilate in
ASSAY
30 mL of water, make alkaline with 5M ammonia and extraet
To a volume eontaining 0.7 g of Etamiphylline Camsilate
with three 25-mL quantities of chloroform, washing eaeh
add 15 mL of water, make alkaline with 5M ammonia and
ehloroform extraet with the same 5-smL quantity of water.
extraet with three 25-mL quantities of chloroform, washing
Evaporate the eombined ehloroform extraets to dryness,
eaeh extraet with the same 5-mL quantity of water. Evaporate
dissolve the residue in 25 mL of water and titrate with
the eombined extraets to dryness, dissolve the residue in
0.05M sulfuric acid VS using bromocresol green solution as
25 mL of water and titrate with 0.05M sulfuric acid VS using
indieator. Eaeh mL of 0.05M sulfuric acid VS is equivalent
bromocresol green solution as indieator. Eaeh mL of
to 51.16 mg of C13HZINsOz,ClOH1604S.
0.05M sulfuric acid VS is equivalent to 51.16 mg of
C13HZINsOz,ClOH1604S.
(1) Mix, with the aid of ultrasound, a quantity of the (2) 0.01 % w/v ofjenbendazole BPCRS in a mixture of
powdered granules containing 0.1 g of Fenbendazole with 1 volume of O.IM hydrochloric acid and 1 volume of
50 mL of O.IM methanolic hydrochloric acid for 30 minutes, methanol (85%).
cool, dilute to 100 mL with methanol (65%), mix and filter CHROMATOGRAPHIC CONDITIONS
through a glass-fibre filter (Whatman GF/C is suitable).
The chromatographic procedure described under Related
(2) Dilute 1 volume of a 0.001 % w/v solution of substances may be used.
jenbendazole impurity A EPCRS (methyl (IH-benzimidazol-
DETERMINATION OF CONTENT
2-yl)carbamate) in O.IM methanolic hydrochloric acid to /
2 volumes with methanol (65%). Calculate the content of ClsH13N30ZS in the granules using
(3) Dilute 1 volume of a 0.001 % w/v solution ofjenbendazole the declared content of ClsH13N30ZS, in
impurity B EPCRS (methyl (5-chloro-lH-benzimidazol- jenbendazole BPCRS.
2-yl)carbamate) in O.lM methanolic hydrochloric acid to IMPURITIES
2 volumes with methanol (65%). The impurities limited by the requirements of this
(4) Dilute 1 volume of a 0.0010% w/v solution of monograph include impurities A and B listed under
jenbendazole impurity 1 BPCRS (5-phenylthio)- Fenbendazole and the following:
2-aminobenzimidª:?:ole) in O.IM methanolic hydrochloric acid
to 2 volumes with methanol (65%). ~
Q~I )--NH'
(5) Dilute 1 volume of a solution containing 0.002% w/v
each of jenbendazole impurity A EPCRS, jenbendazole I ~ ~
impurity B EPCRS, jenbendazole impurity 1 BPCRS and S N
0.20% w/v ojjenbendazole BPCRS ih O.IM methanolic
hydrochloric acid to 2 volumes with methanol (65%). 1. (5-phenylthio )-2-aminobenzimidazole.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
with octadecylsilyl silica gel jor chromatography (5 /lm)
(Nucleosil C18 is suitable).
(b) Use isocratic elution and the mobile phase described
Fenbendazole Oral Suspension
below. Action and use
(c) Use a flow rate of 1 mL per minute. Antihelminthic.
(d) Use an ambient column temperature.
DEFINITION
(e) Use a detection wavelength of 280 nm.
Fenbendazole Oral Suspension is an aqueous suspension of
(f) Inject 20 /lL of each solution. F enbendazole.
MOBILE PHASE The oral suspension complies with the requirements stated under
350 volumes of a 0.5% w/v solution of sodium dihydrogen Oral Liquids and with the jollowing requirements.
orthophosphate and 650 volumes of methanol containing 1.88 g Content of fenbendazole, ClsH13N302S
of sodium hexanesulfonate, the pH of which has been adjusted 95.0 to 105.0% of the stated amount.
to 3.5 with orthophosphoric acid.
IDENTIFICATION
SYSTEM SUIT ABILITY
In the Assay, the retention time of the principal peak in the
The test is not valid unless the chromatogram obtained with chromatogram obtained with solution (1) is the same as that
solution (5) closely resembles the reference chromatogram of the principal peak in the chromatogram obtained with
supplied with jenbendazole BPCRS. solution (2).
LIMITS TESTS
In the chromatogram obtained with solution (1): Related impurities A, B and 1
the areas of any peaks corresponding to fenbendazole Carry out the method for liquid chromatography,
impurity A, fenbendazole impurity B and fenbendazole Appendix III D, using the following solutions.
impurity 1 (5-(phenylthio)-2-aminobenzimidazole) are not (1) Mix with the aid of ultrasound, a quantity of the oral
greater than the areas of the corresponding peaks in the suspension containing 0.1 g of Fenbendazole with 50 mL of
chromatograms obtained with solutions (2), (3) and (4) O.IM methanolic hydrochloric acid for 30 minutes, cool, dilute
respectively (0.5% each). to 100 mL with methanol (65%), mix and filter through a
ASSAY glass-fibre filter (Whatman GF/C is suitable).
Carry out the method for liquid chromatography, (2) Dilute 1 volume of a 0.001 % w/v solution of
Appendix III D, using the following solutions. jenbendazole impurity A EPCRS (methyl (IH-benzimidazol-
(1) Mix with the aid of ultrasound a quantity of the 2-yl) carbamate) in 0.1 M methanolic hydrochloric acid to
powdered granules containing 0.1 g of Fenbendazole with 2 volumes with methanol (65%).
50 mL of O.IM methanolic hydrochloric acid for 30 minutes, (3) Dilute 1 volume of a 0.001 % w/v solution ofjenbendazole
cool, dilute to 100 mL with methanol (65%), mix, filter impurity B EPCRS (methyl(5-chloro-1H-benzimidazol-
through a glass-fibre filter (Whatman GF/C is suitable). 2-yl)carbamate) in O.IM methanolic hydrochloric acid to
Dilute 5 volumes of the resulting solution to 50 volumes 2 volumes with methanol (65%).
with O.IM hydrochloric acid in methanol (85%). (4) Dilute 1 volume of a 0.001 % w/v solution ofjenbendazole
impurity 1 BPCRS ((5-phenylthio)-2-aminobenzimidazole)
in O.IM methanolic hydrochloric acid to 2 volumes with
methanol (65%).
162 Fenbendazole Preparations
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
Serum Gonadotrophin Injection
with octadecylsilyl silica gel for chromatography (5 ¡lm) Action and use
(Nuc1eosil C18 is suitable). Equine serum gonadotrophin.
(b) Use isocratic elution and the mobile phase described
below. DEFINITION
(c) Use a flow rate of 1 mL per minute. Serum Gonadotrophin Injection is a sterile solution of Serum
Gonadotrophin in Water for Injections. It is prepared by
(d) Use an ambient column temperature.
dissolving Serum Gonadotrophin for Injection in the requisite
(e) Use a detection wavelength of 280 nm. amount of Water for Injections irnmediately before use.
(f) Inject 20 ¡lL of each solution. The injection complies with the requirements stated under
MOBILE PHASE Parenteral Preparations.
350 volumes of a 0.5% w/v solution of sodium dihydrogen STORAGE
orthophosphate and 650 volumes of methanol containing 1.88 g Serum Gonadotrophin Injection should be used immediately
of sodium hexanesulfonate, the pH of which has been adjusted after preparation.
to 3.5 with orthophgsphoric acid
SYSTEM SUITABILITY
SERUM GONADOTROPHIN FOR
The test is not valid unless the chromatogram obtained with INJECTION
solution (5) c10sely resembles the reference chromatogram
supplied with fenbendazole BPCRS. DEFINITION
Serum Gonadotrophin for Injection is a sterile material
LIMITS
consisting of Serum Gonadotrophin with or without
In the chromatogram obtained with solution (1): excipients. It is supplied in a sealed container.
the area of any peaks corresponding to fenbendazole The contents of the sealed container comply with the requirements
impurity A (methyl (lH-benzimidazol-2-yl)carbamate), for Powders for Injections or Infusions stated under Parenteral
fenbendazole impurity B (methyl (5-chloro-1H-benzimidazol- Preparations and with the following requirements.
2-yl)carbamate) and fenbendazole impurity 1
((5-phenylthio)-2-aminobenzimidazole) are not greater than CHARACTERISTICS
the areas of the corresponding peaks in the chromatograms A white or pale grey, amorphous powder.
obtained with solutions (2), (3) and (4) respective1y Soluble in water.
(0.5% each). IDENTIFICATION
ASSAY Causes enlargement of the ovaries of immature female rats
Carry out the method for liquid chromatography, when administered as directed in the Assay.
Appendix III D, using the following solutions. TESTS
(1) Mix with the aid of ultrasound a quantity of the oral Clarity, colour and acidity or aIkalinity of solution
paste containing 0.1 g ofFenbendazole with 50 mL of A solution containing 5000 IU per mL is clear,
O.lM methanolic hydrochloric acid for 30 minutes, cool, dilute Appendix IV A, Method 1, and colourless, Appendix IV B,
to 100 mL with methanol (65%), mix, filter through a glass- Method 1, and has a pH of 6.0 to 8.0, Appendix V L.
fibre filter (Whatman GF/C is suitable). Dilute 5 volumes of
Water
the resulting solution to 50 volumes with O.lM hydrochloric
Not more than 10.0% w/w, Appendix IX C. Use 80 mg.
acid in methanol (85%).
(2) 0.01 % w/v offenbendazole BPCRS in a mixture of
Bacterial endotoxins
Carry out the test for bacterial endotoxins, Appendix XIV C,
1 volume of O.lM hydrochloric acid and 1 volume of methanol
using Method C. Dissolve the contents of the sealed
(85%).
container in water BET to give a solution containing 1000 IU
CHROMATOGRAPHIC CONDITIONS of serum gonadotrophin per mL (solution A). The endotoxin
The chromatographic conditions described under Related limit concentration of solution A is 35 IU of endotoxin per
impurities A, B and 1 may be used. mL. Carry out the test using a suitable dilution of solution A
DETERMINATION OF CONTENT as described under Method C.
Calculate the content of ClsH13N302S in the veterinary oral ASSAY
paste from the chromatograms obtained and using the Carry out the Assay described under Serum Gonadotrophin.
dec1ared content of ClsH13N302S infenbendazole BPCRS. For each container tested, the estimated potency is not les s
than 80% and not more than 125% ofthe stated potency.
IMPURITIES
The fiduciallimits of error are not less than 64% and not
The impurities limited by the requirements of this
more than 156% ofthe stated potency.
monograph inc1ude impurities A and B listed under
Fenbendazole and the following: STORAGE
The sealed container should be protected from light and
0
stored at a temperature not exceeding 8 Under these
•
1. (5-phenylthio )-2-aminobenzimidazole.
164 Griseofulvin Preparations
obtained with solution (3) (1 %) and the sum of the areas of (1) dissolve a quantity of the preparation being examined
any such peaks is not greater than 6 times the area of the in sufficient methanol to produce a solution containing
principal peak in the chromatogram obtained with solution 0.04% w/v of Ivermectin. Solutions (2), (3) and (4) contain
(3) (6%). Disregard any peak with an area less than the area 0.04% w/v, 0.0004% w/v and 0.00002% w/v respectively of
of the principal peak in the chromatogram obtained with ivennectin BPCRS in methanol. Inject 20 ¡..tL of each solution.
solution (4) (0.05%). The chromatographic procedure may be carried out using
ASSAY (a) a stainless steel column (25 cm x 4.6 mm) packed with
Carry out the method for liquid chromatography, octadecylsilyl silica gel for chromatography (5 ¡..tm) (Apex ODS 1
Appendix nI D, using the following solutions. For solution is suitable), (b) as the mobile phase at a flow rate of 1.5 mL
(1) disperse a quantity of the oral paste in methanol with the per minute a mixture of 39 volumes of water, 55 volumes of
aid of ultrasound and add sufficient methanol to produce a methanol and 106 volumes of acetonitrile and (c) a detection
solution containing 0.04% w/v of Ivermectin. Solution (2) wavelength of 245 nm.
contains 0.04% w/v of ivennectin BPCRS in methanol. The test is not valid unless, in the chromatogram obtained
Inject 20 ¡..tL of each solution. with solution (2), the resolution factor between the first peak
The chromatographic conditions described under Re1ated (component H 2B 1b) and the second peak (component
substances may be used. H 2B 1a) is at least 3.0.
Calculate the content of ivermectin (H2B 1a + H 2B 1b) in the In the chromatogram obtained with solution (1) the area of
oral paste and the ratio H 2B 1a / (H2B 1a + H 2B 1b) using as any peak with a retention time of 1.3 to 1.5 relative to that of
the declared content the contents of C4sH74014 (H2B1J and the principal peak is not greater than 2.7 times the area of
C47H72014 (H2B 1b) in ivennectin BPCRS. the principal peak in the chromatogram obtained with
solution (3) (2.7%), the area of any other secondary peak is
not greater than the area of the principal peak inthe
chromatogram obtained with solution (3) (1%) and the sum
of the areas of any such peaks is not greater than 6 times the
Ivermectin Pour-on area of the principal peak in the chromatogram obtained with
solution (3) (6%). Disregard any peak with an area less than
Action and use the area of the principal peak in the chromatogram obtained
Antihe1minthic. with solution (4) (0.05%).
DEFINITION ASSAY
Ivermectin Pour-on is a pour-on solution. It contains Carry out the method for liquid chromatography,
Ivermectin in a suitable non-aqueous vehicle. Appendix In D, using the following solutions. For solution
The pour-on complies with the requirements stated under (1) dissolve a quantity of the preparation being examined in
Veterinary Liquid Preparations for Cutaneous Application and sufficient methanol to produce a solution containing
with the following requirements. 0.04% w/v of Ivermectin. Solution (2) contains 0.04% w/v of
ivennectin BPCRS in methanol. Inject 20 ¡..tL of each solution.
Content of ivermectin, calculated as the sum of
component H 2B 1a (C48H74014) and The chromatographic conditions described under Related
component H2Blb (C47Hn014) substances may be used.
95.0 to 105.0% of the stated amount. Calculate the content of ivermectin (H2B 1a + H 2B 1b) in the
The ratio of the contents H 2B1a / (H2B 1a + H 2B 1b) is preparation being examined and the ratio
at least 90.0%. H 2B 1a / (H2B 1a + H 2B 1b) using as the declared content the
contents of C4sH74014 (H2B 1J and C47H72014 (H2B 1b) in
IDENTIFICATION ivennectin BPCRS.
A. Carry out the method for thin-layer chromatography,
Appendix In A, using a silica gel 60 F 254 precoated plate
(Merck silica gel 60 F 254 plates are suitable) and a mixture of
1 volume of concentrated ammonia Rl, 9 volumes of methanol
and 90 volumes of dichloromethane as the mobile phase. Levamisole Injection
Apply separately to the plate 2 ¡..tL of each of the following
solutions. For solution (1) dissolve a quantity of the Action and use
preparation being examined in sufficient methanol to produce Immunostimulant; antihelminthic.
a solution containing 0.05% w/v of Ivermectin. Solution (2)
contains 0.05% w/v of ivennectin BPCRS in methanol. After DEFINITION
removal of the plate, allow it to dry in air and examine under Levamisole Injection is a sterile solution of Levamisole
ultraviolet light (254 nm and 366 nm). The principal spot in Hydrochloride in Water for Injections. It may contain
the chromatogram obtained with solution (1) is similar in suÍtable colouring matter.
position, colour and size to that in the chromatogram The injection complies with the requirements stated under
obtained with solution (2). Parenterdl Preparations and with the following requirements.
B. In the Assay, the chromatogram obtained with solution Content oflevamisole hydroch1oride, C ll H 12N2 S,HCI
(1) shows two principal peaks with retention times similar to 92.5 t9 107.5% ofthe stated amount.
those of the two principal peaks in the chromatogram
IDENTIFICATION
obtained with solution (2).
A. Carry out the method for thin-layer chromatography,
TESTS Appendix In A, using silica gel G as the coating substance
Related substances and a mixture of 100 volumes of ethyl acetate, 10 volumes of
Carry out the method for liquid chromatography, methanol and 1 volume of 13.5M ammonia as the mobile
Appendix nI D, using the following solutions. For solution phase. Apply separately to the plate 1 ¡..tL of each of the
Levamisole Preparations 167
DETERMINATION OF CONTENT
Lincomycin Premix Ca1culate the content of ClsH34Nz06S in the premix using .
Action and use the declared content of ClsH34Nz06S in lincomycin
Lincosamide antibacterial. hydrochloride BPCRS.
LABELLING
DEFINITION
The quantity of active ingredient is stated in terms of the
Lincomycin Premix contains Lincomycin Hydrochloride. equivalent amount of lincomycin.
The premix complies with the requirements stated under Premixes
and with the following requirements.
Content of lincomycin, C18H3~206S
90.0 to 105.0% ofthe stated amount.
Lincomycin Tablets
IDENTIFICATION
In the Assay the chromatogram obtained with solution (2) Action and use
shows a peak with the same retention time as the peak due Lincosamide antibacterial.
to the trimethylsilyl derivative of lincomycin in the
chromatogram obtained with solution (1). DEFINITION
Lincomycin Tablets contains Lincomycin Hydrochloride.
TESTS
Lincomycin B The tablets comply with the requirements stated under T ablets and
Examine solution (3) as described in the Assay but increasing with the following requirements.
the sensitivity by eight to ten times while recording the peak Content of lincomycin, C18H34N206S
due to the trimethylsilyl derivative of lincomycin B, which is 90.0 to 105.0% of the stated amount.
eluted immediately before the trimethylsilyl derivative of IDENTIFICATION
lincomycin. A. Mix a quantity of the powdered tablets containing the
LIMITS equivalent of 200 mg of lincomycin with 5 mL of a mixture
The area of the peak due to the trimethylsilyl derivative of of 4 volumes of chloroform and 1 volume of methanol, filter
lincomycin B, when corrected for the sensitivity factor, is not and evaporate the filtrate. Dissolve the oily residue in 1 mL
more than 5 % of the area of the peak due to the of water, add acetone until precipitation begins and add a
trimethylsilyl derivative of lincomycin. further 20 mL of acetone. Filter, wash with two 10-mL
quantities of acetone, dissolve the residue in the chloroform-
ASSAY methanol mixture, evaporate to dryness and dry at 60° at a
Carry out the method for gas chromatography, pressure of 2 kPa for 1 hour. The infrared absorption spectrum
Appendix III B, using the following solutions. of the residue, Appendix II A, is concordant with the reference
(1) Add 10 mL of a 0.8% w/w solution of dotriacontane spectrum of lincomycin hydrochloride (RSV 52).
(internal standard) in chloroform to 0.1 g of lincomycin B. In the Assay, the chromatogram obtained with solution
hydrochloride BPCRS, dilute to 100 mL with a 2% w/v (2) shows a peak with the same retention time as the peak
solution of imidazole in chloroform, shake to dissolve and filter. due to the trimethylsilyl derivative of lincomycin in the
Place 4 mL of the filtrate in a 15 mL ground-glass-stoppered chromatogram obtained with solution (1).
centrifuge tube, add 1 mL of a mixture of 99 volumes of
N,O-bis(trimethylsilyl)acetamide and 1 volume of Lincomycin B
trimethylchlorosilane and swirl gently. Loosen the glass stopper Examine solution (3) as described under the Assay but
and heat at 65° for 30 minutes. increasing the sensitivity by eight to ten times while recording
the peak due to the trimethylsilyl derivative of lincomycin B,
(2) Prepare in the same manner as solution (1) but omitting
which is eluted immediately before the trimethylsilyl
the internal standard and using a quantity of the premix
derivative of lincomycin. The area of the peak due to the
containing the equivalent of 90 mg of lincomycin in place of
trimethylsilyl derivative of lincomycin B, when corrected for
the lincomycin hydrochloride BPCRS.
the sensitivity factor, is notmore than 5% of the area of the
(3) Prepare in the same manner as solution (1) but using a peak due to the trimethylsilyl derivative of lincomycin.
quantity of the premix containing the equivalent of 90 mg of
lincomycin in place of the lincomycin hydrochloride BPCRS. ASSAY
Carry out the Assay described under Lincomycin Premix but
CHROMATOGRAPHIC CONDITIONS
using in the preparation of solutions (2) and..(3) a quantity of
(a) Use a glass column (1.5 m x 3 mm) packed with powdered tablets containing the equivalent of 90 mg of
acid-washed silanised diatomaceous support impregnated with lincomycin.
3% w/w of phenyl methyl silicone fluid (50% phenyl)
(OV-17 is suitable) and maintained at 260°. LABELLING
The quantity of active ingredient is stated in terms of the
(b) U se helium as the carrier gas at a flow rate of about
equivalent, amount of lincomycin.
45 mL per minute. 1,
(3) Add 0.3 mL of O.4Msodium hydroxide to 40 mg of B. Disperse a quantity of the oral suspension containing
meloxicam impurity standard BPCRS and dilute with 1.5 mg of Meloxicam in 5 mL of O.lM sodium hydroxide,
methanol (40%) to produce 10 mL. dilute to 100 mL with methanol and filter. The light absorption
CHROMATOGRAPHIC CONDITIONS
of the filtrate, Appendix n B, in the range 340 to 450 nm
exhibits a maximum at 362 nm.
The chromatographic procedure described under Related
substances may be used with a detection wavelength of ASSAY
350 nm. Carry out the method for liquid chromatography,
Appendix nI D, using the following solutions.
SYSTEM SUIT ABILITY
(1) Mix a quantity of the oral suspension containing 15 mg
The test is not valid unless the chromatogram obtained with.
of Meloxicam with sufficient of the mobile phase to produce
solution (3):
200 mL, mix with the aid of ultrasound for 30 minutes and
closely resembles the chromatogram supplied with meloxicam filter.
impurity standard BPCRS at 350 nm;
(2) 0.0075% w/v of meloxicam BPCRS in the mobile phase.
the resolution factor between the peaks due to meloxicam and
impurity A at 350 nm is at least 3.0. CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (10 cm x 4 mm) packed
DETERMINATION OF CONTENT
with octadecylsilyl silica gel for chromatography (10 Jlm)
Calculate the content of C14Hl}N304SZ using the declared (Nucleosil C18 is suitable) and a pre-column 1-cm long
content of C14H13N304SZ in meloxicam BPCRS. packed with the same material.
IMPURITIES (b) Use isocratic elution and the mobile phase described
The impurities limited by the requirements of this below.
monograph include those listed under Meloxicam. (c) Use a fiow rate of 0.8 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a detection wavelength of 254 nm.
(f) Inject 20 JlL of each solution and continue the
Meloxicam Oral Suspension chromatography for twice the retention time of the
principal peak.
Action and use
Cyclo-oxygenase inhibitor; analgesic; anti-infiarnmatory. MOBILE PRASE
35 volumes of a mixture containing 10 parts of propan-2-01
DEFINITION and 65 parts of methanol and 65 volumes of a 0.2% w/v
Meloxicam Oral Suspension is a suspension of Meloxicam solution of diammonium hydrogen orthophosphate previously
in a suitable vehicle. adjusted to pH 7.0 with orthophosphoric acid.
The oral suspension complies with the requirements stated under DETERMINATION OF CONTENT
Oral Liquids and with the following requirements. Determine the weight per mL of the oral suspension,
Content ofmeloxicam, C1J113N304S2 Appendix V G, and calculate the content of C14H13N304SZ,
95.0 to 105.0% of the stated amount. weight in volume, from the dec1ared content of
IDENTIFICATION C14H13N304SZ in meloxicam BPCRS.
A. Carry out the method for thin-Iayer chromatography,
Appendix nI A, using the following solutions.
(1) Dilute a quantity of the oral suspension containing 3 mg
of Meloxicam to 10 mL with acetone, stir for 10 minutes, Mepivacaine Injection
filter and use the filtrate.
(2) Dissolve 3 mg of meloxicam BPCRS in about 5 mL of Action and use
acetone, add 0.5 mL of water and dilute to 10 mL with Local anaesthetic.
acetone.
DEFINITION
CHROMATOGRAPHIC CONDITIONS Mepivacaine Injection is a sterile solution of Mepivacaine
(a) Use as the coating high-performance silica gel F 254 Hydrochloride in Water for Injections.
(Merck silica gel 60 F Z54 HPTLC plates are suitable). The injection complies with the requirements stated under
(b) Use the mobile phase as described below. Parenteral Preparations and with the following requirements.
(c) Apply 5 JlL of each solution. Content of mepivacaine hydroch1oride,
(d) Develop the plate to 8 cm. C 15H 22N 20 2,HCl
(e) After removal of the pI ate, allow it to dry in air and 95.0 to 105.0% ofthe stated amount.
examine under ultraviolet light (254 and 365 nm). IDENTIFICATION
MOBILE PRASE A. Carry out the method for thin-Iayer chromatography,
1 volume of 13.5M ammonia, 20 volumes of methanol and Appendix lIT A, using a TLC silica gel F254 plate and a
80 volumes of dichloromethane. mixture of 1 volume of 13.5M ammonia, 5 volumes of
methanol and 100 volumes of ether as the mobile phase, but
CONFIRMATION
allowing the solvent front to ascend 12 cm above the line of
By each method of visualisation the principal spot in the application. Apply separately to the plate 10 JlL of each of
chromatogram obtained with solution (1) is similar in the following solutions. For solution (1) dilute a quantity of
position, colour and size to that in the chromatogram the injection with sufficient ethanol (96%) to produce a
obtained with solution (2). solution containing 0.4% w/v of Mepivacaine Hydrochloride.
Moxidectin Preparations 171
DEFINITION DEFINITION
Oxfendazole Oral Suspension is an aqueous suspension of Oxyclozanide Oral Suspension is an aqueous suspension of
Oxfendazole. Oxyclozanide containing suitable suspending and dispersing
The oral suspension complies with the requirements stated under agents.
Oral Liquids and with the jollowing requirements. The oral suspension complies with the requirements stated under
Content of oxfendazole, ClsH13N303S Oral Liquids and with the jollowing requirements.
90.0 to 110.0% of the stated amount. Content of oxyc1ozanide, C 13H 6 Cl sN0 3
IDENTIFICATION 95.0 to 105.0% of the stated amount.
Shake a quantity of the oral suspension containing 0.1 g of IDENTIFICATION
Oxfendazole with 50 mL of methanol for 15 minutes, In test A for Re1ated substances the principal spot in the
centrifuge, evaporáte the supernatant liquid to a volume of chromatogram obtained with 10 IlL of solution (1)
about 2 mL, cool and filter. Wash the residue with a little corresponds to that in the chromatogram obtained with
water and dry at 105° at a pressure not exceeding 2.7 kPa for solution (3).
1 hour. The residue complies with the following tests. Related substances
A. The infrared absorption spectrum, Appendix 11 A, is A. Carry out the method for thin-layer chromatography,
concordant with the rejerence spectrum of oxfendazole Appendix 111 A, using silica gel G as the coating substance
(RSV 32). and a mixture of 5 volumes of glacial acetic acid, 20 volumes
B. The light absorption, Appendix 11 B, in the range 220 to of acetone and 60 volumes of petroleum spirit (boiling rangeJ 60°
350 nm of a 0.001 % w/v solution in 1M hydrochloric acid to 80°) as the mobile phase. Apply separately to the plate
exhibits three maxima, at 226, 284 and 291 nm. 40 IlL and 10 IlL of solution (1), 4 IlL of solution (2) and
10 IlL of solution (3). For solution (1) dilute the oral
TESTS
suspension with acetone to contain 1.0% w/v of Oxyclozanide,
Acidity
centrifuge and use the supernatant liquido Solution (2)
pH, 4.3 to 5.3, Appendix V L.
contains 0.050% w/v of 3J 5J 6-trichloro-2-hydroxybenzoic
Related substances acid BPCRS in acetone. Solution (3) contains 1.0% w/v of
Carry out the method for thin-layer chromatography, oxyclozanide BPCRS in acetone. After removal of the plate,
Appendix 111 A, using silica gel G as the coating substance allow it 10 dry in air and spray with a 3.0% w/v solution of
and a mixture of 5 volumes of glacial acetic acid and iron(m) chloride hexahydrate in methanol. In the chromatogram
95 volumes of ethyl acetate as the mobile phase. Apply obtained with 40 IlL of solution (1) any spot corresponding
separately to the plate 20 IlL of each of the following to 3,5,6-trichloro-2-hydroxybenzoic acid is not more intense
solutions. For solution (1) shake a quantity of the oral than the spot in the chromatogram obtained with solution (2)
suspension containing 0.1 g of Oxfendazole with 20 mL of a (0.5%).
mixture of 4 volumes of ethyl acetate and 1 volume of glacial B. Carry out the method for thin-layer chromatography,
acetic acid and filter. For solution (2) dilute 1 volume of Appendix 111 A, using silica gel G as the coating substance
solution (1) to 50 volumes with the same solvent mixture. and a mixture of 1 volume of 13.5M ammonia as the mobile
Solution (3) contains 0.005% w/v ofjenbendazole BPCRS. phase, 10 volumes of methanol and 100 volumes of ethyl
After removal of the plate, allow it to dry in air and examine acetate. Apply separate1y to the plate 40 IlL of solution (1)
under ultraviolet light (254 nm). Any spot in the and 4 IlL of solution (2). For solution (1) dilute the oral
chromatogram obtained with solution (1) corresponding to suspension with acetone to contain 1.0% w/v of Oxyclozanide,
methyl 5-phenylthio-1H-benzimidazol-2-ylcarbamate is not centrifuge and use the supernatant liquido Solution (2)
more intense than the spot in the chromatogram obtained contains 0.050% w/v of 2-amino-4J 6-dichlorophenol
with solution (3) (1%) and any other secondary spot is not hydrochloride BPCRS in acetone. After removal of the plate,
more intense than the spot in the chromatogram obtained allow it to dry in air and spray with phosphomolybdotungstic
with solution (2) (2%). reagent. In the chromatogram obtained with solution (1) any
ASSAY spot corresponding to 2-amino-4,6-dichlorophenol is not
Disperse a quantity of the well-mixed oral suspension more intense than the spot in the chromatogram obtained
containing 0.1 g of Oxfendazole in 15 mL of water. with solution (2) (0.4%).
Add 200 mL of methanol and mix with the aid of ultrasound ASSAY
for 15 minutes, cool, add sufficient methanol to produce Protect the solutions from light throughout the Assay.
500 mL and filter. Dilute 4 mL of the filtrate to 100 mL To a quantity of the oral suspension containing 60 mg of
with methanol and measure the absorbance of the resulting Oxyclozanide add 60 mL of acidified methanol and boil gent1y
solution at the maximum at 296 nm, Appendix 11 B. on a water bath. Shake continuously for 20 minutes, cool to
Calculate the content of ClsH13N303S taking 550 as the 2° and dilute to 100 mL with acidified methanol. Filter, dilute
value of A(l %, 1 cm) at the maximum at 296 nm. 5 mL of the filtrate to 100 mL with acidified methanol and
measure the absorbance of the resulting solution at the
maximum at 300 nm, Appendix 11 B. Calculate the content
of C 13 H 6 CIsN0 3 taking 254 as the value of A(1 %, 1 cm) at
the maximum at 300 nm.
174 Oxytetracycline Preparations
ASSAY
Oxytetracycline Veterinary Oral Powder Carry out the method for liquid chromatography,
Action and use Appendix 111 D, using the following solutions.
Tetracyc1ine antibacterial. (1) Dissolve a quantity of the oral powder in sufficient
O.OlM hydrochloric acid to produce a solution containing
DEFINITION 0.005% w/v of Oxytetracyc1ine Hydrochloride.
Oxytetracyc1ine Veterinary Oral Powder is a mixture of (2) 0.005% w/v of oxytetracycline BPCRS in
Oxytetracyc1ine Hydrochloride and Lactose or other suitable O. O1M hydrochloric acid.
diluent.
(3) 0.1 % w/v of 4-epioxytetracycline EPCRS in
Content of oxytetracycline hydrochloride, O. O1M hydrochloric acid.
C22H24N209,HCl (4) 0.1 % w/v of tetracycline hydrochloride BPCRS in
90.0 to 110.0% ofthe stated amount. O.OlM hydrochloric acid.
The veterinary oral powder complies with the requirements stated (5) Dilute a mixture containing 1.5 mL of a 0.1 % w/v
under Veterinary Oral Powders and with the following solution of oxytetracycline BPCRS in O.OlM hydrochloric acid,
requirements. 1 mL of solution (3) and 3 mL of solution (4) to 25 mL
IDENTIFICATION with O.OlM hydrochloric acid.
A. Carry out the method for thin-layer chromatography, CHROMATOGRAPHIC CONDITIONS
Appendix 111 A, using the following solutions.
(a) Use a stainless steel column (25 cm x 4.6 mm) packed
(1) Extract a quantity of the oral powder containing 10 mg with styrene-divinylbenzene copolymer (8 to 1O ~m) (Polymer
of Oxytetracycline Hydrochloride with 20 mL of methanol, Laboratories, PLRP-S 100A, is suitable).
centrifuge and use the supernatant liquido
(b) Use isocratic elution and the mobile phase described
(2) 0.05% w/v of oxytetracycline hydrochloride BPCRS in belo~ .
methanol.
(c) Use a fiow rate of 1 mL per minute.
(3) 0.05% w/v of each of oxytetracycline hydrochloride BPCRS
(d) Use a column temperature of 60 0
•
and demeclocycline hydrochloride BPCRS in methanol.
(e) Use a detection wavelength of 254 nm.
CHROMATOGRAPHIC CONDITIONS
(f) Inject 20 ~L of each solution.
(a) Use a silica gel precoated plate (Merck silica gel 60 plates
MOBILE PHASE
are suitable). Adjust the pH of a 10% w/v solution of
disodium edetate to 7.0 with 10M sodium hydroxide and spray To 50.0 g of 2-methylpropan-2-ol add 200 mL of water,
the solution evenly onto the plate (about 10 mL for a plate 60 mL of O.33M phosphate buffer pH 7.5, 50 mL of a
100 mm x 200 mm). Allow the plate to dry in a horizontal 1.0% w/v solution of tetrabutylammonium hydrogen sulfate
position for at least 1 hour. Before use, dry the plate at 110 0
previously adjusted to pH 7.5 with 2M sodium hydroxide and
for 1 hour. 10 mL of a 0.04% w/v solution of disodium edetate previously
(b) Use the mobile phase as described below. adjusted to pH 7.5 with 2M sodium hydroxide and dilute to
1 litre with water.
(c) Apply 1 ~L of each solution.
SYSTEM SUITABILITY
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry it in a current of air and The Assay is not valid unless, in the chromatogram obtained
with solution (5):
examine under ultraviolet light (365 nm).
the resolution factor between the first peak
MOBILE PHASE
(4-epioxytetracyc1ine) and the second peak (oxytetracyc1ine)
6 volumes of water, 35 volumes of methanol and 59 volumes is at least 4.0;
of dichloromethane.
the resolution factor between the second peak and the third
SYSTEM SUIT ABILITY peak (tetracyc1ine) is at least 5.0 (if necessary reduce the
The test is not valid unless the chromatogram obtained with content of 2-methylpropan-2-o1 in the mobile phase to
solution (3) shows two c1early separated spots. increase the resolution);
CONFIRMATION the symmetry factor of the peak due to oxytetracycline is not
The principal spot in the chromatogram obtained with more than 1.25.
solution (1) is similar in position, colour and size to that in DETERMINATION OF CONTENT
the chromatogram obtained with solution (2). Ca1culate the content of C22H24N209,HcCln the oral
B. To a quantity of the powder containing 0.4 mg of powder using the declared content of C22H24N209 in
Oxytetracyc1ine Hydrochloride add 5 mL of a 1% w/v oxytetracycline BPCRS. Each mg of C22H24N209 is equivalent
solution of sodium carbonate, shake and add 2 mL of to 1.079 mg of C22H24N209,HCl.
diazobenzenesulfonic acid solution. A light brown colour is
produced.
C. Shake a quantity ofthe powder containing 0.1 g of
Oxytetracyc1ine Hydrochloride with 10 mL of 2M nitric acid
and filter. Decolourise the filtrate with activated charcoal and
filter again. The filtrate yields the reactions characteristic of
chlorides, Appendix VI.
Phenylbutazone Preparations 175
DEFINITION DEFINITION
Pentobarbital Injection is a sterile 1 solution of Pentobarbital Phenylbutazone Tablets contain Phenylbutazone. Theyare
Sodium in a suitable vehic1e. coated.
The injection complies with the requirements stated under The tablets comply with the requirements stated under T ablets and
Parenteral Preparations and with the following requirements. with the following requirements.
Content ofpentobarbital sodium, CUH17N2Na03'i, Content of phenylbutazone, C19H2oN202
95.0 to 105.0% of the stated amount. 95.0 to 105.0% of the stated amount.
CHARACTERISTICS IDENTIFICATION
A c1ear, colourless or almost colourless solution. Extract a quantity of the powdered tablets containing 0.2 g of
Phenylbutazone with 40 mL of warm acetone, filter and
IDENTIFICATION
evaporate the filtrate to dryness. The residue complies with
A. The infrared absorption spectrum of the residue obtained in
the following tests.
the Assay, Appendix II A, is concordant with the reference
spectrum of pentobarbital (RSV 34). A. The infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum of phenylbutazone
B. Melting point of the residue obtained in the Assay, about
(RSV 35).
128°, Appendix V A.
B. To 0.1 g ofthe residue add 1 mL of glacial acetic acid and
C. When introduced on a platinum wire into the flame of a
2 mL of hydrochloric acid and heat on a water bath for
Bunsen burner, imparts a yellow colour to the flameo
30 minutes. Cool, add 10 mL of water and filter. Add to the
TESTS filtrate 3 mL of O.IM sodium nitrite; a yellow colour is
AIkalinity produced. Add 1 mL of this solution to 5 mL of 2-naphthol
pH, 10.0 to 11.5, Appendix V L. solution; a brownish red precipitate is produced which
Isomer dissolves on the addition of ethanol (96%) yielding a red
To a volume containing 0.3 g of Pentobarbital Sodium solution.
diluted, if necessary, to 5 mL with water, add 0.3 g of TESTS
4-nitrobenzyl bromide dissolved in 10 mL of ethanol (96%) and Dissolution
heat under a reflux condenser for 30 minutes. Cool to 25°, Comply with the requirements for Monographs of the British
filter, wash the residue with four 5 mL quantities of water Pharmacopoeia in the dissolution test for tablets and capsules,
and transfer as completely as possible to a small flask. Appendix XII Bl.
Add 25 mL of ethanol (96%) and heat under a reflux
TEST CONDITIONS
condenser for 10 minutes. The residue, after drying at 105°
for 30 minutes, melts completely between 136° and 155°. (a) Use Apparatus 1, rotating the basket at 100 revolutions
per minute.
ASSAY
(b) Use 900 mL of a 0.68% w/v solution of potassium
To a volume containing 0.5 g of Pentobarbital Sodium
dihydrogen orthophosphate adjusted to pH 7.5 with 1M sodium
diluted to 15 mL with water add 5 mL of 2M hydrochloric
hydroxide, at a temperature of 37°, as the medium.
acid, extract with 50 mL of ether and then with successive
25 mL quantities of ether until complete extraction is PROCEDURE
effected. Wash the combined extracts with two 5 mL After 45 minutes withdraw a 10 mL sample of the medium
quantities of water and wash the combined aqueous washings and measure the absorbance of the filtered sample, suitably
with 10 mL 6f ether. Add the ether to the main ether extract, diluted with the dissolution medium if necessary, at the
evaporate to low volume, add 2 mL of absolute ethanol, maximum at 264 nm, Appendix II B using the dissolution
evaporate to dryness and dry the residue to constant weight medium in the reference cell.
at 105°. Each g of residue is equivalent to 1.097 g of DETERMINATION OF CONTENT
CUH17N2Na03.
Calculate the total content ofphenylbutazone, C19H20N202,
When pentobarbitone injection is prescribed or demanded, in the medium taking 653 as the value of A(1 %, 1 cm) at the
Pentobarbital Injection shall be dispensed or supplied. maximum at 264 nm.
1 Solutions containing 20% w/v of Pentobarbital Sodium in 100 mL and
Related substances
500 mL quantities are also available for purposes other than injection; such Carry out the method for thin-layer chromatography,
solutions are not necessarily sterile but comply with all the other requirements Appendix III A, using the following solutions.
of the monograph; they may be coloured. (1) Shake a quantity of the powdered tablets containing 0.1 g
of Phenylbutazone with 3 mL of chloroform containing
0.02% w/v of butylated hydroxytoluene, centrifuge and use the
supernatant liquido
(2) Dilute 1 volume of solution (1) with sufficient of the
same solvent mixture to produce a solution containing
0.5 mg ofPhenylbutazone per mL.
176 Piperazine Preparations
MOBILE PHASE
Ca1culate the content of C13H20N202 and of
C13H20N202,C16HlSN204S,H20 in the injection from the
250 volumes of acetonitrile, 250 volumes of water and chromatograms obtained and using the dec1ared content of
500 volumes of a freshly prepared solution containing
C13H20N202 and of C13H20N202,C16HlSN204S,H20 in
1.4% w/v of potassium dihydrogen orthophosphate and procaine benzylpenicillin BPCRS.
0.65% w/v of tetrabutylammonium hydroxide, adjusted to
pH 7.0 with 1M potassium hydroxide. Adjust the pH of the 3 g of Procaine Benzylpenicillin is approximately equivalent
mixture to 7.2 with 2M orthophosphoric acid, if necessary. to 2 g of benzylpenicillin.
For solution (3), when the chromatogram is recorded under
the prescribed conditions the substances elute in the
following order: 4-aminobenzoic acid, procaine, Pyrethrum Extract
benzylpenicillin.
DEFINITION
SYSTEM SUITABILITY Pyrethrum Extract is prepared from Pyrethrum Flower.
The test is not valid unless, in the chromatogram obtained Extemporaneous preparation
with solution (3), the resolution factor between the peaks due Exhaust Pyrethrum Flower, in coarse powder, by percolation
to 4-aminobenzoic acid and procaine is at least 2.0. with a suitable hydrocarbon solvent; remove the solvent and
If necessary, adjust the concentration of acetonitrile in the concentrate at a low temperature. The resulting product may
mobile phase. be decolourised by a suitable procedure. Determine the
proportion of pyrethrins in a portion of the extract by the
Assay. To the remainder add, if necessary, sufficient Light
Liquid Paraffin or deodorised kerosene to produce an extract of
the required strength.
178 Pyrethrum Preparations
The extract complies with the requirements for Labelling stated absorbent cotton into the second separating funnel. Shake
under Extracts and with the following requirements. the combined petroleum spirit extracts and water for about
Content of pyrethrins 30 seconds and allow to separate; remove the lower layer and
24.5% to 25.5% w/w, of which not less than half consists of add it to the aqueous liquid reserved for the assay of
pyrethrin 1. pyrethrin II. Wash the combined petroleum spirit extracts
with a further 10 mL of water, adding the washings to the
CHARACTERISTICS reserved aqueous liquido
A dark olive green or brown viscous liquid or, if To the petroleum spirit extracts add 5 mL of O.IM sodium
decolourised, a pale amber liquido hydroxide, shake vigorously for 1 minute, allow to separate
ASSAY and remove the clear lower layer, washing the stem of the
To 0.5 g of the well-mixed extract add 20 mL of separating funnel with 1 mL of water. Repeat the extraction
O.5M ethanolic potassium hydroxide and boil under a refiux by shaking for about 30 seconds with two quantities of
condenser for 45 minutes. Transfer the solution to a beaker 2.5 mL and 1.5 mL of O.IM sodium hydroxide and add the
and wash the fiask with sufficient hot water, adding the extracts to the alkaline extract. Add to the fiask 10 mL of
washings to the beaker, to produce a total volume of mercury(lI) sulfate solution, stopper, swirl and allow to stand in
200 mL. Boil until the volume is reduced to 150 mL, cool the dark at 25 0 ± 0.5 0 for exactly 60 minutes after the
rapidly and transfer the solution to a stoppered fiask, washing addition of the mercury(n) sulfate solution. Add 20 mL of
the beaker with three 20 mL quantities of water and acetone and 3 mL of a saturated solution of sodium chloride,
transferring any gummy residue to the fiask. Add 1 g of heat to boiling on a water bath, allow the precipitate to settle
diatomaceous earth (Filtercel is suitable) and 10 mL of and decant the supematant liquid through a filter paper
barium chloride solution, swirl gently and add sufficient water (Whatman No. 1 is suitable), retaining most of the
to produce 250 mL. Stopper the fiask, shake vigorously until precipitate in the fiask. Wash the precipitate with 10 mL of
the separating liquid is clear and filter the suspension through acetone, again boil, allow to settle and decant through the
a filter paper (Whatman No. 1 is suitable). same filter papero Repeat the washing and decanting with
If the pyrethrum extract is coloured, carry out the following three 10 mL quantities of hot chloroform. Transfer the filter
preliminary treatment. Transfer 0.5 g of the well-mixed paper to the fiask, add 50 mL of a cooled mixture of three
extract to a stoppered fiask, add 50 mL of aromatic-free volumes of hydrochloric acid and two volumes of water, 1 mL
petroleum spirit (boiling range, 400 to 600 ) , swirl, add 1 g of of strong iodine monochloride reagent and 6 mL of chloroform.
diatomaceous earth (Filtercel is suitable), swirl to mix Titrate with O.OIMpotassium iodate VS, running almost all the
completely, stopper the fiask and allow to stand at 20 0 to 22 0 required volume of titrant into the fiask in one portion.
for 16 hours. Mix the contents of the fiask thoroughly, filter Continue the titration, shaking the fiask vigorously for
with gentle suction through a sintered-glass filter (ISO 4793, 30 seconds after each addition of the titrant, until the
porosity grade 4, is suitable) and wash the residue with five chloroform is colourless. Repeat the operation without the
10 mL quantities of aromatic-free petroleum spirit (boiling range, extract; the difference between the titrations represents the
400 to 600 ) . Remove the solvent from the combined filtrate amount of potassium iodate required. Each mL of
and washings and evaporate to a volume of 1 to 2 mL. O.OIMpotassium iodate VS is equivalent to 5.7 mg of
Add 20 mL of O.5M ethanolic potassium hydroxide and boil pyrethrin 1.
under a refiux condenser for 45 minutes. Transfer the For pyrethrin 11
solution to a beaker and wash the fiask with sufficient hot Transfer the combined aqueous liquids reserved in the Assay
water, adding the washings to the beaker, to produce a total for pyrethrin I to a beaker, cover with a watch glass and
volume of 200 mL. Boil until the volume is reduced to evaporate to 50 mL within 35 to 45 minutes. Cool, washing
150 mL, cool rapidly and transfer the solution to a stoppered the underside of the watch glass with not more than 5 mL of
fiask, washing the beaker with three 20 mL quantities of water and adding the washings to the beaker. Filter through
water and transferring any gummy residue to the fiask. ab¡sorbent cotton into a separating funnel, washing with
Add 1 g of diatomaceous earth (Filtercel is suitable) and successive quantities of 10, 7.5, 7.5, 5 and 5 mL of water.
10 mL of barium chloride solution, swirl gently and add Saturate the aqueous liquid with sodium chloride, add 10 mL
sufficient water to produce 250 mL. Stopper the fiask, shake of hydrochloric acid and 50 mL of ether, shake for 1 minute,
vigorously until the separating liquid is clear and filter the allow to separate, and remove the lower layer. Repeat the
suspension through a filter paper (Whatman No. 1 is extraction successively with 50, 25 and 25 mL of ether. Wash
suitable). the combined ether extracts with three 10 mL quantities of a
For pyrethrin 1 saturated solution of sodium chloride and transfer the ether
Transfer 200 mL of the filtrate to a separating funnel, rinsing layer to a fiask with the aid of 10 mL of ether. Remove the
the measuring vessel with two 5 mL quantities of water, and bulk of the ether by distillation and remove the remainder
add 0.05 mL of phenolphthalein solution Rl. Neutralise the with a gentle current of air and dry the residue at 100 0 for
solution by the drop wise addition of hydrochloric acid and 10 minutes, removing any residual acid fumes with a gentle
add 1 mL of hydrochloric acid in excess. Add 5 mL of a current of airo Add 2 mL of ethanol (96%) previously
saturated solution of sodium chloride and 50 mL of aromatic- neutralised to phenolphthalein solution Rl and 0.05 mL of
free petroleum spirit (boiling range, 400 to 600 ) , shake vigorously phenolphthalein solution Rl, swirl to dissolve the residue, add
for 1 minute, allow to separate, remove and retain the lower 20 mL of carbon dioxide-free water and titrate rapidly with
layer. Filter the petroleum spirit extract through absorbent 0.02M sodium hydroxide VS until the colour changes to
cotton into a second separating funnel containing 10 mL of brownish pink and persists for 30 seconds, keeping the fiask
water. Retum the aqueous layer to the first separating funnel stoppered between additions of alkali. Repeat the operation
and repeat the extraction with 50 mL and then with 25 mL using the aqueous liquid reserved for the repeat operation in
of aromatic-free petroleum spirit (boiling range, 400 to 600 ) , the Assay for pyrethrin 1. The difference between the
reserving the aqueous layer for the assay of pyrethrin II, and titrations represents the volume of O.02M sodium hydroxide VS
filtering the petroleum spirit extracts through the same
Sulfadimidine Preparations 179·
DEFINITION
Sodium Ca1cium Edetate Intravenous Infusion for Veterinary Sulfadimidine Injection
Use is a sterile solution of Sodium Ca1cium Edetate. It is
prepared immediately before use by diluting Sterile Sodium Action and use
Ca1cium Edetate Concentrate for Veterinary Use with a Sulfonamide antibacterial.
suitable diluent in accordance with the manufacturer's
instructions. DEFINITION
The intravenous infusion complies with the requirements stated Sulfadimidine Injection is a sterile solution of sulfadimidine
under Parenteral Preparations and with the following requirement. sodium in Water for Injections free from dissolved airo It is
prepared by the interaction of Sulfadimidine and Sodium
LABELLING Hydroxide.
The quantity of active ingredient is stated in terms of the
The injection complies with the requirements stated under
equivalent amount of anhydrous sodium ca1cium edetate in a
Parenteral Preparations and with the following requirements.
suitable dose-volume.
Content of sulfadimidine sodium, C12H13N4Na02S
95. O to 105. O% of the stated amount.
STERILE SODIUM CALCIUM EDETATE
IDENTIFICATION
CONCENTRATE FOR VETERINARY USE
A. Acidify a volume containing 0.1 g of sulfadimidine sodium
DEFINITION with 6M acetic acid, filter, reserving me filtrate, wash the
Sterile Sodium Ca1cium Edetate Concentrate for Veterinary residue with water and dry at 105 0 • The infrared absorption
Use is a sterile solution of Sodium Ca1cium Edetate in Water spectrum of the residue, Appendix n A, is concordant with
for Injections containing the equivalent of 25.0% w/v of the reference spectrum of sulfadimidine (RSV 50).
anhydrous sodium ca1cium edetate. B. The residue obtained in test A yields the reaction
The concentrate complies with the requirements for Concentra tes characteristic of primary aromatic amines, Appendix VI,
for Injections or Infusions stated under Parenteral Preparations producing a bright orange-red precipitate.
and with the following requirements.
TESTS
Content of anhydrous sodium calcium edetate, Alkalinity
ClOH12CaN2Na20s pH, 10.0 to 11.0, Appendix V L.
22.5 to 27.5% w/v.
Colour of solution
CHARACTERISTICS An injection containing 1 g of sulfadimidine sodium in 3 mL
A colourless solution. is not more intensely coloured than reference solution Y4'
IDENTIFICATION Appendix IV B, Method 1.
A. Dilute 2.5 mL with 7.5 mL of water, make alkaline to Related substances
litmus paper with 5M ammonia and add 5 mL of a 2.5% w/v Carry out the method for thin-Iayer chromatography,
solution of ammonium oxalate. Not more than a trace of Appendix nI A, using silica gel H as the coating substance
precipitate is produced. and a mixture of 18 volumes of 10M ammonia and
B. To 10 mL add 2 mL of a 10% w/v solution of lead(Il) 90 volumes of butan-I-ol as the mobile phase. Apply
nitrate, shake and add 5 mL of dilute potassium iodide solution; separately to the plate 10 J.lL of each of the following
no yellow precipitate is produced. Make alkaline to litmus solutions. For solution (1) use the injection being examined
paper with 5M ammonia and add 5 mL of a 2.5% w/v solution diluted with water to contain 0.20% w/v of sulfadimidine
of ammonium oxalate; a white precipitate is produced. sodium. Solution (2) contains 0.0020% w/v of sulfanilamide
in a mixture of 1 volume of 13.5M ammonia and 9 volumes
C. Evaporate to dryness and ignite. The residue yields the 0
of ethanol (96%). After removal of the plate, heat it at 105
reactions characteristic of sodium salts and of calcium salts,
for 10 minutes and spray with a 0.1 % w/v solution of
Appendix VI.
4-dimethylaminobenzaldehyde in ethanol (96%) containing
TESTS 1% v/v of hydrochloric acid. Any secondary spot in the
Acidity or alkalinity chromatogram obtained with solution (1) is not more intense
pH, 6.5 to 8.0, Appendix V L. than the spot in the chromatogram obtained with solution (2)
(1%).
180 Sulfadoxine Preparations
Itntnunological Products
Veterinary Irnrnunosera 185
tested to establish that they are free from the list of agents
VETERINARV IMMUNOSERA relevant for the donor animals. It may be necessary to test
(Immunosera for Veterinary Use, the animals in quarantine for freedom from additional agents,
Ph Eur monograph 0030) depending on their known breeding and rearing history or
Veterinary Immunosera comply with the requirements of the any lack of information on their source.
European Pharmacopoe-ia monograph for Immunosera for Any routine or therapeutic medicinal treatment administered
Veterinary Use. These requirements are reproduced below. to the animals in quarantine or thereafter must be recorded.
The provisions of this monograph apply to the following IMMUNISING ANTIGEN
immunosera. The principIes described in the Production section of
Antitoxic sera Vaccines for veterinary use (0062) are applied to the
Clostridium Novyi Alpha Antitoxin* production of the immunogen. The antigen used is identified
and characterised. The starting materials used for antigen
Clostridium Perfringens Antitoxins preparation mustbe controlled to minimise the risk of
(incorporating Clostridium Perfringens Beta Antitoxin* contamination with extraneous agents. The antigen may be
and Clostridium Perfringens Epsilon Antitoxin*) blended with a suitable adjuvant. The immunogen is
Clostridium TetanLAntitoxin* produced on a batch basis. The batches must be prepared
and tested in such a manner that assures that each batch will
*Monographs oi the European Phannacopoeia be equally safe and free from extraneous agents and will
PhEuf _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ produce a satisfactory, consistent immune response.
IMMUNISATION
DEFINITION
The donor animals are immunised according to a defined
Immunosera for veterinary use are preparations containing schedule. For each animal, the details of the dose of
immunoglobulins, purified immunoglobulins or immunising antigen, route of administration and dates of
immunoglobulin fragments obtained from serum or plasma administration are recorded. Animals are kept under general
of immunised animals. They may be preparations of crude health surveillance and the development of specific antibodies
polyc1onal antisera or purified preparations. are monitored at appropriate stages of the immunisation
The immunoglobulins or immunoglobulin fragments have process.
the power of specifically neutralising the antigen used for
COLLECTION OF BLOOD OR PLASMA
immunisation. The antigens inc1ude microbial or other
Animals are thoroughly examined before each collection.
toxins, bacteria! and viral antigens, venoms of snakes and
Only healthy animals may be used as a donor animal.
hormones. The preparation is intended for parenteral
Collection of blood is made by venepuncture or
administration to provide passive immunity.
plasmapheresis. The puncture area is shaved, c1eaned and
PRODUCTION disinfected. The method of collection and the volume to be
GENERAL PROVISIONS collected on each occasion are specified. The blood or
Immunosera are obtained from the serum or plasma of plasma is collected in such a manner as to maintain sterility
healthy animals immunised by administration of one or more of the producto If the serum or plasma is stored before
suitable antigens. further processing, precautions are taken to avoid microbial
The production method shall have been shown to yield contamination.
consistently batches of immunosera of acceptable safety The blood or plasma collection is conducted at a site
(5.2.6) and efficacy (5.2.7). separate from the area where the animals are kept or bred
DONOR ANIMAL S and the area where the immunoserum is further processed.
The animals used are exc1usively reserved for production of Clear criteria are established for determining the time
immunoserum. They are maintained under conditions between immunisation and first collection of blood or plasma
protecting them from the introduction of disease, as far as as well as the time between subsequent collections and the
possible. The donor animals, and any animals in contact with length of time over which collections are made. The criteria
them, are tested and shown 10 be free from a defined list of applied must take into account the effect of the collections
infectious agents and re-tested at suitable intervals. The list on the health and welfare of the animal as well as the effect
of agents for testing inc1udes not only those agents that are on the consistency of production of batches of the finished
relevant to the donor animal, but also those that are relevant product, over time.
to the recipient target species for the producto Where the The rate of c1earance of any residues that may arise from the
donor animals have not been demonstrated to be free from a immunising antigen or medication given needs to be taken
relevant pathogen, a justification must be provided and a into account. In the case of the risk of residues from
validated inactivation or purification procedure must be chemical substances, consideration could be given to the
inc1uded in the manufacturing procedure. The feed originates inc1usion of a withdrawal period for the finished producto
from a controlled source. Where the donor animals are If the immunising agent consists of a live organism, the time
chickens, use chickens from a flock free from specified between immunisation and collection may need to take into
pathogens (5.2.2). Where applicable for the species used, account the time required for the donor to eliminate the
measures are taken to avoid contamination with agents of immunogen, particularly if any residual live organisms might
transmissible spongiform encephalopathies. be harmful to the recipient.
As far as possible, animals being introduced into the herd are PREPARATION OF THE FINISHED PRODUCT
from a known source and have a known breeding and rearing Several single plasma or serum collections from one or more
history. The introduction of animals into the herd follows animals may be pooled to form a bulk for preparation of a
specified procedures, inc1uding defined quarantine measures. batch. The number of collections that may be used to
During the quarantine period the animals are observed and produce a bulk and the size of the bulk are defined. Where
186 Veterinary Irnrnunosera
pooling is not undertaken, the production procedure must be inactivation of extraneous agents, the test for extraneous
very carefully controlled to ensure that the consistency of the agents can be omitted on that product with the agreement ()f
product is satisfactory. the competent authority. If a product is treated by a
The active substance is subjected to a purification andlor validated procedure for inactivation of mycoplasmas, the test
inactivation procedure unless omission of such a step has for mycoplasmas can be omitted on that product with the
been justifed and agreed with the competent authority. agreement of the competent authority.
The procedure applied must have been validated and be Only a batch that complies with each of the relevant
shown not to adversely impair the biological activity of the requirements given below under Identification, Tests and
producto The validation studies must address the ability of Potency andlor in the relevant specific monograph may be
the procedure to inactivate or remove any potential released for use. With the agreement of the competent
contaminants such as pathogens that could be transmitted authority, certain tests may be omitted where in-process tests
from the donor to the recipient target species and infectious give an equal or better guarantee that the batch would
agents such as those that cause ubiquitous infections in the comply or where alternative tests validated with respect to
donor animals and cannot be readily eliminated from these the Pharmacopoeia method have been carried out.
donor animals. Certain tests, e.g. for antimicrobial preservatives, for foreign
For purified immunosera, the globulins containing the proteins and for albumin, may be carried out by the
immune substances may be obtained from the crude manufacturer on the final bulk rather than on the batch,
immunoserum by enzyme treatment and fractional batches or sub-batches of finished product prepared from it.
precipitation or by other suitable chemical or physical In sorne circumstances, e.g. when collections are made into
methods plasmapheresis bags and each one is, essentially, a batch,
Antimicrobial preservatives pools of samples may be tested, with the agreement of the
Antimicrobial preservatives are used to prevent spoilage or competent authority.
adverse effects caused by microbial contamination occurring It is recognised that, in accordance with General Notices
during use of a producto Antimicrobial preservatives are not (section 1.1. General statements), for an established
included in freeze-dried products but, if justified, taking into antiserum the routine application of the safety test will be
account the maximum recommended period of use after waived by the competent authority in the interests of animal
reconstitution, they may be included in the diluent for welfare when a sufficient number of consecutive batches have
multidose freeze-dried products. For single-dose liquid been produced and found to comply with this test, thus
preparations, inclusion of antimicrobial preservatives is not demonstrating consistency of the manufacturing process.
normally acceptable, but may be acceptable, for example Significant changes to the manufacturing process may require
where the same product is filled in single-dose and multidose resumption of routine testing to re-establish consistency.
containers and is for use in non-food producing species. The number of consecutive batches to be tested depends on
For multidose liquid preparations, the need for effective a number of factors such as the type of antiserum, the
antimicrobial preservation is evaluated taking into account frequency of production of batches, and experience with the
likely contamination during use and the maximum immunoserum during developmental safety testing and
recommended period of use after broaching of the container. during application of the batch safety test. Without prejudice
During development studies the effectiveness of the to the decision of the competent authority in the light of
antimicrobial preservative throughout the period of validity information available for a given antiserum, testing of
shall be demonstrated to the satisfaction of the competent 10 consecutive batches is likely to be sufficient for the
authority. majority of products. For products with an inherent safety
risk, it may be necessary to continue to conduct the safety
The efficacy of the antimicrobial preservative is evaluated as
test on each batch.
described in chapter 5.1.3; for a multidose preparation,
additional samples are taken, to monitor the effect of the Animal tests In accordance with the provisions of the
antimicrobial preservative over the proposed in-use shelf-life. Eu.ropean Convention for the Protection of Vertebrate
If neither the A criteria nor the B criteria can be met, then in Animals Used for Experimental and Other Scientific
justified cases the following criteria are applied to antisera for Purposes, tests must be carried out in such a way as to use
veterinary use: bacteria, no increase at 24 h and 7 days, 3 log the minimum number of animals and to cause the least pain,
reduction at 14 days, no increase at 28 days; fungi, no suffering, distress or lasting harm. The criteria for judging
increase at 14 days and 28 days. tests in monographs must be applied in the light of this.
For example, if it is indicated that an animal is considered to
Addition of antibiotics as antimicrobial preservative is not
show positive, infected etc. when typical clinical signs occur
acceptable.
then as soon as sufficient indication of a positive result is
U nless otherwise prescribed in the monograph, the final bulk obtained the animal in question shall be either euthanised or
is distributed aseptically into sterile, tamper-proof containers given suitable treatment to prevent unnecessary suffering.
which are then closed so as to exclude contamination. In accordance with the General Notices, alternative test
The preparation may be freeze-dried. methods may be used to demonstrate compliance with the
In-process tests Suitable tests are carried out in-process, such monogra'ph and the use of such tests is particularly
as on samples from collections before pooling to form a bulk. encouraged when this leads to replacement or reduction of
BATCH TESTS animal use or reduction of suffering.
The tests that are necessary to demonstrate the suitability of pH (2:2.3)
a batch of a product will vary and are influenced by a The pH of crude and purified immunosera is shown to be
number of factors, including the detailed method of within the limits set for the products.
production. The tests to be conducted by the manufacturer
on a particular product are agreed with the competent
authority. If a product is treated by a validated procedure for
Veterinary Immunosera 187
Determination of test dose of toxin 0.1 IU in 0.2 mL. Repeat the determination at least once
Prepare a solution of the reference preparation in a suitable and calculate the average of aH valid estimates. Estimates are
liquid so that it contains 1 IU/rnL. Prepare a solution of the valid only if the reference preparation gives a result within
test toxin in a suitable liquid so that 1 mL contains a 20 per cent of the expected value.
precise1y known amount such as 1 mg. Prepare mixtures of The confidence limits (P = 0.95) have been estimated to be:
the solution of the reference preparation and the solution of -- 85 per cent and 114 per cent when 2 animals per dose
the test toxin such that each mixture contains 1.0 mL of the are used;
solution of the reference preparation (1 IU), one of a series -- 91.5 per cent and 109 per cent when 4 animals per dose
of graded volumes of the solution of the test toxin and are used;
sufficient of a suitable liquid to bring the total volume to - 93 per cent and 108 per cent when 6 animals per dose
2.0 mL. AHow the mixtures to stand at room tempeflature for are used.
60 mino Using not fewer than 2 mice, each weighing 17-22 g, The potency of the finished product is expressed in
for each mixture, inject adose of 0.2 mL intramuscular1y or Intemational Units per millilitre and is shown to be not les s
subcutaneously into each mouse. Observe the mice for 72 h. than the minimum number stated on the label.
If aH the mice die, the amount of toxin present in 0.2 mL of ___________________________________________ PhE~
only if the reference preparation gives a result within The potency of the finished product is expressed in
20 per cent of the expected value. Intemational Units per millilitre and is shown to be not less
The confidence limits (P = 0.95) have been estimated to be: than the minimum number stated on the label.
~ 85 per cent and 114 per cent when 2 animals per dose IDENTIFICATION
are used; The antitoxin is shown, by a suitable immunochemical
- 91.5 per cent and 109 per cent when 4 animals per dose method (2.7.1), to react specifically with the epsilon toxin
are used; formed by C. perjringens.
- 93 per cent and 108 per cent when 6 animals per dose
are used. POTENCY
The potency of the finished product is expressed in The potency of Clostridium perfringens epsilon antitoxin is
Intemational Units per millilitre and is shown to be not less determined by comparing the dos e necessary to protect mice
than the minimum number stated on the label. or other suitable animals against the toxic effects of a fixed
___________________________________________ PhE~
dose of C. perjringens epsilon toxin with the quantity of a
reference preparation of Clostridium perfringens epsilon
antitoxin, calibrated in International Units, necessary to give
the same protection. F or this comparison, a suitable
preparation of C. perjringens epsilon toxin for use as a test
toxin is required. The dos e of the test toxin is determined in
Clostridium Perfringens Epsilon relation to the reference preparation, the potency of the
Antitoxin antitoxin to be examined is determined in relation to the
(Clostridium Perjringens Epsilon Antitoxin lor reference preparation using the test toxin.
Veterinary Use, Ph Eur monograph 0341) Preparation of test toxin
~E~ ___________________________________________ Prepare the test toxin from a sterile filtrate of an earlY culture
in liquid medium of C. perjringens type D and dry by a
DEFINITION suitable method. Select the test toxin by determining for
Clostridium perfringens epsilon antitoxin for veterinary use is mice the L+/10 dose and the LD so, the observation period
a preparation containing the globulins that have the power of being 72 h. A suitable epsilon toxin contains not les s than
specifically neutralising the epsilon toxin formed by one L+/10 dose in 0.005 mg and not less than 20 LD so in
Clostridium perjringens type D. It consists of the serum or a each L+/10 dose.
preparation obtained from the serum of animals immunised
Determination of test dose of toxin
against C. perjringens epsilon toxin.
Prepare a solution of the reference preparation in a suitable
PRODUCTION liquid so that it contains 0.5 IU/mL. Prepare a solution of
CHOICE OF COMPOSITION the test toxin in a suitable liquid so that 1 mL contains a
The antitoxin is shown to be satisfactory with respect to precisely known amount such as 1 mg. Prepare mixtures of
safety (5.2.6) and efficacy (5.2.7). For the latter, it shall be the solution of the reference preparation and the solution of
demonstrated, for each target species, that the product, when the test toxin such that each mixture contains 2.0 mL of the
administered at the minimum recommended dos e and solution of the reference preparation (1 IV), one of a series
according to the recommended schedule(s), provides a of graded volumes of the solution of the test toxin and
response or responses consistent with the claims made for the sufficient of a suitable liquid to bring the total volume to
producto 5.0 mL. Allow the mixtures to stand at room temperature for
Batch potency test 30 mino Using not fewer than 2 mice, each weighing 17-22 g,
The test described under Potency is not necessarily carried for each mixture, inject adose of 0.5 mL intravenously or
out for routine testing of batches of antitoxin. It is carried intraperitoneally into each mouse. Observe the mice for 72 h.
out on one or more occasions as decided by or with the If all the mice die, the amount of toxin present in 0.5 mL of
agreement of the competent authority. Where the test is not the mixture is in excess of the test dose. If none of the mice
carried out, a suitable validated altemative test is carried out, die, the amount of toxin present in 0.5 mL of the mixture is
the criteria for acceptance being set with reference to a batch less than the test dose. Prepare similar fresh mixtures such
of antitoxin that has given satisfactory results in the test that 5.0 mL of each mixture contains 2.0 mL of the solution
described under Potency and that has been shown to be of the reference preparation (1 IV) and 1 of a series of
satisfactory with respect to immunogenicity in the target graded volumes of the solution of the test toxin, separated
species. The following test may be used after a satisfactory from each other by steps of not more than 20 per cent and
correlation with the test described under Potency has been covering the expected end-point. Allow the mixtures to stand
established. at room temperature for 30 mino Using not fewer than
2 mice for each mixture, inject adose of 0.5 mL
Determine the level of antibodies against C. perjringens
intravenously or intraperitoneally into each mouse. Observe
epsilon toxin in the batch of antitoxin using a suitable
the mice for 72 h. Repeat the determination at least once and
method such as an immunochemical method (2.7.1) or
combine the results of the separate tests that have been made
neutralisation in cell cultures. Use a homologous reference
with mixtures of the same composition so that a series of
serum calibrated in International Units of Clostridium
totals is obtained, each total representing the mortality due to
perfringens epsilon antitoxin.
a mixture of a given composition. The test dose of the toxin
The Intemational Unit is the specific neutralising activity for is the amount present in 0.5 mL of that mixture which
C. perjringens epsilon toxin contained in a stated amount of causes the death of one half of the total number of mice
the International Standard, which consists of a quantity of injected with it.
dried immune horse serum. The equivalence in Intemational
Units of the Intemational Standard is stated by the World
Health Organisation.
192 Veterinary Antisera
at least once and calculate the test dose as the mean of the Intemational Standard which consists of a quantity of dried
different tests. The test dos e of the toxin is the amount immune horse serum. The equivalence in International Units
present in that mixture which causes tetanic paralysis in one of the Intemational Standard is stated by the World Health
half of the total number of animals injected with it. Organisation.
Determination of the neutralising ability of the The potency of the finished product is expressed in
antitoxin to be examined Intemational Units per millilitre and is shown to be not less
Preliminary test Measure or weigh a quantity of the test than the minimum number stated on the label.
toxin and dilute with or dissolve in a suitable liquid so /that _____________________________________________ PhE~
maintained for each master seed loto Each master seed lot is contaminants of the seed virus and is shown to be free of any
assigned a specific code for identification purposes. non-specific inhibiting effects on the ability of virus es to
2-1-3-1-2 Propagation. The minimum and maximum infect and propagate within cells (or eggs, where applicable).
number of sub cultures of each master seed lot prior to the If such a serum cannot be obtained, other methods are used
production stage are specified. The methods used for the to remove or neutralise the seed virus specifidllly.
preparation of seed cultures, preparation of suspensions for If the seed lot virus would interfere with the conduct and
seeding, techniques for inoculation of seeds, titre and sensitivíty of a test for extraneous virus es, a sample of the
concentration of inocula and the media used, are master seed lot is treated with a minimum amount of the
documented. It shall be demonstrated that the characteristics monoclonal or polyclonal antibody so that the vaccine virus
of the seed material (for example, dissociation or antigenicity) is neutralised as far as possible or removed. The final virus-
are not changed by these subcultures. The conditions under serum mixture shall, if possible, contain at least the virus
which each seed lot is stored are documented. content of 10 doses ofvaccine per 0.1 mL for avían vaccines
2-1-3-1-3 Identity and purity. Each master seed lot is shown and per millilitre for other vaccines. For avían vaccines, the
to contain only the species and strain of bacterium stated. testing to be carried out on seed lots is given in chapter
A brief description of the method of identifying each strain 2.6.24. For mammalian vaccines, the seed lot or the mixture
by biochemical, serological and morphological characteristics of seed lot and antiserum is tested for freedom from
and distinguishing it as far as possible from related strains is extraneous agents as follows.
recorded, as is also the method of determining the purity of The mixture is inoculated onto cultures of at least 70 cm2 of
the strain. If the master seed lot is shown to contain living the required cell types. The cultures may be inoculated at
organisms of any kind other than the species and strain any suitable stage of growth up to 70 per cent confiuency.
stated, then it is unsuitable for vaccine production. At least 1 monolayer of each type must be retained as a
2-1-3-2 Virus seed lots control. The cultures must be monitored daily for a week.
2-1-3-2-1 General requirements. Virus es used in manufacture At the end of this period the cultures are freeze thawed
are handled in a seed-Iot system. Each master seed lot is 3 times, centrifuged to remove cell debris and re-inoculated
tested as described below. A record of the origin, date of onto the same cell type as above. This is repeated twice.
isolation, passage history (including purification and The final passage must produce sufficient cells in appropriate
characterisation procedures) and storage conditions is ves seIs to carry out the tests below.
maintained for each seed loto Each master seed lot is assigned Cytopathic and haemadsorbing agents are tested for using
a specific code for identification purposes. Production of the methods described in the relevant sections on testing cell
vaccine is not normally undertaken using virus more than cultures (5.2.4) and techniques such as immuno-fiuorescence
5 passages from the master seed loto In the tests on the are used for detection of specific contaminants for the tests in
master seed lot described below, the organisms used are not cell cultures. The master seed lot is inoculated onto:
normally more than 5 passages from the master seed lot at - primary cells of the species of origin of the virus;
the start of the tests, unless otherwise indicated. - cells sensitive to viruses pathogenic for the species for
Where the master seed lot is contained within a permanently which the vaccine is intended;
infected master cell seed, the following tests are carried out - cells sensitive to pestiviruses.
on an appropriate volume of virus from disrupted master cell If the master seed lot is shown to contain living organisms of
seed. Where relevant tests have been carried out on disrupted any kind, other than the virus of the species and strain stated,
cells to validate the suitability of the master cell seed, these or foreign viral antigens, then it is unsuitable for vaccine
tests need not be repeated. production.
2-1-3-2-2 Propagation. The master seed lot and all 2-1-4 Inactivation
subsequent passages are propagated on cells, on embryonated Inactivated vaccines are subjected to a validated inactivation
eggs or in animal s that have been shown to be suitable for procedure. The testing of the inactivation kinetics described
vaccine production (se e above), and, where applicable, using beiow is carried out once for a given production process.
substances of animal origin that meet the requirements The rest of this section applies to each production runo When
prescribed in chapter 5.2.5. conducting tests for inactivation, it is essential to take
2-1-3-2-3 Identification. A suitable method to identify the account of the possibility that under the conditions of
vaccine strain and to distinguish it as far as possible from manufacture, organisms may be physically protected from
related strains must be used. inactivant.
2-1-3-2-4 Bacteria and fungí. The master seed lot complies 2-1-4-1 Inactivation kinetics The inactivating agent and the
with the test for sterility (2.6.1). inactivation procedure shall be shown, underconditions of
manufacture, to inactivate the vaccine micro-organismo
2-1-3-2-5 Mycoplasmas (2.6.7). The master seed lot
Adequate data on inactivation kinetics shall be obtained.
complies with the test for mycoplasmas (culture method and
Normally, the time required for inactivation shall be not
indicator cell culture method).
more than 67 per cent of the duration of the inactivation
2-1-3-2-6 Absence of extraneous viruses. Monographs may process.¡
contain requirements for freedom from extraneous agents,
2-1-4-2 Aziridine If an aziridine compound is used as the
otherwise the requirements stated below apply.
inactivating agent then it shall be shown that no inactivating
Preparations of monoclonal or polyclonal antibodies agent r~mains at the end of the inactivation procedure. This
containing high levels of neutralising antibody to the virus of may be accomplished by neutralising the inactivating agent
the seed lot are made on a batch basis, using antigen that is with thiosulfate and demonstrating residual thiosulfate in the
not derived from any passage level of the virus isolate giving inactivated harvest at the completion of the inactivation
rise to the master seed virus. Each batch of serum is procedure.
maintained at 56 oC for 30 min to inactivate complemento
Each batch is shown to be free of antibodies to potential
Veterinary Vaccines 197
During development studies the effectiveness of the For inactivated vaccines, if the test described under Potency
antimicrobial preservative throughout the period of validity is not used for routine testing, a batch potency test is
shall be demonstrated to the satisfaction of the competent established during development. The aim of the batch
authority. potency test is to ensure that each batch of vaccine would, if
The efficacy of the antimicrobial preservative is evaluated as tested, comply with the test described under Potency and
described in chapter 5.1.3 and in addition samples are tested Immunogenicity. The acceptance criteria for the batch
at suitable intervals over the proposed in-use shelf-life. potency test are therefore established by correlation with the
If neither the A criteria nor the B criteria can be met, then in test described under Potency. Where a batch potency test is
justified cases the following criteria are applied to vaccines for described in a monograph, this is given as an example of a
veterinary use: bacteria, no increase from 24 h to 7 days, test that is considered suitable, after establishment of
3 log reduction at 14 days, no increase at 28 days; fungi, no correlation with the potency test; other test models can also
increase at 14 days and 28 days. be used.
Addition of antib)otics as antimicrobial preservative is 2-3-3 Batch
generally not acceptable. Unless otherwise prescribed in the monograph, the final bulk
vaccine is distributed aseptically into sterile, tamper-proof
2-2-6 Stability
containers which are then closed so as to exclude
Evidence of stability is obtained to justify the proposed
contamination.
period of validity. This evidence takes the form of the results
of virus titrations, bacterial counts or potency tests carried Only a batch that complies with each of the requirements
out at regular intervals until 3 months beyond the end of the given below under 3 Batch tests or in the relevant individual
shelf life on not fewer than 3 representative consecutive monograph may be released for use. With the agreement of
batches of vaccine kept under recommended storage the competent authority, certain of the batch tests may be
conditions together with results from studies of moisture omitted where in-process tests give an equal or better
content (for freeze-dried products), physical tests on the guarantee that the batch would comply or where altemative
adjuvant, chemical tests on substances such as the adjuvant tests validated with respect to the Pharmacopoeia method
constituents and preservatives, and pH, as appropriate. have been carried out.
Where applicable, studies on the stability of the reconstituted The identification test can often be conveniently combined
vaccine are carried out, using the product reconstituted in with the batch potency test to avoid unnecessary use of
accordance with the proposed recommendations. animals. For a given vaccine, a validated in vitro test can be
used to avoid the unnecessary use of animals.
2-3 MANUFACTURER'S TESTS
Certain tests may be carried out on the final bulk vaccine It is recognised that, in accordance with the General Notices
rather than on the batch or batches prepared from it; such (section 1.1. General statements), for an established vaccine
tests include those for antimicrobial preservatives, free the routine application of the safety test will be waived by the
formaldehyde and the potency determination for inactivated competent authority in the interests of animal welfare when a
vaccines. sufficient number of consecutive production batches have
been produced and found to comply with the test, thus
2-3-1 Residuallive viruslbacteria andlor detoxification demonstrating consistency of the manufacturing process.
testing Significant changes to the manufacturing process may require
For inactivated vaccines, where the auxiliary substances resumption of routine testing to re-establish consistency.
would interfere with a test for inactivation and/or The number of consecutive batches to be tested depends on
detoxification, a test for inactivation or detoxification is a number of factors such as the type of vaccine, the
carried out during preparation of the final bulk, after the frequency of production of batches and experience with the
different batches of antigen have been combined but before vaccine during development safety testing and during
addition of auxiliary substances; the test for inactivation or application of the batch safety test. Without prejudice to the
detoxification may then be omitted on the final bulk and the decision of the competent authority in the light of
batch. information available for a given vaccine, testing of
Where there is a risk of reversion to toxicity, the test for 10 consecutive batches is likely to be sufficient for most
detoxification performed at the latest stage of the production products. For products with an inherent safety risk, it may be
process at which the sensitivity of the test is not necessary to continue to conduct the safety test on each
compromised (e.g. after the different batches of antigen have batch.
been combined but before the addition of auxiliary 2-3-3-1 Animal tests In accordance with the provisions of
substances) is important to demonstrate a lack of reversion to the European Convention for me Protection of Vertebrate
toxicity.
Animals Used for Experimental and Other.Scientific
2-3-2 Batch potency test Purposes, tests must be carried out in such a way as to use
For most vaccines, the tests cited under Potency or the minimum number of animals and to cause the least pain,
Irnmunogenicity are not suitable for the routine testing of suffering, distress or lasting harm. The criteria for judging
batches. tests in monographs must be applied in light of this.
For live vaccines, the minimum acceptable virus titre or For examp,le, if it is indicated that an animal is considered to
bacterial count that gives satisfactory results in the potency be positive, infected etc. when typical clinical signs occur
test and other efficacy studies is established during then as soon as it is clear that result will not be affected the
development. For routine testing it must be demonstrated for animal in question shall be either euthanised or given suitable
each batch that the titre or count at release is such that at the treatment to prevent unnecessary suffering. In accordance
end of the period of validity, in the light of stability studies, with the General Notices, alternative test methods may be
the vaccine, stored in the recommended conditions, will used to demonstrate compliance with the monograph and the
Contain not les s than the minimum acceptable virus titre or use of such tests is particularly encouraged when this leads to
bacterial count determined during development studies.
Veterinary Vaccines 199
replacement or reduction of animal use or reduction of 2) extensive testing of seed lots and cell seed for extraneous
suffering. agents;
2-3-3-2 Physical tests A vaccine with an oily adjuvant is 3) requirements for SPF flocks used for providing substrates
tested for viscosity by a suitable method and shown to be for vaccine production;
within the limits set for the producto The stability of the 4) testing of substances of animal origin, which must,
emulsion shall be demonstrated. wherever possible, undergo an inactivation procedure;
2-3-3-3 Chemical tests Tests for the concentrations of 5) for live vaccines, testing of the final product for infectious
appropriate substances such as aluminium and preservatives extraneous agents; such tests are less extensive than those
are carried out to show that these are within the limits set for carried out at earlier stages because of the guarantees given
the producto by in-process testing.
2-3-3-4 pH The pH of liquid products and diluents is In case of doubt, the tests intended for the seed lot of a live
measured and shown to be within the limits set for the vaccine may also be applied to the final producto If an
product. extraneous agentis found in such a test, the vaccine does not
2-3-3-5 Water Where applicable, the freeze-drying process comply with the monograph.
is checked by a determination of water and shown to be Avian live viral vaccines comply with the tests for extraneous
within the limits set for the producto agents in batches of finished product (2.6.25).
3 BATCH TESTS 3-6 Mycoplasmas (2.6.7)
The monographs also indica te tests to be carried out on each Live viral vaccines comply with the test for mycoplasmas
partzcular vaccine. (culture method).
All hen eggs, chickens and chicken cell cultures for use in 3-7 Safety
quality control tests shall be derived from an SPF flock In general, 2 doses of an inactivated vaccine ami/or 10 doses
(5.2.2). of a live vaccine are injected by a recommended route.
3-1 Identification It may be necessary to reduce the prescribed number of
For inactivated vaccines, the identification prescribed in doses under certain circumstances or amend the method of
monographs is usually an antibody induction test since this is re-constitution and injection, for example for a combined
applicable to all vaccines. vaccine, where it is difficult to reconstitute 10 dos es of the
live component in 2 doses of the inactivated component.
3-2 Formaldehyde
The animals are observed for the longest period stated in the
(2. 4.18j use Method B ij sodium metabisulfite has been used to
monographs. No abnormallocal or systemic reaction occurS.
neutralise excess formaldehyde)
Where several batches are prepared from the same final bulk,
Where formaldehyde has been used in the preparation, the the safety test is carried out on the first batch and then
concentration of free formaldehyde is not greater than omitted for further batches prepared from the same final
0.5 giL, unless a higher amount has been shown to be safe. bulk.
3-3 Phenol (2.5.15) During development studies, the type and degree of reactions
When the vaccine contains phenol, the concentration is not expected with the vaccine are defined in light of safety
greater than 5 gIL. testing. This definition is then used as part of the operating
3-4 Sterility (2.6.1) procedure for the batch safety test to evaluate acceptable and
Vaccines comply with the test for sterility. Where the volume unacceptable reactions.
of liquid in a container is greater than 100 mL, the method The immune status of animals to be used for the safety test
of membrane filtration is used wherever possible. Where the is specified in the individual monograph. F or most
method of membrane filtration cannot be used, the method monographs, one of the 3 following categories is specified:
of direct inoculation may be used. Where the volume of 1) the animal s must be free from antibodies against the
liquid in each container is at least 20 mL, the minimum viruslbacteriumltoxin etc. contained in the vaccine;
volume to be used for each culture medium is 10 per cent of
2) the animals are preferably free from antibodies but
the contents or 5 mL, whichever is less. The appropriate
animals with a low level of antibody may be used as long as
number of items to be tested (2.6.1) is 1 per cent of the
the animals have not been vaccinated and the administration
batch with a minimum of 4 and a maximum of 10.
of the vaccine does not cause an anamnestic response;
For live bacterial and for live fungal vaccines, the absence of
3) the animals must not have been vaccinated against the
micro-organisms other than the vaccine strain is
disease that the vaccine is intended to prevent.
demonstrated by suitable methods such as microscopic
examination and inoculation of suitable media. As a general rule, category 1 is specified for live vaccines.
For other vaccines, category 2 is usually specified but where
For avian live viral vaccines, for non-parenteral use only, the
most animal s available for use in tests would comply with
requirement for sterility is usually replaced by requirements
category 1, this may be specified for inactivated vaccinesalso.
for absence of pathogenic micro-organisms and for a
Category 3 is specified for sorne inacfIvated vaccines where
maximum of 1 non-pathogenic micro-organism per dose.
determination of antibodies prior to testing is unnecessary or
3-5 Extraneous agents impractical. For poultry vaccines, as a general rule the use of
Monographs prescribe a set of measures that, taken together, SPF birds is specified.
give an acceptable degree of assurance that the final product
For avian vaccines, the safety test is generally carried out
does not contain infectious extraneous agents. These
using 10 SPF chickens (5.2.2), except that for vaccines not
mea sures include:
recommended for use in chickens it is carried out using 10
1) production within a seed-lot system and a cell-seed birds of one of the species for which the vaccine is
system, wherever possible; recommended, the birds being free from antibodies against
200 Veterinary Vaccines
The vaccine complies with the requirements of the test Ph Eur monograph 0441)
mentioned under Immunogenicity (section 2-2-1) when PhEw ____________________________________________
administered by a recommended route and method.
1 DEFINITION
Expiry date Unless otherwise stated, the expiry date is Anthrax spore vaccine (live) for veterinary use is a
calculated from the beginning of the virus titration or preparation of live spores of a suitable attenuated,
bacterial count (for live vaccines) or the beginning of the non-capsulated strain of Bacillus anthracis. This monograph
potency test (for other vaccines). For combined vaccines, the applies to vaccines intended for the active immunisation of
expiry date is tha~of the component which expires first. animals against disease caused by B. anthracis.
For vaccines stored by the manufacturer at a temperature
lower than that stated on the label, the stability for the entire 2 PRODUCTION
storage period is demonstrated by an appropriate study. 2-1 PREPARATION OF THE VACCINE
The expiry date is then calculated from the date that the B. anthracis is grown in an appropriate medium. At the end
vaccine is stored in the conditions stated on the label. of growth the spores are suspended in a stabilising solution
The expiry date applies to vaccines stored in the prescribed and counted. The vaccine may be adjuvanted.
conditions. 2-2 CHOICE OF VACCINE STRAIN
The strain used is:
4STORAGE
- not lethal to the guinea-pig or the mouse,
Store protected from light at a temperature of 5 ± 3 oC,
- or lethal to the guinea-pig but not to the rabbit,
unless otherwise indicated. Liquid preparations are not to be
- or lethal to sorne rabbits.
allowed to freeze, unless otherwise indicated.
The vaccine strain is shown to be satisfactory with respect to
5 LABELLING safety (5.2.6) and efficacy (5.2.7) for the animals for which it
The label states: is intended.
-- that the preparation is for veterinary use; The following test for Immunogenicity (2-2-1) may be used
-- the volume of the preparation and the number of doses in during the demonstration of efficacy. ~
the container;
-- the route of administration; 2-2-1 Immunogenicity
-- the type or types of bacteria (and where applicable the For a strain of B. anthracis which is not lethal to the guinea-
antigenic components) or viruses used and for live pig or the mouse, the test may be carried out in guinea-pigs.
vaccines the minimum and the maximum number of live For a strain which is lethal to the guinea-pig but not to the
bacteria or the minimum and the maximum virus titre; rabbit, the test may be carried out in rabbits. For a strain
-- where applicable, for inactivated vaccines, the minimum which is lethal to sorne rabbits, carry out the test in sheep.
potency in International Units; If the test is carried out in guinea-pigs or in rabbits, use not
-- where applicable, the name and amount of antimicrobial fewer than 13 healthy animals (group a). Inject by the
preservative or other substance added to the vaccine; subcutaneous or the intradermal route into each of not fewer
-- the name of any substance that may cause an adverse than 10 animals 1/10th of the smallest dose to be
reaction; recommended for sheep. Maintain not fewer than 3 animals
-- for freeze-dried vaccines: of the same species and the same origin as controls. Observe
- the name or composition and the volume of the the animals at least daily for 21 days. If more than 2 animals
reconstituting liquid to be added; die from non-specific causes, repeat the test.
- the period within which the vaccine is to be used after If the test is carried out in sheep, use not fewer than
reconstitution; 8 healthy sheep (group b). Vaccinate by the subcutaneous or
-- for vaccines with an oily adjuvant, that if the vaccine is the intradermal route each of not fewer than 5 sheep 1/10 of
accidentally injected into man, urgent medical attention is the smallest dose of the vaccine stated on the label for sheep.
necessary; Maintain not fewer than 3 sheep of the same origin as
- the animal species for which the vaccine is intended; controls. Observe the sheep at least daily for 21 days.
-- the indications for the vaccine; Challenge each vaccinated animal of group (a) or group (b)
-- the instructions for use; by a subcutaneous route with at least 100 MLD, and
any contra-indications to the use of the product inc1uding challenge each control animal by a SUbcutlineous route with
any required warning on the dangers of administration of at least 10 MLD of a strain of B. anthracis pathogenic for the
an overdose; species of animal used in the test. Observe all the animals at
-- the doses recommended for different species. least daily for 10 days after challenge.
_________________________________ ~ _________ ~Ew
3-2 Bacteria and fungi intended. The following tests for safety (section 2-3-1) and
Carry out the test by microscopic examination and by irnmunogenicity (section 2-3-2) may be used during the
inoculation of suitable media. The vaccine does not contain demonstration of safety and efficacy.
contaminating bacteria and fungi. 2-3-1· Safety.
3-3 Safety 2-3-1-1 Labaratory tests Carry out the tests for each route
Carry out the test on one of the species of animals for which and method of administration to be recommended for
the vaccine is intended. If the vaccine is intended for several vaccination and where applicable, in pigs of each category for
species, including goat, carry out the test on goats. which the vaccine is intended (sows, fattening pigs). Use a
U se 2 animals of the minimum age recommended for batch of vaccine containing not less than the maximum
vaccination and that do not have antibodies against B. potency that may be expected in a batch of vaccine.
anthracis. Administer by the subcutaneous or the intrladermal 2-3-1-1-1. General safety. For each test, use not fewer than
route to each animal twice the dose stated on the label for 10 pigs that do not have antibodies against Aujeszky's disease
the species used. Observe the animals at least daily for virus or against a fraction of the virus. Administer to each pig
14 days. a double dose of the vaccine, then one dose after 14 days.
The vaccine complies with the test if no animal shows Observe the pigs at least daily until 14 days after the last
notable signs of dis.ease or dies from causes attributable to administration.
the vaccine. A local reaction may occur at the site of The vaccine complies with the test if no pig shows abnormal
injection. The severity of the local reaction may vary local or systemic reactions or dies from causes attributable to
according to the strain of the spores and the adjuvants used the vaccine during the 28 days of the test.
in the preparation, but necrosis does not occur. 2-3-1-1-2. Safety in pregnant sows. If the vaccine is intended
3-4 Live spore for use in pregnant sows, use for the test not fewer than 10
Make a count of live spores by plate count. The vaccine pregnant sows at the stage or at different stages of pregnancy
complies with the test if the number of live spores is not less according to the schedule to be recommended. Administer to
than 80 per cent of that stated on the labe!' each sow a double dose of the vaccine, then one dos e after
3-5 Potency 14 days. Observe the sows at least daily until farrowing.
The vaccine complies with the requirements of the test The vaccine complies with the test if no sow shows abnormal
prescribed under Immunogenicity (section 2-2-1). It is not local or systemic reactions or dies from causes attributable to
necessary to carry out the potency test for each batch of the the vaccine and if no adverse effects on the pregnancy or the
vaccine if it hasbeen carried out on a representative batch offspring are noted.
using a vaccinating dose containing not more than the 2-3-1-1-3. Safety in the pigs used in tests 2-3-2 for
minimum number of live spores stated on the labe!' irnmunogenicity. The pigs used in the tests for
_____________________________________________ ~E~
immunogenicity are also used to evaluate safety. Measure the
rectal temperature of each vaccinated pig at the time of
vaccination and 6 h, 24 h and 48 h latero Examine the
injection site at slaughter for local reactions.
Aujeszky's Disease Vaccine, *** The vaccine complies with the test if no pig shows:
** ** -- a temperature rise greater than 1.5 oC and the number of
Inactivated ***** pigs showing a temperature greater than 41 °C does not
(Aujeszky's Disease Vaccine (Inactivated) far Pigs, exceed 10 per cent of the group;
Ph Eur managraph 0744)
-- other systemic reactions (for example, anorexia);
PhE~ _____________________________________________
-- abnormal local reactions attributable to the vaccine.
2-3-1-2 Field studies The pigs used for field trials are also
1 DEFINITION used to evaluate safety. Carry out a test in each category of
Aujeszky's disease vaccine (inactivated) for pigs is a pigs for which the vaccine is intended (sows, fattening pigs).
preparation of a suitable strain of Aujeszky's disease virus, Use not fewer than 3 groups each of not fewer than 20 pigs
inactivated while maintaining adequate irnmunogenic with corresponding groups of not fewer than 10 controls.
properties or a preparation of an inactivated fraction of the Measure the rectal temperature of each vaccinated pig at the
virus having adequate immunogenic properties. This time of vaccination and 6 h, 24 h and 48 h latero Examine
monograph applies to vaccines intended for the active the injection site at slaughter for local reactions.
immunisation of pigs and for passive protection of their The vaccine complies with the test if no pig shows:
progeny against Aujeszky's disease. -- a temperature rise greater than 1.5 oC and the number of
2 PRODUCTION pigs showing a temperature greater than 41 oC does not ~
2-1 PREPARATION OF THE VACCINE exceed 25 per cent of the group;
The vaccine virus is grown in cell cultures. The viral -- abnormal local reactions attributable to the vaccine.
suspension is harvested and inactivated; it may be treated to 2-3-2 Immunogenicity
fragment the virus and the viral fragments may be purified A test is carried out for each route and method of
and concentrated. The vaccine may be adjuvanted. administration to be recommended, using in each case pigs
2-2 SUBSTRATE FOR VIRUS PROPAGATION of the age to be recommended for vaccination. The vaccine
administered to each pig is of minimum potency.
2-2-1 Cell cultures
The cell cultures comply with the requirements for cell 2-3-2-1 Vaccines intended far active immunisatian Use for the
cultures for production of veterinary vaccines (5.2.4). test not fewer than 15 fattening pigs that do not have
antibodies against Aujeszky's disease virus or against a
2-3 CHOICE OF VACCINE COMPOSITION
fraction of the virus. The body mass of none of the pigs
The vaccine is shown to be satisfactory with respect to safety differs from the average body mass of the group by more
(5.2.6) and efficacy (5.2.7) for the pigs for which it is
202 Veterinary Vaccines
than 20 per cent. Vaccinate not fewer than 10 pigs, 3 BATCH TESTS
according to the schedule to be recommended. Maintain not 3-1 Identification
fewer than 5 pigs as controls. At the end of the fattening In animal s that do not have antibodies against Aujeszky's
period (80-90 kg), weigh and challenge each pig by the disease virus or against a fraction of the virus, the vaccine
intranasal route with a sufficient quantity of virulent stimulates the production of specific antibodies against
Aujeszky's disease virus (challenge with at least 10 6 CCID so Aujeszky's disease virus or the fraction of the virus used in
of a virulent strain having undergone not more than 3 the production of the vaccine.
passages and administered in not les s than 4 mL of diluent 3-2 Bacteria and fungi
has been found to be satisfactory). The titre of excreted virus The vaccine and, where applicable, the liquid supplied with it
is determined in swabs taken from the nasal cavity of each comply with the test for sterility prescribed in the monograph
pig daily from the day before challenge until virus is no Vaccines for veterinary use (0062).
longer detected. Each pig is weighed 7 days after challenge or
at the time of death if this occurs earlier and the average 3-3 Safety
daily gain is calculated as a percentage. For each group Use not fewer than 2 piglets of the minimum age
(vaccillated and controls), the average of the average daily recommended for vaccination and that do not have
gains is calculated. antibodies against Aujeszky's disease virus or against a
fraction of the virus. Administer to each piglet by a
The test is invalid unless all the control pigs display signs of
recommended route a double dose of the vaccine, then one
Aujeszky's disease and the average of their daily gains is les s
dose after 14 days. Observe the piglets at least daily until
than - 0.5 kg. The vaccine complies with the test if:
14 days after the last administration.
- all the vaccinated pigs survive and the difference between
the average s of the daily gains for the 2 groups is not les s The vaccine complies with the test if no piglet shows notable
than 1.5 kg, signs of disease or dies from causes attributable to the
- the geometrical mean titres and the duration of excretion vaccine.
of the challenge virus are significantly lower in vaccinates 3-4 Residuallive virus
than in controls. Wherever possible, carry out a suitable test for residual live
2-3-2-2 Vaccines in tended for passive immunisation If the Aujeszky's disease virus using 2 passages in the same type of
vaccine is intended for use in sows for the passive protection cell culture as used in the production of the vaccine or cells
of piglets, the suitability of the strain for this purpose may be shown to be at least as sensitive. Otherwise, inject one dose
demonstrated by the following method. of the vaccine subcutaneously into each of 5 healthy non-
immunised rabbits. Observe the rabbits for 14 days after the
U se for the test not fewer than 12 sows that do not have
injection. The vaccine complies with the test if no abnormal
antibodies against Aujeszky's disease virus or against a
reaction (in particular a local rash) occurs. If the vaccine
fraction of the virus. Vaccinate not fewer than 8 sows,
strain is not pathogenic for the rabbit, carry out the test in 2
according to the schedule to be recommended. Maintain not
sheep.
fewer than 4 sows as controls. At 6-10 days of age, challenge
the piglets from the sows with a sufficient quantity of virulent 3-5 Extraneous agents
Aujeszky's disease virus. Observe the piglets at least daily for On the pigs used for the safety test carry out tests for
21 days. antibodies. The vaccine complies with the test if it do es not
The test is invalid if the average number of piglets per litter stimulate the formation of antibodies, other than those
for each group is less than 6. The vaccine complies with the against Aujeszky's disease virus, against virus es pathogenic for
test if not less than 80 per cent protection against mortality is pigs or against virus es that could interfere with the diagnosis
found in the piglets from the vaccinated sows compared to of infectious diseases of pigs (inc1uding the virus es of the
those from the control sows. pestivirus group).
controls. For the piglets from vaccinated sows: carry out tests operation not fewer than 5 times, the last time in not fewer
for serum antibodies against Aujeszky's disease virus; carry than 5 piglets; verify the presence of the virus at each
out tests for Aujeszky's disease virus antigen in the liver and passage. If the virus is not found at a passage level, carry out
lungs of those piglets showing abnormalities and in a quarter a second series of passages.
0t the remaining healthy piglets. The vaccine virus complies with the test if no piglet die s or
The vaccine virus complies with the test if: shows neurological disorders from causes attributable to the
- the number of piglets born to the vaccinated sows, any vaccine virus and no indication of increased virulence of the
abnormalities in the piglets and the duration of gestation maximally passaged virus compared with the unpassaged
do not differ significantly from those of the controls; virus is observed. If virus is not recovered at any passage level
- no Aujeszky's disease virus antigen is found in piglets in the first and second series of passages, the vaccine virus
born to the vaccinated sowS; also complies with the test.
- no antibodies against Aujeszky's disease virus are found in 2-3-5 Immunogenicity
the serum taken before ingestion of the colostrum. A test is carried out for each route and method of
2-3-2 Virus excretion administration to be recommended for vaccination.
U se for the test not fewer than 18 pigs, 3-4 weeks old and The quantity of vaccine virus to be administered to each pig
that do not have antibodies against Aujeszky's disease virus is not greater than the minimum virus titre to be stated on
or against a fraction of the virus. Administer to not fewer the label and the virus is at the most attenuated passage level
than 14 pigs a quantity of the vaccine virus equivalent to not that will be present in a batch of vaccine.
less than the maximum virus titre likely to be contained in 2-3-5-1 Vaccines intended for active immunisation Use for the
one dose of the vaccine by a route and a site to be test not fewer than 15 fattening pigs of the age to be
recommended. Maintain not fewer than 4 pigs as contact recommended and that do not have antibodies against
controls. Carry out suitably sensitive tests for the virus Aujeszky's disease virus or against a fraction of the virus.
individually on the nasal and oral secretions as follows: The body mas s of non e of the pigs differs from tlie average
collect nasal and oral swabs daily from the day before body mass of the group by more than 20 per cent. Vaccinate
vaccination until 10 days after vaccination. not fewer than 10 pigs, according to the schedule to be
The vaccine complies with the test if the virus is not isolated recommended. Maintain not fewer than 5 pigs as controls.
from the secretions collected. At the end of the fattening period (80-90 kg), weigh and
2-3-3 Transmissibility chaHenge each pig by the intranasal route with a sufficient
Carry out the test on 4 separate occasions. Each time, quantity of virulent Aujeszky's disease virus (challenge with at
administer to not fewer than 4 piglets, 3-4 weeks old and least 10 6 CCID so of a virulent strain having undergone not
that do not have antibodies against Aujeszky's disease virus more than 3 passages and administered in not les s than 4 mL
or against a fraction of the virus, by a route to be of diluent has been found to be satisfactory). Determine the
recommended, a quantity of the vaccine virus equivalent to titre of virus in swabs taken from the nasal cavity of each pig
not les s than the maximum virus titre likely to be contained daily from the day before chaHenge until virus is no longer
in one dose of the vaccine. After one day, keep not fewer detected. Weigh each pig 7 days after chaHenge or at the
than 2 other piglets of the same age and that do not have time of death if this occurs earlier and calculate the average
antibodies against Aujeszky's disease virus or against a daily gain as a percentage. For each group (vaccinated and
fraction of the virus close together with them. After 5 weeks, controls), calculate the average of the average daily gains.
test aH the piglets for the presence of antibodies against The test is invalid unless aH the control pigs display signs of
Aujeszky's disease virus. Aujeszky's disease and the average of their average daily gains
The test is invalid if any vaccinated piglet does not show an is less than - 0.5 kg. The vaccine complies with the test if:
antibody response. The vaccine virus complies with the test if - aH the vaccinated pigs survive and the difference between
no antibodies against Aujeszky's disease virus are detected in the average s of the average daily gains for the 2 groups is
any group of contact controls and if all the vaccinated piglets not less than 1.5 kg,
show an antibody response. - the geometrical mean titres and the duration of excretion
of the challenge virus are significantly lower in vaccinates
2-3-4 Increase in virulence than in controls.
The test for increase in virulence consists of the
administration of the vaccine virus at the least attenuated 2-3-5-2 Vaccines intended for passive protection If the vaccine
passage level that will be present between the master seed lot is intended for use in sows for the passive protection of
and a batch of the vaccine to 2 piglets, 3-5 days old, that do piglets,the suitability of the vaccine virus for this purpose
not have antibodies against Aujeszky's disease virus or against may be demonstrated by the following method.
a fraction of the virus, sequential passages, 5 times where Use for the test not fewer than 12 sows that do not have
possible, to further similar groups and testing of the final antibodies against Aujeszky's disease virus or against a
recovered virus for increase in virulence. Passage as described fraction ofthe virus. Vaccinate not fewer than 8 sows,
below is carried out and the maximally passaged virus that according to the schedule to be recommended. Maintain not
has been recovered is tested for increase in virulence. Care fewer th~n 4 sows as controls. At 6-10 days of age, chaHenge
must be taken to avoid contamination by virus from previous the piglets from the sows with a sufficient quantity of virulent
passages. Aujeszky's disease virus. Observe the piglets at least daily for
Administer to each piglet by the intranasal route a quantity 21 daYJ.
of the vaccine virus that will aHow recovery of virus for the The test is invalid if the average number of piglets per litter
passages described below. After 3-5 days, prepare a for each group is less than 6.
suspension from the brain, lung, tonsils and locallymph The vaccine complies with the test if not less than
glands of each piglet and pool the samples. Administer 1 mL 80 per cent protection against mortality is found in the
of the pooled samples by the intranasal route to each of 2 piglets from the vaccinated sows compared to those from the
other piglets of the same age. Carry out this passage control sows.
Veterinary Vaccines 205
2-4 MANUFACTURER'S TESTS group (vaccinated and contro1s), calcu1ate the average of the
2-4-1 Batch potency test average dai1y gains.
It is not necessary to carry out the Potency test (section 3-7) The test is inva1id un1ess all the control pigs display signs of
for each batch of the vaccine if it has been carried out on a Aujeszky's disease and the average of their average dai1y gains
representative batch using a vaccinating dose containing not is les s than - 0.5 kg. The vaccine complies with the test if all
more than the minimum virus titre stated on the 1abel. the vaccinated pigs survive and the difference between the
The test described under Potency is carried out for a given averages of the average dai1y gains for the 2 groups is not 1ess
vaccine, on one or more occasions, as decided by or with the than 1.6 kg.
agreement of the competent authority. Where this test is not 4 LABELLING
carried out, an altemative va1idated method is used, .the The 1abel states the substrate used for production of the
criteria for acceptance being set with reference to a batch of vaccine (cell cultures or eggs).
vaccine that has given satisfactory results in the test described
under Potency. ________________-----------------------------PhE~
3 BATCH TESTS
3-1 Identification
In anima1s that do--not have antibodies against Aujeszky's
disease virus or against a fraction of the virus, the vaccine
Avian Infectious Bronchitis
stimu1ates the production of specific neutra1ising antibodies. Vaccine (Inactivated)
3-2 Bacteria and fungi Infectious Bronchitis Vaccine, Inactivated
The vaccine and, where app1icab1e, the liquid supp1ied with it (Ph Eur monograph 0959)
comp1y with the test for steri1ity prescribed in the monograph CAUTION Accidental injection of oily vaccine can cause serious
Vaccines for veterinary use (0062). local reactions in mano Expert medical advice should be sought
3-3 Mycoplasmas (2.6.7) immediately and the doctor should be informed that the vaccine is
The vaccine complies with the test for mycop1asmas. an oil emulsiono
3-4 Extraneous agents ~E~ _____________________________________________
Neutralise the vaccine virus with a suitab1e monospecific
1 DEFINITION
antiserum or monoc1ona1 antibodies against Aujeszky's
Avian infectious bronchitis vaccine (inactivated) is a
disease virus or against a fraction of the virus and inocu1ate
preparation of one or more suitab1e strains of one or more
into cell cultures known for their susceptibi1ity to virus es
serotypes of avian infectious bronchitis virus, inactivated
pathogenic for pigs and to pestiviruses. Carry out at 1east one
whi1e maintaining adequate immunogenic properties. This
passage and maintain the cultures for 14 days. The vaccine
monograph applies to vaccines intended to protect birds
comp1ies with the test if no cytopathic effect deve10ps and
against a drop in egg production or quality; for vaccines a1so
there is no sign of the presence of haemadsorbing agents.
intended for protection against respiratory sígns, a
Carry out a specific test for pestiviruses.
demonstration of efficacy additiona1 to that described under
3-5 Safety Potency is required.
U se 2 pigs of the minimum age recommended for
vaccination and that do not have antibodies against 2 PRODUCTION
Aujeszky's disease virus or against a fraction of the virus. 2-1 PREPARATION OF THE VACCINE
Administer to each pig by a recommended route 10 doses of The vaccine virus is propagated in ferti1ised hens' eggs or in
the vaccine. Observe the pigs at 1east dai1y for 14 days. cell cultures. The vaccine may be adjuvanted.
The vaccine comp1ies with the test if no pig shows notable 2-2 SUBSTRATE FOR VIRUS PROPAGATION
signs of disease or dies from causes attributab1e to the 2-2-1 Embryonated hens' eggs
vaccine virus. If the vaccine virus is grown in embryonated hens' eggs, they
3-6 Virus titre are obtained from healthy flocks.
Titrate the vaccine virus on the same substrate as used for 2-2-2 Cell cultures
production (cell cultures or inocu1ation into the allantoic If the vaccine is grown in cell cultures, they comp1y with the
cavity of ferti1ised hen eggs). The vaccine comp1ies with the requirements for cell cultures for production of veterinary
test if one dose contains not les s than the minimum virus vaccines (5.2.4).
titre stated on the 1abel.
2-3 SEED LOTS
3-7 Potency 2-3-1 Extraneous agents
The vaccine comp1ies with the requirements of the test The master seed 10t comp1ies with the test for eXtraneous
described below when administered by a recommended route agents in seed 10ts (2.6.24). In these tests on the master seed
and method. 10t, the organisms used are not more than 5 passages from
Use not fewer than 10 pigs weighing 15-35 kg, that do not the master seed 10t at the start of the test.
have antibodies against Aujeszky's disease virus or against a
2-4 CHOICE OF VACCINE COMPOSITION
fraction of the virus. The body mas s of non e of the pigs
The vaccine is shown to be satisfactory with respect to safety
differs from the average body mas s of the group by more
(5.2.6) and efficacy (5.2.7) for the birds for which it is
than 25 per cent. Vaccinate not fewer than 5 pigs with one
intended.
dose of the vaccine. Maintain not fewer than 5 pigs as
contro1s. After 3 weeks, weigh each pig, then challenge them The following test for Irnmunogenicity (section 2-4-1) may
by the intranasa1 route with a sufficient quantity of viru1ent be used during the demonstration of efficacy.
Aujeszky's disease virus. Weigh each pig 7 days after 2-4-1 Immunogenicity
challenge or at the time of death if this occurs earlier and A test is carried out for each route and method of
calcu1ate the average dai1y gain as a percentage. For each administration to be recommended, using in each case
206 Veterinary Vaccines
chickens from a flock free from specified pathogens (SPF) from detectable specific antibody. The vaccine complies with
(5.2.2) and for each serotype in the vaccine. The vaccine the test if the antibody levels are not significantly les s than
administered to each chicken is of minimum potency. those obtained with a batch that has given satisfactory results
Use for the test 4 groups of not fewer than 30 chickens in the test described under Potency.
treated as follows. 3 BATCH TESTS
Group A: unvaccinated controls. 3-1 Identification
Group B: vaccinated with inactivated avian infectious When injected into chickens that do not have antibodies
bronchitis vaccine. against each of the virus serotypes in the vaccine, the vaccine
Group C: vaccinated with live avian infectious bronchitis stimulates the production of such antibodies, detectable by
vaccine and inactivated avian infectious bronchitis vaccine virus neutralisation.
according to the recommended schedule. 3-2 Bacteria and fungi
Group D: vaccinated with live avian infectious bronchitis The vaccine and, where applicable, the liquid supplied with it
vaccine. comply with the test for sterility prescribed in the monograph
Vaccines for veterinary use (0062).
Monitor egg production and quality in all chickens from
point of lay until at least 4 weeks after challenge. At the peak 3-3 Safety
of lay, challenge all groups with a quantity of virulent avian U se 10 chickens, 14 to 28 days old, from an SPF flock
infectious bronchitis virus sufficient to cause a drop in egg (5.2.2). Inject a double dose of vaccine by a recommended
production or quality over 3 consecutive weeks during the route into each chicken. Observe the chickens at least daily
4 weeks following challenge. The test is invalid unless there is for 21 days.
a drop in egg production in group A compared to the normal The vaccine complies with the test if no chicken shows
level noted before challenge of at least 35 per cent where notable signs of disease or dies from causes attributable to
challenge has been made with a Massachusetts-type strain; the vaccine.
where it is necessary to carry out a challenge with a strain of 3-4 Residuallive virus
another serotype for which there is documented evidence that A test for residual live virus is carried out to confirm
the strain will not cause a 35 per cent drop in egg inactivation of avian infectious bronchitis virus.
production, the challenge must produce a drop in egg
A. For vaccine prepared with embryo-adapted strains of
production commensurate with the documented evidence
virus, inject 2/5 th of adose into the allantoic cavity of ten
and in any case not les s than 15 per cent. The vaccine
9- to ll-day-old embryonated hens' eggs from an SPF flock
complies with the test if egg production or quality is
(5.2.2) and incubate. Observe for 5 to 6 days and pool
significantly better in group C than in group D and
separately the allantoic liquid from eggs containing live
significantly better in group B than in group A.
embryos and that from eggs containing dead embryos,
2-5 MANUFACTURER'S TESTS excluding those that die within the first 24 h after injection.
2-5-1 Residuallive virus Examine for abnormalities all embryos which die after 24 h
An amplification test for residuallive avian infectious of injection or which survive 5 to 6 days. No death or
bronchitis virus is carried out on each batch of antigen abnormality attributable to the vaccine virus occurs. Inject
immediately after inactivation and on the final bulk vaccine into the allantoic cavity of each of ten 9- to ll-day-old
or, if the vaccine contains an adjuvant, on the bulk antigen or embryonated hens' eggs from an SPF flock (5.2.2) 0.2 mL of
mixture of bulk antigens irnmediately before the addition of the pooled allantoic liquid from the live embryos and into
adjuvant; the test is carried out in embryonated hen eggs each of 10 similar eggs 0.2 mL ofthe pooled liquid from the
from SPF flocks (5.2.2) or in suitable cell cultures (5.2.4), dead embryos and incubate for 5 to 6 days. Examine for
whichever is the most sensitive for the vaccine strain. abnormalities all embryos which die after 24 h of injection or
The quantity of inactivated virus harvest used in the test is which survive 5 to 6 days. If more than 20 per cent of the
equivalent to not less than 10 doses of vaccine. The vaccine embryos die at either stage repeat the test from that stage.
complies with the test if no live virus is detected. The vaccine complies with the test if there is no death or
2-5-2 Batch potency test abnormality attributable to the vaccine virus.
It is not necessary to carry out the Potency test (section 3-6) B. For vaccine prepared with cell-culture-adapted strains of
for each batch of vaccine if it has been carried out using a virus, inoculate 10 doses of the vaccine into suitable cell .
batch of vaccine with a minimum potency. Where the test is cultures. If the vaccine contains an oil adjuvant, eliminate it
not carried out, an alternative validated method is used, the by suitable means. Incubate at 38 ± 1 oC for 7 days. Make
criteria for acceptance being set with reference to a batch of a passage on another set of cell cultures and incubate at 38
vaccine that has given satisfactory results in the test described ± 1 oC for 7 days. The vaccine complies with the test if
under Potency. The following test may be used. none of the cultures show signs of infection.
Administer 1 dos e of vaccine by the intramuscular route to 3-5 Extraneous agents
each of not fewer than 10 chickens, between 2 weeks of age U se the chickens from the safety test. 21 days after injection
and the minimum age stated for vaccination and from an of the dquble dos e of vaccine, inject 1 dos e by the same
SPF flock (5.2.2), and maintain 5 hatch mates as route intb each chicken. Collect serum samples from each
unvaccinated controls. Collect serum samples from each chicken 2 weeks later and carry out tests for antibodies
chicken just before administration of the vaccine and after against the following agents by the methods prescribed in
the period defined when testing the reference vaccine; general chapter 5.2.2. Chicken fiocks free from specijied
determine the antibody titre of each serum, for each serotype pathogens for the production and quality control of vaccines: avian
in the vaccine, by a suitable serological method, for example, encephalomyelitis virus, avian leucosis viruses, egg-drop
serum neutralisation. The test is invalid unless the sera syndrome virus, avian infectious bursal disease virus, avian
collected from the unvaccinated control s and from the infectious laryngotracheitis virus, influenza A virus, Marek's
chickens just before the administration of the vaccine are free disease virus, Newcastle disease virus. The vaccine complies
Veterinary Vaccines 207
with the test if it does not stimulate the formation of contained in 1 dose of the vaccine. On each of days 5, 7 and
antibodies against these agents. 10 after adrninistration of the virus, euthanise not fewer than
3-6 Potency 5 of the chickens and take samples of trachea and kidney.
The vaccine complies with the requirements of the test Fix kidney samples for histological exarnination. Remove the
mentioned under Imrnunogenicity (section 2-4-1) when tracheas and prepare 3 transverse sections from the upper
adrninistered by a recommended route and method. part, 4 from the middle part and 3 from the lower part of the
trachea of each chicken; examine all tracheal explants as soon
4LABELLING as possible and at the latest 2 h after sampling by low-
The label states whether the strain in the vaccine is eníhryo- magnification microscopy for ciliary activity. Score for
adapted or cell-culture-adapted. ciliostasis on a scale from O (100 per cent ciliary activity) to 4
________________________________________ ~-~E~
(no activity, complete ciliostasis); calculate the mean
ciliostasis score (the maximurn for each trachea being 40) for
the 5 chickens euthanised on each of days 5, 7 and 10.
The test is not valid if more than 10 per cent of the chickens
die from causes not attributable to the vaccine virus.
Avian Infectious Bronchitis The vaccine virus complies with the test if:
Vaccine, Living - no chicken shows notable clinical signs of avian infectious
(Avian Infeetiaus Branehitis Vaeeine (Live), bronchitis or dies from causes attributable to the vaccine
Ph Eur managraph 0442) virus;
PhE~ ___________________________________________ - any inflamrnatory lesions seen during the kidney
histological examination are, at most, moderate.
1 DEFINITION A risk/benefit analysis is carried out, taking into account the
Avian infectious bronchitis vaccine (live) is a preparation of average ciliostasis scores obtained and the benefits expected
one or more suitable strains of different types of avian from the use of the vaccine.
infectious bronchitis virus. This monograph applies 10 2-4-1-2 Safety far the repraduetive traet If the
vaccines intended for administration to chickens for active recommendations for use state or imply that the vaccine may
immunisation against respiratory disease caused by avian be used in females less than 3 weeks old that are
infectious bronchitis virus. subsequently kept to sexual maturity, it shall be
2 PRODUCTION demonstrated that there is no damage to the development of
2-1 PREPARATION OF THE VACCINE the reproductive tract when the vaccine is given to chickens
The vaccine virus is grown in embryonated hens' eggs or in of the minimurn age to be recommended for vaccination.
cell cultures. The following test may be carried out: use not fewer than 40
2-2 SUBSTRATE FOR VIRUS PROPAGATION female chickens from an SPF flock (5.2.2) that are not older
than the minimum age recommended for vaccination; use the
2-2-1 Embryonated hens' eggs
vaccine virus at the least attenuated passage level that will be
If the vaccine virus is grown in embryonated hens' eggs, they
present in a batch of vaccine; adrninister to each chicken by a
are obtained from flocks free from specified pathogens (SPF)
recommended route a quantity of virus equivalent to not less
(5.2.2).
than the maximurn titre likely to be present in 1 dos e of
2-2-2 Cell cultures vaccine; at least 10 weeks after administration of the vaccine
If the vaccine virus is grown in cell cultures, they comply virus, euthanise the chickens and carry out a macroscopic
with the requirements for cell cultures for production of examination of the oviducts. The vaccine virus complies with
veterinary vaccines (5.2.4). the test if abnorrnalities are present in not more than
2-3 SEED LOTS 5 per cent of the oviducts.
2-3-1 Extraneous agents 2-4-2 Increase in virulence
The master seed lot complies with the tests for extraneous The test for increase in virulence consists of the
agents in seed lots (2.6.24). In these tests on the master seed adrninistration of the vaccine virus, at the least attenuated
lot, the organisms used are not more that 5 passages from virus passage level that will be present between the master
the master seed lot at the start of the test. seed lot and a batch of the vaccine, to a group of 5 two-
2-4 CHOICE OF VACCINE VIRUS week-old chickens from an SPF flock (5.2.2); sequential
The vaccine virus shall be shown to be satisfactory with passages, 5 times where possible, 10 further similar groups
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens and testing of the final recovered virus for increase in
for which it is intended. virulence. If the properties of the vaccine virus allow
sequential passage to 5 groups via natural spreading, this
The following tests for safety (section 2-4-1), increase in
method may be used, otherwise, passage as described below
virulence (section 2-4-2) and imrnunogenicity (section 2-4-3)
is carried out and the maximally passaged virus that has been
may be used during the demonstration of safety and efficacy.
recovered is tested for increase in virulence. Care must be
2-4-1 Safety taken to avoid contamination by virus from previous
2-4-1-1 Safety far the respiratory tract and kidneys Carry out passages.
the test in chickens not older than the minimum age to be Administer to each chicken by eye-drop a quantity of the
recommended for vaccination. Use vaccine virus at the least vaccine virus that will allow recovery of virus for the passages
attenuated passage level that will be present between the described below. 2-4 days after administration of the vaccine
master seed lot and a batch of the vaccine. virus, prepare a suspension from the mucosa of the trachea of
Use not fewer than 15 chickens of the same origin and from each chicken and pool these samples. Administer 0.05 mL of
an SPF flock (5.2.2). Adrninister to each chicken by the the pooled samples by eye-drop to each of 5 other two-week-
oculonasal route a quantity of the vaccine virus equivalent to old chickens from an SPF flock (5.2.2). Carry out this
not les s than 10 times the maximurn virus titre likely to be
208 Veterinary Vaccines
passage operation not fewer than 5 times; verify the presence allantoic cavity of each of 5 embryonated hens' eggs,
of the virus at each passage. If the virus is not found at a 9-11 days old, from an SPF fiock (5.2.2). Incubate the eggs
passage level, carry out a second series of passages. Carry out for 6-8 days after inoculation. Eggs that after 1 day of
the test for safety for the respiratory tract and kidney (section incubation do not contain a live embryo are eliminated and
2-4-1-1) and, where applicable, the test for safety for the considered as non-specific deaths. Record the other eggs
reproductive tract (section 2-4-1-2) using the unpassaged containing a dead embryo and after 6-8 days' incubation
vaccine virus and the maximally passaged virus that has been examine each egg containing a live embryo for lesions
recovered. Administer the virus by the route to be characteristic of avían infectious bronchitis; Make
recommended for vaccination that is likely to be the least successively 3 such passages. If 1 embryo of a series of eggs
safe. dies or shows characteristic lesions, the inoculum is
The vaccine virus complies with the test if no indication of considered to be a carrier of avian infectious bronchitis virus.
an increase in virulence of the maximally passaged virus The examination of a series of eggs is considered to be
compared with the unpassaged virus is observed. If virus is definitely negative if no inoculum concerned is a carrier.
not recovered at any passage level in the 1st and 2nd series of The test is not valid if:
passages, the vaccine virus also complies with the test. - the challenge virus is re-isolated from fewer than
80 per cent of the control chickens;
2-4-3 Irnmunogenicity
- and/or during the period between vaccination and
Irnmunogenicity is demonstrated for each strain of virus to
challenge, more than 10 per cent of the vaccinated or
be included in the vaccine. A test is carried out for each
control chickens show abnormal clinical signs or die from
route and method of administration to be recommended
causes not attributable to the vaccine;
using in each case chickens from an SPF fiod: (5.2.2) that
- and/or more than 1 egg in any group is eliminated
are not older than the minimum age to be recommended for
because of non-specific embryo death.
vaccination. The quantity of the vaccine virus administered
to each chicken is not greater than the minimum virus titre The vaccine virus complies with the test if the challenge virus
to be stated on the label and the virus is at the most is re-isolated from not more than 20 per cent of the
attenuated passage level that will be present in a batch of the vaccinated chickens.
vaccine. 3 BATCH TESTS
Either or both of the tests below may be used during the 3-1 Identification
demonstration of immunogenicity. 3-1-1 Vaccines containing one type of virus The vaccine,
2-4-3-1 Ciliary activity of tracheal explants Use not fewer diluted if necessary and mixed with avian infectious
than 25 chickens of the same origin and from an SPF fiod: bronchitis virus antiserum specific for the virus type, no
(5.2.2). Vaccinate by a recommended route not fewer than longer infects embryonated hens' eggs from an SPF fiock
20 chickens. Maintain not fewer than 5 chickens as controls. (5.2.2) or susceptible cell cultures (5.2.4) into which it is
Challenge each chicken after 21 days by eye-drop with a inoculated.
sufficient quantity of virulent avían infectious bronchitis virus 3-1-2 Vaccines containing more than one type ofvirus The
of the same type as the vaccine virus to be tested. Euthanise vaccine, diluted if necessary and mixed with type-specific
the chickens 4-7 days after challenge and prepare antisera against each strain present in the vaccine except that
3 transverse sections from the upper part, 4 from the middle to be identified, infects embryonated hens' eggs from an SPF
part, and 3 from the lower part of the trachea of each fiock (5.2.2) or susceptible cell cultures (5.2.4) into which it
chicken. Examine all tracheal explants as soon as possible is inoculated, whereas after further admixture with type-
and at the latest 2 h after sampling by low-magnification specific antiserum against the strain to be identified it no
microscopy for ciliary activíty. For a given tracheal section, longer produces such infection.
ciliary activity is considered as normal when at least 3-2 Bacteria and fungi
50 per cent of the internal ring shows vigorous ciliary V~ccines intended for administration by injection comply
movement. A chicken is considered not affected if not fewer with the test for sterility prescribed in the monograph
than 9 out of 10 rings show normal ciliary activity. Vaccines for veterinary use (0062).
The test is not valid if: Vaccines not intended for administration by injection comply
- fewer than 80 per cent of the control chickens show either with the test for sterility prescribed in the monograph
cessation or extreme loss of vigour of ciliary activity; Vaccines for veterinary use (0062) or with the following test:
and/or during the period between the vaccination and carry out a quantitative test for bacterial and fungal
challenge, more than 10 per cent of vaccinated or control contamination; carry out identification tests for micro-
chickens show abnormal clinical signs or die from causes organisms detected in the vaccine; the vacClne does not
not attributable to the vaccine. contain pathogenic micro-organisms and contains not more
The vaccine virus complies with the test if not fewer than than 1 non-pathogenic micro-organism per dose.
80 per cent of the vaccinated chickens show normal ciliary Any liquid supplied with the vaccine complies with the test
activity. for sterility prescribed in the monograph Vaccines for
2-4-3-2 Virus recovery from tracheal swabs Use not fewer than veterinarj{ use (0062).
30 chickens of the same origin and from an SPF fiock 3-3 Mycoplasmas
(5.2.2). Vaccinate by a recommended route not fewer than The vaccine complies with the test for mycoplasmas (2.6.7).
20 chickens. Maintain not fewer than 10 chickens as
controls. Challenge each chicken after 21 days by eye-drop 3-4 EXtraneous agents
with a sufficient quantity of virulent avian infectious The vaccine complies with the tests for extraneous agents in
bronchitis virus of the same type as the vaccine virus to be batches of finished product (2.6.25).
tested. Euthanise the chickens 4-7 days after challenge and 3-5 Safety
prepare a suspension from swabs of the tracheal mucosa of Use not fewer than 10 chickens from an SPF fiock (5.2.2)
each chicken. Inoculate 0.2 mL of the suspension into the and of the minimum age recommended for vaccination.
Veterinary Vaccines 209
found at a passage level, carry out a second series of Any liquid supplied with the vaccine complies with the test
passages. Carry out the test for safety (section 2-4-1) using for sterility prescribed in the monograph Vaccines for
the unpassaged vaccine virus and the maximally passaged veterinary use (0062).
vaccine virus that has been recovered. The vaccine virus 3-3 Mycoplasmas
complies with the test if no indication of increase in virulence The vaccine complies with the test for mycoplasmas (2.6.7).
of the maximally passaged virus compared with the
unpassaged virus is observed. If the virus is not recovered at 3-4 Extraneous agents
any passage level in the first and second series of passages, The vaccine complies with the tests for extraneous agents in
the vaccine virus also complies with the test. batches of finished product (2.6.25).
2-3-1 Safety and the virus is at the most attenuated passage level that will
Carry out the test for each route and method of be present in a batch of vaccine.
administration to be recommended Jor vaccination. U se for the test not fewer than 10 calves that do not have
Use vaccine virus at the least attenuated passage level that antibodies against bovine parainfluenza 3 virus; calves having
will be present between the master seed lot and a batch of low levels of such antibodies may be used if it has been
the vaccine. demonstrated that valid results are obtained in these
For each test, use not fewer than 5 calves of the minimum conditions. Collect sera from the calves before vaccination,
age to be recommended for vaccination and preferably that 7 days and 14 days after the time of vaccination and just
do not have antibodies against bovine parainfluenza 3 virus before challenge. Vaccinate not fewer than 5 calves,
or, where justified, use calves with a very low level of such according to the schedule to be recommended. Maintain not
antibodies as long as they have not been vaccinated against fewer than 5 calves as controls. Challenge each calf after
bovine parainfluenza virus and administration of the vaccine 21 days by a respiratory tract route with a sufficient quantity
do es not cause an anamnestic response. Administer to each of a suspension of a low-passage virulent bovine
calf a quantity of the vaccine virus equivalent to not less than parainfluenza 3 virus. Observe the calves at least daily for
10 times the maximum virus titre likely to be contained in 14 days after challenge and monitor each of them for signs,
one dose of the vaccine. Observe the calves at least daily for in particular respiratory signs and virus shedding (by nasal
21 days. Measure the body temperature of each calf on the swabs or tracheobronchial washing).
day before vaccination, at the time of vaccination and for the The test is invalid if tests for antibodies against bovine
4 subsequent days. parainfluenza 3 virus on the sera indicate that there was
The vaccine virus complies with the test if no abnormal effect intercurrent infection with the virus during the test or if,
on body temperature occurs and if no calf shows abnormal, during the observation period after challenge, more than 2 of
local or systemic reactions or dies from causes attributable to the 5 control calves show no excretion of the challenge virus,
the vaccine virus. as shown by nasal swabs or samples harvested by
2-3-2 Increase in virulence tracheobronchial washing. The vaccine virus complies with
The test for increase in virulence consists of the the test if during the observation period after challenge, in
administration of the vaccine virus at the least attenuated vaccinated calves compared to controls there is (a) a
passage level that will be present between the master seed significant reduction in mean titre and in mean duration of
lot and a batch of the vaccine to 2 calves that do not have virus excretion and (b) a notable reduction in general and
antibodies against bovine parainfluenza 3 virus, sequential local signs (if the challenge virus used produces such signs).
passages, 5 times where possible, to further similar groups 3 BATCH TESTS
and testing of the final recovered virus for increase in 3-1 Identification
virulence. If the properties of the vaccine virus allow Carry out an irnmunostaining test in suitable cell cultures,
sequential passage to 5 groups via natural spreading, this using a monospecific antiserum.
method may be used, otherwise passage as described below
3-2 Bacteria and fungi
is carried out and the maximally passaged virus that has been
The vaccine and, where applicable, the liquid supplied with it
recovered is tested for increase in virulence. Care must be
comply with the test for sterility prescribed in the monograph
taken to avoid contamination by virus from previous
Vaccines far veterinary use (0062).
passages.
3-3 Mycoplasmas (2.6.7)
Administer to each calf by the intranasal route a quantity of
The vaccine complies with the test for mycoplasmas.
the vaccine virus that will allow recovery of virus for the
passages described below. On each of days 3 to 7 after 3-4 Extraneous agents
administration of the virus, take nasal swabs from each calf Neutralise the vaccine virus with a suitable monospecific
and collect in not more than 5 mL of a suitable medium al1tiserum against bovine parainfluenza 3 virus and inoculate
which is then used to inoculate cell cultures to verify the into cell cultures known for their susceptibility to viruses
presence of virus. Administer about 1 mL of the suspension pathogenic for cattle. Carry out at least one passage and
from the swabs that contain the maximum amount of virus, maintain the cultures for 14 days. The vaccine complies with
as indicated by the titration of cell cultures, to each of 2 the test if no cytopathic effect develops and there is no sign
other calves of the same age. Carry out this passage operation of the presence of haemadsorbing agents. Carry out a specific
not fewer than 5 times; verify the presence of the virus at test for pestiviruses.
each passage. If the virus is not found at a passage level, 3-5 Safety
carry out a second series of passages. U se 2 calves of the minimum age recommended for
The vaccine virus complies with the test if no calf shows vaccination and preferably that do not have antibodies
signs attributable to the vaccine virus and no indication of against bovine parainfluenza 3 virus or, where justified, use
increased virulence of the maximally passaged virus calves with a very low level of such antibodies as long as they
compared with the unpassaged virus is observed; account is have not been vaccinated against bovine parainfluenza virus
taken of the titre of excreted virus in the nasal swabs. If virus and administration of the vaccine does not cause an
is not recovered at any passage level in the first and second anamnestic response. Administer to each calf by a
series of passages, the vaccine virus also complies with the recommended route 10 dos es of the vaccine. Observe the
test. calves ~t least daily for 21 days.
2-3-3 Irnmunogenicity The vaccine complies with the test if no calf shows notable
A test is carried out for each route and method of signs of disease or dies from causes attrjbutable to the
administration to be recommended for vaccination using in vaccine.
each case calves of the minimum age to be recommended.
The quantity of vaccine to be administered to each calf is not
greater than the minimum virus titre to be stated on the label
Veterinary Vaccines 213
3-6 Virus titre 2-3-1-2 Field studies The calves used for the field trials are
Titrate the vaccine virus in suitable cell cultures. The vaccine also used to evaluate the incidence of hypersensitivity
complies with the test if one dose contains not less than the reactions in vaccinated calves following subsequent exposure
minimum virus titre stated on the label. to the vaccine or to wild virus. The vaccine complies with the
3-7 Potency test if it is not associated with an abnormal inciden ce of
The vaccine complieswith the requirements of the test immediate hypersensitivity reactions.
prescribed under Immunogenicity (section 2-3-3) when 2-3-2 Increase in virulence
administered by a recommended route and method. It is not The test for increase in virulence consists of the
necessary to carry out the potency test for each batch of the administration of the vaccine virus at the least attenuated
vaccine if it has been carried out on a representative batch passage level that will be present between the master seed lot
using a vaccinating dose containing not more than !\le and a batch of the vaccine to 2 calves that do not have
minimum virus titre stated on the label. antibodies against bovine respiratory syncytial virus,
_____________________________________________ ~E~ sequential passages, 5 times where possible, to further similar
groups and testing of the final recovered virus for increase in
virulence. If the properties of the vaccine virus allow
sequential passage to 5 groups via natural spreading, this
method may be used, otherwise passage as described below is
Bovine Respiratory Syncytial Virus carried out and the maximally passaged virus that has been
recovered is tested for increase in virulence. Care must be
Vaccine, Living taken to avoid contamination by virus from previous
(Bovine Respiratory Syncytial Virus Vaccine (Live)~ passages.
Ph Eur monograph 1177)
Administer to each calf by the intranasal route a quantity of
PhE~ _____________________________________________
the vaccine virus that will allow recovery of virus for the
1 DEFINITION passages described below. On each of days 3 to 7 after
Bovine respiratory syncytial virus vaccine (live) is a administration of the virus, take nasal swabs from each calf
preparation of a suitable strain of bovine respiratory syncytial and collect in not more than 5 mL of a suitable medium
virus. This monograph applies to vaccines intended for the which is then used to inoculate cell cultures to veri:fy the
active immunisation of catde against infection with bovine presence of virus. Administer about 1 mL of the suspension
respiratory syn~ytial virus. from the swabs that contain the maximum amount of virus,
as indicated by the titration of cell cultures, to each of 2
2 PRODUCTION other calves of the same age. Carry out this passage operation
2-1 PREPARATION OF THE VACCINE not fewer than 5 times; veri:fy the presence of the virus at
The vaccine virus is grown in cell cultures. each passage. If the virus is not found at a passage level,
2-2 SUBSTRATE FOR VIRUS PROPAGATION carry out a second series of passages.
2-2-1 Cell cultures The vaccine virus complies with the test if no calf shows
The cell cultures comply with the requirements for cell signs attributable to the vaccine virus and no indication of
cultures for production of veterinary vaccines (5.2.4). increased virulence of the maximally passaged virus
compared with the unpassaged virus is observed; account is
2-3 CHOICE OF VACCINE VIRUS
taken of the titre of excreted virus in the nasal swabs. If virus
The vaccine virus is shown to be satisfactory with respect to
is not recovered at any passage level in the first and second
safety (5.2.6) and efficacy (5.2.7) for the catde for which it is
series of passages, the vaccine virus also complies with the
intended.
test.
The following tests for safety (section 2-3-1), increase in
virulence (section 2-3-2) and immunogenicity (2-3-3) may be 2-3-3 Immunogenicity
used during the demonstration of safety and efficacy. A test is carried out for each route and method of
administration to be recommended for vaccination using in
2-3-1 Safety each case calves of the minimum age to be recommended.
Carry out the test for each route and method of The quantity of vaccine to be administered to each calf is not
administration to be recommended for vaccination, using in greater than the minimum virus titre to be stated on the label
each case calves of the minimum age to be recommended. and the virus is at the most attenuated passage level that will
Use vaccine virus at the least attenuated passage level that be present in a batch of vaccine.
will be present between the master seed lot and a batch of
U se for the test not fewer than 10 calves that do not have
the vaccine.
antibodies against bovine respiratory syncytial virus. Collect
2-3-1-1 Laboratory test For each test, use not fewer than 5 sera from the calves before the time of vaccination, 7 and
calves that do not have antibodies against bovine respiratory 14 days after the time of vaccination and just before
syncytial virus. Administer to each calf a quantity of the challenge. Vaccinate not fewer than 5 calves, according to the
vaccine virus equivalent to not less than 10 times the schedule to be recommended. Maintain not fewer than 5
maximum virus titre likely to be contained in one dose of the calves as controls. Challenge each calf after 21 days by a
vaccine. Observe the calves at least daily for 21 days. respiratory tract route with a sufficient quantity of a
Measure the rectal temperature of each calf on the day suspension of a low-passage virulent bovine respiratory
before vaccination, at the time of vaccination and daily for syncytial virus. Observe the calves at least daily for 14 days
the following 7 days. after challenge and monitor each of them for signs, in
The vaccine virus complies with the test if no abnormal effect particular respiratory signs and virus shedding (by nasal
on body temperature occurs and if no calf shows abnormal swabs or tracheobronchial washing).
local or systemic reactions or dies from causes attributable to The test is invalid if antibodies against bovine respiratory
the vaccine virus. syncytial virus are detected in any sample from control calves
214 Veterinary Vaccines
before challenge or if more than 2 of the 5 control calves the active immunisation of heifers and cows for protection of
show no excretion of the challenge virus, as shown by nasal their progeny against transplacental infection.
swabs or samples harvested by tracheobronchial washing. 2 PRODUCTION
The vaccine virus complies with the test if during the
2-1 PREPARATION OF THE VACCINE
observation period after challenge, there is: (a) a significant
The vaccine virus is grown in cell cultures. The viral
reduction in mean titre and in mean duration of virus
suspensions of each vaccine virus are harvested separately
excretion in vaccinates compared to controls and (b) a
and inactivated by a method that maintains immunogenicity.
notable reduction in general and local signs in vaccinated
The viral suspensions may be purified and concentrated.
calves (if the challenge virus used produces such signs).
The vaccine may be adjuvanted.
3 BATCH TESTS 2-2 SUBSTRATE FOR VIRUS PROPAGATION
3-1 Identification
2-2-1 Cell cultures
Identify the vaccine by an irnmunostaining test in suitable
The cell cultures comply with the requirements for cell
cell cultures using a monospecific antiserum.
cultures for production of veterinary vaccines (5.2.4).
3-2 Bacteria and fungi
2-3 CHOICE OF VACCINE COMPOSITION
The vaccine and, where applicable, the liquid supplied with it
The vaccine is shown to be satisfactory with respect to safety
comply with the test for sterility prescribed in the monograph
Vaccines for veterinary use (0062).
(5.2.6) and efficacy (5.2.7) for the cattle for which it is
intended.
3-3 Mycoplasmas (2.6.7)
The following tests for safety (section 2-3-1) and
The vaccine complies with the test for mycoplasmas.
irnmunogenicity (section 2-3-2) may be used during the
3-4 Extraneous agents demonstration of safety and efficacy.
Neutralise the vaccine virus with a suitable monospecific
2-3-1 Safety
antiserum against bovine respiratory syncytial virus and
Carry out the test for each route and method of
inoculate into cell cultures known for their susceptibility to
administration to be recommended for vaccination and in
virus es pathogenic for cattle. Carry out at least one passage
each category of cattle for which the vaccine is intended.
and maintain the cultures for 14 days. The vaccine complies
U se a batch of vaccine containing not less than the
with the test if no cytopathic effect develops and there is no
maximum potency that may be expected in a batch of
sign of the presence of haemadsorbing agents. Carry out a
vaccine.
specific test for pestiviruses.
2-3-1-1 General safety For each test, use not fewer than 10
3-5 Safety cattle of the minimum age to be recommended for
U se 2 calves of the minimum age recommended for vaccination and that do not have bovine diarrhoea virus nor
vaccination and that do not have antibodies against bovine antibodies against the virus. Administer to each animal a
respiratory syncytial virus. Administer to each calf by a double dose of the vaccine. Observe the cattle at least daily
recommended route 10 doses ofthe vaccine. Observe the for 14 days.
calves at least daily for 21 days.
The vaccine complies with the test if no animal shows
The vaccine complies with the test if no calf shows notable abnormallocal or systemic reactions or dies from causes
signs of disease or dies from causes attributable to the attributable to the vaccine.
vaccine.
2-3-1-2 Safety in pregnant cattle If the vaccine is intended for
3-6 Virus titre use in pregnant cattle, use not fewer than 10 cattle at the
Titrate the vaccine virus in suitable cell cultures. The vaccine beginning of each semester for which use is not
complies with the test if one dose contains not less than the contraindicated. Administer to each animal a double dose of
mínimum virus titre stated on the label. the vaccine. Observe the cattle at least daily until calving.
3-7 Potency The vaccine complies with the test if no animal shows
The vaccine complies with the requirements of the test abnormal local or systemic reactions or die s from causes
prescribed under Immunogenicity (section 2-3-3) when attributable to the vaccine and if no adverse effects on the
administered by a recommended route and method. It is not pregnancy or the offspring are noted.
necessary to carry out the potency test for each batch of the
2-3-1-3 Examination of reproductive performance If the vaccine
vaccine if it has been carried out on a representative batch
is intended for administration shortly before or at
using a vaccinating dose containing not more than the
insemination, absence of undesirable effects on conception
minimum virus titre stated on the label.
rate must be demonstrated.
_____________________________________________ ~Ew
2-3-2 Irnmunogenicity
The following test is suitable to demonstrate the
irnmunogenicity of the vaccine with respect to bovine
diarrhoea virus of genotype 1; if protection against bovine
Bovine Viral Diarrhoea Vaccine diarrhoea virus of genotype 2 is claimed, an additional test,
similar to \.that described below, but using bovine diarrhoea
(Inactivated) virus of genotype 2 for challenge, is carried out.
(Ph Bur monograph 1952) A test is carried out for each route and method of
PhEw _____________________________________________ administration to be recommended. The vaccine
1 DEFINITION administered to each heifer is of minimum potency.
Bovine viral diarrhoea vaccine (inactivated) is a preparation U se for the test not fewer than 20 heifers free from bovine
of one or more suitable strains of bovine diarrhoea virus diarrhoea virus and that do not have antibodies against
inactivated while maintaining adequate immunogenic bovine diarrhoea virus. Vaccinate not fewer than 13 heifers
properties. This monograph applies to vaccines intended for according to the schedule to be recommended. Maintain not
Veterinary Vaccines 215
fewer than 7 heifers as controls. Keep all the animals as one 3 BATCH TESTS
group. Inseminate the heifers. Take a blood sample from 3-1 Identification
non-vaccinated heifers shortly before challenge. The test is When administered to animals that do not have specific
discontinued if fewer than 10 vaccinated heifers or 5 non- neutralising antibodies against bovine diarrhoea virus, the
vaccinated heifers are pregnant at the time of challenge. vaccine stimulates the production of such antibodies.
Challenge each heifer between the 60th and 90th days of 3-2 Bacteria and fungi
gestation. For both test models described (observation until The vaccine and, where applicable, the liquid supplied with it
calving and harvest of foetuses at 28 days), challenge may be comply with the test for sterility prescribed in the monograph
made by the intranasal route with a sufficient quantity of a Vaccines jor veterinary use (0062).
non-cytopathic strain of bovine diarrhoea virus or
altematively, where the heifers are observed until ca,lving, 3-3 Safety
challenge may be made by contact with a persistent1y U se 2 cattle not older than the minimum age recommended
viraemic animal. Observe the heifers clinically at least daily for vaccination and that do not have bovine diarrhoea virus
from challenge either until the end of gestation or until nor antibodies against the virus. Administer to each animal
harvest of foetuses after 28 days. If abortion occurs, examine by a recommended route a double dose of the vaccine.
the aborted foetus for bovine diarrhoea virus by suitable Observe the cattle at least daily for 14 days.
methods. If cattle-are observed until calving, immediately The vaccine complies with the test if no animal shows
after birth and prior to ingestion of colostrum, examine all notable signs of disease or dies from causes attributable to
calves for viraemia and antibodies against bovine diarrhoea the vaccine.
virus. If foetuses are harvested 28 days after challenge, 3-4 Residuallive virus
examine the foetuses for bovine diarrhoea virus by suitable Carry out a test for residuallive bovine diarrhoea virus by
methods. Transplacental infection is considered to have inoculating not less than 10 doses onto cells known to be
occurred if virus is detected in foetal organs or in the blood sensitive to bovine diarrhoea virus; passage the cells after
of newbom calves or if antibodies are detected in precolostral 7 days and observe the second culture for not less than
sera of calves. 7 days. The vaccine complies with the test if no live virus is
The test is invalid if any of the control heifers have detected. If the vaccine contains an adjuvant, separate the
neutralising antibody before challenge or if transplacental adjuvant if possible from the liquid phase by a method that
infection fails to occur in more than 10 per cent of the calves does not interfere with the detection of possible live virus.
from the control heifers. The vaccine complies with the test if 3-5 Potency
at least 90 percent of the calves from the vaccinated heifers The vaccine complies with the requirements of the test
are protected from transplacental infection. prescribed under Immunogenicity (section 2-3-2) when
2-4 MANUFACTURER'S TESTS administered by a recommended route and method.
___________________________________________
2-4-1 Residuallive virus PhE~
The following test for irnmunogenicity (section 2-2-1) may 3-3 Determination of dissociation phase
be used during the demonstration of efficacy. Examine not fewer than 200 colonies by a suitable technique~
2-2-1 Irnrnmunogenicity The culture of the vaccine strain is seen to be in the smooth
Each test is carried out for each route and method of (S) phase.
administration to be recommended, using in each case ewe The vaccine complies with the test if not fewer than
lambs of 4-5 months old. The quantity of the vaccine strain 95 per cent of the colonies are of the smooth type.
to be administered to each ewe lamb is not greater than the 3-4 Extraneous micro-organisms
minimum live bacteria titre to be stated on the label. The vaccine complies with the test if it does not contain
Use for the test not fewer than 40 ewe lambs from a non- extraneous micro-organisms. Verify the absence of micro-
vaccinated flock free from brucellosis. For the challenge organisms other than Brucella melitensis Rev. 1 strain as
strain, a 24-hour culture of B. melitensis strain H38 in described in the test for sterility prescribed in the monograph
trypticase agar is used. Carry out a preliminary test using ewe on Vaccines for veterinary use (0062).
lambs of the same breed and age as for the main test to 3-5 Live bacteria
determine the challenge dose, which is between 10 7 and Make a count of live bacteria on a solid medium suitable for
108 colony-forming units and is chosen so as to produce the culture of Brucella melitensis Rev. 1 strain.
abortion in all non-vaccinated ewes. Vaccinate not fewer than
The vaccine complies with the test if it contains not fewer
20 ewe lambs at 4-6 months of age, according to the
than 0.5 x 109 and not more than 4 x 109 live bacteria per
schedule to be recommended. Maintain not fewer than 20
dose.
ewe lambs as controls. Oestrus is synchronised in the 40
ewes which are then inseminated at 10-12 months of age. 3-6 Fifty per cent persistence time
Make a diagnosis of pregnancy on all ewes 2-3 months after Use 32 female CDl mice, 5-6 weeks old. Vaccinate each
insemination and exc1ude all non-gravid ewes from the test. mouse by the subcutaneous route with a suspension
Challenge each gravid ewe by the conjunctival route with a containing 108 live bacteria of the vaccine to be examined.
sufficient quantity of B. melitensis strain H38. Note the Euthanise the mice in groups of 8, se1ected at random, 3, 6,
occurrence of abortion and confirm aetiology by testing for 9 and 12 weeks latero Remove the spleens and homogenise
the presence of the challenge strain in the aborted foetus and individually and aseptically in 10 volumes of phosphate
in the ewe (using selective media of Kuzdas and Morse or of buffered saline pH 6.8 R. Spread the suspension on plates
Farrell). Carry out tests for the presence of the challenge containing a suitable culture medium, using 0.4 mL per plate
strain on each ewe at lambing. Carry out tests for the and not fewer than 3 plates per spleen (lower limit of
presence of the challenge strain in the prescapular and detection: 5 bacteria per spleen). Carry out in parallel a
retromammary lymph nodes at slaughter of the ewes similar test using Brucella melitensis Rev. 1 strain BRP
4-6 weeks after lambing. (reference strain). Calculate the 50 per cent persistence time
The test is invalid unless at least 70 per cent of non- by the usual statistical methods (5.3) for probit analysis.
vaccinated ewes: The vaccine complies with the test if the 50 per cent
- show abortion caused by the challenge strain; persistence time for the vaccine strain does not differ
- show infection with the challenge strain at lambing; significandy from that of the reference strain.
- show infection of the prescapular and retromarnmary 4LABELLING
lymph nodes at slaughter. The labe1 states:
The vaccine complies with the test if: - that the vaccine may be dangerous for man;
- not more than 30 per cent of vaccinated ewes show - that the vaccine is not to be used in pregnant or lactating
abortion caused by the challenge strain; animals;
- the challenge strain is found in not more than 50 per cent - that the vaccine may be dangerous for catde and that they
of vaccinated ewes at lambing; are not to be kept in contact with vaccinated animals.
- the challenge strain is found in not more than 40 per cent ___________________________________________ PhE~
liquid phase of the vaccine by a method that does not vaccine, then one dos e after the interval to be recommended.
inactivate virus nor interfere in any other way with detection After each injection, measure the body temperature on the --
of live viruses. day of the injection and on the 4 following days. Observe the
3-5 Extraneous agents cows at least daily until calving.
Carry out tests for antibodies on the cattle used for the safety The vaccine complies with the test if no cow shows abnormal
test. Take a blood sample at the end of the second local or systemic reactions or dies from causes attributable to
observation periodo The vaccine complies with the test if it the vaccine and if no adverse effects on the pregnancy or the
does not stimulate the formation of antibodies against bovine offspring are noted.
herpesvirus 1 (BHV1), bovine leukaemia virus (BLV) and 2-3-2 Immunogenicity
bovine viral diarrhoea virus (BVDV). A test is carried out for each route and method of
3-6 Potency administration to be recommended. The vaccine
The vaccine complies with the requirements of the test administered to each cow is of minimum potency.
prescribed under Irnmunogenicity (section 2-3-2) when Use for the test not fewer than 15 pregnant cows, preferably
administered by a recommended route and method. that do not have antibodies against bovine rotavirus. Where
4 LABELLING such cows are not available, use cows that: have not been
The label states the recommended schedule for administering vaccinated against bovine rotavirus; come from a farm where
colostrum and milk, post-partum. there is no recent history of infection with bovine rotavirus;
_____________________________________________ ~E~
and have a low level of antibodies against bovine rotavirus,
the levels being comparable in all cows. Vaccinate not fewer
than 10 pregnant cows according to the schedule to be
recommended. Maintain not fewer than 5 pregnant cows as
controls. Starting at calving, take the colostrum and then
milk from each cow and keep it in suitable conditions.
Calf Rotavirus Diarrhoea Vaccine Determine individually the protective activity of the
(Inactivated) colostrum and milk from each cow using calves born from
(Ph Eur monograph 1954) healthy cows, and which may be born by Caesarean section,
PhE~ _____________________________________________ and maintained in an environment where they are not
exposed to infection by bovine rotavirus. Feed colostrum and
1 DEFINITION then milk to each calf every 6 h or according to the
Calf rotavirus diarrhoea vaccine (inactivated) is a preparation recommended schedule. At 5-7 days after birth, challenge
of one or more suitable strains of bovine rotavirus, each calf by the oral route with a sufficient quantity of a
inactivated while maintaining adequate immunogenic virulent strain of bovine rotavirus. Observe the calves at least
properties. This monograph applies to vaccines intended for daily for 7 days. Note the incidence, severity and duration of
the active irnmunisation of dams for passive protection of diarrhoea and the duration and quantity of virus excretion.
their progeny against rotavirus diarrhoea during the first few The vaccine complies with the test if there is a significant
weeks of life. reduction in diarrhoea and virus excretion in calves given
2 PRODUCTION colostrum and milk from vaccinated cows compared to those
2-1 PREPARATION OF THE VACCINE given colostrum and milk from controIs.
Each vaccine virus is grown separately in cell cultures. 2-4 MANUFACTURER'S TESTS
The viral suspensions of each vaccine virus are harvested 2-4-1 Residuallive virus
separately and inactivated by a method that maintains The test for residual live virus is carried out using 2 passages
immunogenicity. The viral suspensions may be purified and in cell cultures of the same type as those used for production
concentrated. The vaccine may be adjuvanted. or in cells shown to be at least as sensitive. The quantity of
2-2 SUBSTRATE FOR VIRUS PROPAGATION inactivated virus harvest used in the test is equivalent to not
2-2-1 Cell cultures less than 100 doses of vaccine. The inactivated viral harvest
The cell cultures comply with the requirements for cell complies with the test if no live virus is detected.
cultures for production ofveterinary vaccines (5.2.4). 2-4-2 Batch potency test
2-3 CHOICE OF VACCINE COMPOSITION It is not necessary to carry out the Potency test (section 3-6)
The vaccine is shown to be satisfactory with respect to safety for each batch of vaccine if it has been carried out using a
(5.2.6) and efficacy (5.2.7) for the pregnant cows for which it batch of vaccine with a minimum potency. Where the test is
is intended. not carried out, an alternative validated method is used, the
The following tests for safety (section 2-3-1) and criteria for acceptance being set with reference to a batch of
immunogenicity (section 2-3-2) may be used during the vaccine that has given satisfactory results in the test described
demonstration of safety and efficacy. under Potency. The following test may be used.
To obtain a valid assay, it may be necessary to carry out a
2-3-1 Safety
test using several groups of animals, each receiving a different
Carry out the test for each route and method of
dose. For each dose required, carry out the test as follows.
administration to be recommended for vaccination, using in
Use for the test not fewer than 7 animals of a suitable species
each case cows that have not been vaccinated against bovine
that do not have antibodies against bovine rotavirus.
rotavirus. U se a batch of vaccine containing not less than the
Vaccinate not fewer than 5 animals, using 1 injection of a
maximum potency that may be expected in a batch of
suitable dose. Maintain not fewer than 2 animals as controls.
vaccine.
Where the recommended schedule requires a booster
For each test, use not fewer than 10 cows at the stage or at injection to be given, a booster vaccination may also be given
different stages of pregnancy according to the schedule to be in this test provided it has been demonstrated that this will
recommended. Administer to each cow a double dose of the
Veterinary Vaccines 219
quantity of the vaccine virus equivalent to not less than The test is invalid if during the observation period after
10 times the maximum virus titre likely to be contained in challenge, fewer than 100 per cent of the control dogs die or
one dose of the vaccine. Observe the bitches at least daily show notable signs of canine adenovirosis. The vaccine virus
until one day after whelping. complies with the test if during the observation period after
The vaccine virus complies with the test if no bitch shows challenge, all the vaccinated dogs survive and show no signs
abnormal local or systemic reactions, signs of disease or dies of disease except for a possible transient elevated rectal
from causes attributable to the vaccine virus and if no temperature.
adverse effects on the pregnancy or the offspring are nóted. 2-3-3-2 Vaccine intended to protect against respiratory signs Use
2-3-2 Increase in virulence for the test not fewer than 20 dogs that do not have
The test for increase in virulence consists of the I
antibodies against canine adenovirus. Vaccinate not fewer
administration of the vaccine virus at the least attem.iated than 10 dogs, according to the schedule to be recommended.
passage level that will be present between the master seed lot Maintain not fewer than 10 dogs as controls. Challenge each
and a batch of the vaccine to 2 dogs 5-7 weeks-old, that do dog after 21 daysby the intranasal route with a quantity of a
not have antibodies against canine adenovirus, sequential suspension of virulent canine adenovirus 2 sufficient to cause
passages, 5 times where possible, to further similar groups typical signs of respiratory disease in a dog that does not
and testing of the -final recovered virus for increase in have antibodies against canine adenovirus. Observe the dogs
virulence. If the properties of the vaccine virus allow at least daily for 10 days after challenge. Record the
sequential passage to 5 groups via natural spreading,this incidence of signs of respiratory and general disease in each
method may be used, otherwise passage as described below is dog (for example, sneezing, coughing, nasal and lachrymal
carried out and the maximally passaged virus that has been discharge, los s of appetite). Collect nasal swabs or washings
recovered is tested for increase in virulence. Care must be from each dog daily from days 2 to 10 after challenge and
taken to avoid contamination by virus from previous test these samples to determine the presence and titre of
passages. excreted virus.
Administer to each dog by a route to be recommended a The vaccine complies with the test if there is a notable
quantity of the vaccine virus that will allow recovery of virus decrease in the incidence and severity of signs and in virus
for the passages described below. Administer the virus by the excretion in vaccinates compared to controls.
route to be recommended for vaccination most likely to lead 3 BATCH TESTS
to reversion of virulence. After 4-6 days, prepare a 3-1 Identification
suspension froro the nasal and pharyngeal mucosa, tonsils, The vaccine mixed with monospecific antiserum against
lung, spleen and if they are likely to contain virus, liver and canine adenovirus 2 no longer infects susceptible cell
kidney of each dog and pool the samples. Administer 1 mL cultures.
of the pooled samples by a suitable route - for example, the
3-2 Bacteria and fungi
intranasal route - to each of 2 other dogs of the same age.
The vaccine and where applicable, the liquid supplied with it,
Carry out this passage operation not fewer than 5 times;
comply with the test for sterility prescribed in the monograph
verify the presence of the virus at each passage. If the virus is
on Vaccines for veterinary use (0062).
not found at a passage level, carry out a second series of
passages. 3-3 Mycoplasmas (2.6. 7)
The vaccine complies with the test for mycoplasmas.
Carry out the test for safety (section 2-3-1) using unpassaged
vaccine virus and the maximally passaged virus that has been 3-4 Extraneous agents
recovered. Neutralise the vaccine virus with a suitable monospecific
The vaccine virus complies with the test if no indication of antiserum against canine adenovirus 2 and inoculate into cell
increased virulence of the maximally passaged virus cultures known for their susceptibility to virus es pathogenic
compared with the unpassaged virus is observed. If virus is for the dogo Carry out a passage after 6-8 days and maintain
not recovered at any passage level in the first and second the cultures for a total of 14 days. The vaccine complies with
series of passages, the vaccine virus also complies with the the test if no cytopathic effect develops and there is no sign
test. of the presence of haemadsorbing agents.
representative batch using a vaccinating dos e containing not from causes attributable to the vaccine virus and if no
more than the minimum virus titre stated on the label. adverse effects on the pregnancy or the offspring are noted.
_____________________________________________ PhE~
2-3-2 Increase in virulence
The test for increase in virulence consists of the
administration of the vaccine virus at the least attenuated
passage level that will be present between the master seed lot
Canine Distemper Vaccine, Living and a batch of the vaccine to 2 dogs 5-7 weeks-old, that do
not have antibodies against canine distemper virus, sequential
(Canine Distemper Vaccine (Live)~ passages, 5 times where possible, to further similar groups
Ph Eur monograph 0448) and testing of the final recovered virus for increase in
PhE~ _____________________________________________ virulence. If the properties of the vaccine virus allow
sequential passage to 5 groups via natural spreading, this
1 DEFINITION method may be used, otherwise passage as described below is
Canine distemper vaccine (live) is a preparation of a suitable carried out and the maximally passaged virus that has been
strain of distemper virus. This monograph applies to vaccines recovered is tested for increase in virulence. Care must be
intended for the active immunisation of dogs against canine taken to avoid contamination by virus from previous
distemper. passages.
2 PRODUCTION Administer to each dog by a route to be recommended a
2-1 PREPARATION OF THE VACCINE quantity of the vaccine virus that will allow recovery of virus
The vaccine virus is grown in embryonated hens' eggs or in for the passages described below. Administer the virus by the
cell cultures. route to be recommended for vaccination most likely to lead
2-2 SUBSTRATE FOR VIRUS PROPAGATION to reversion to virulence. After 5-10 days, prepare a
suspension from the nasal mucosa, tonsils, thymus, spleen
2-2-1 Embryonated hens' eggs
and the lungs and their local lymph nodes of each dog and
If the vaccine virus is grown in embryonated hens' eggs, they
pool the samples. Administer 1 mL of the pooled samples by
are obtained from flocks free from specified pathogens (SPF)
the intranasal route to each of 2 other dogs of the same age.
(5.2.2).
Carry out this passage operation not fewer than 5 times;
2-2-2 Cell cultures verify the presence of the virus at each passage. If the virus is
If the vaccine virus is grown in cell cultures, they comply not found at a passage level, carry out a second series of
with the requirements for cell cultures for production of passages.
veterinary vaccines (5.2.4). Carry out the test for safety (section 2-3-1) using unpassaged
2-3 CHOICE OF VACCINE VIRUS vaccine virus and the maximally passaged virus that has been
The vaccine virus is shown to be satisfactory with respect to recovered.
safety (5.2.6) and efficacy (5.2. 7) for the dogs for which it is The vaccine virus complies with the test if no indication of
intended. increased virulence of the maximally passaged virus
The following tests for safety (section 2-3-1), increase in compared with the unpassaged virus is observed. If virus is
virulence (section 2-3-2) and immunogenicity (2-3-3) may be not recovered at any passage level in the first and second
used during the demonstration of safety and efficacy. series of passages, the vaccine virus also complies with the
2-3-1 Safety test.
Carry out the test for each route and method of 2-3-3 Immunogenicity
administration to be recommended for vaccination. A test is carried out for each route and method of
U se vaccine virus at the least attenuated passage level that administration to be recommended for vaccination using in
will be present between the master seed lot and a batch of each case dogs 8-16 weeks old. The quantity of vaccine virus
the vaccine. to be administered to each dog is not greater than the
2-3-1-1 General safety For each test, use not fewer than 5 minimum virus titre to be stated on the label and the virus is
dogs of the minimum age to be recommended for at the most attenuated passage level that will be present in a
vaccination, that do not have antibodies against canine batch of vaccine.
distemper virus. Administer to each dog a quantity of the U se for the test not fewer than 7 dogs that do not have
vaccine virus equivalent to not less than 10 times the antibodies against canine distemper virus. Vaccinate not
maximum virus titre likely to be contained in one dose of the fewer than 5 dogs according to the schedule to be
vaccine. Observe the dogs at least daily for 42 days. recommended. Maintain not fewer than id.ogs as controls.
The vaccine virus complies with the test if no dog shows Challenge each dog after 21 days by the intravenous route
abnormallocal or systemic reactions, signs of disease or dies with a sufficient quantity of a suspension of virulent canine
from causes attributable to the vaccine virus. distemper virus. Observe the dogs at least daily for 21 days
2-3-1-2 Safety in pregnant bitches If the vaccine is intended after challenge. Dogs displaying typical signs of serious
for use in pregnant bitches, use not fewer than 5 bitches at infection\ with canine distemper virus are euthanised to avoid
the stage of pregnancy to be recommended or at a range of unneces~ary suffering.
stages of pregnancy according to the schedule to be The test is invalid if during the observation period after
recommended. Administer to each bitch a quantity of the challenge, fewer than 100 per cent of the control dogs die or
vaccine virus equivalent to not less than 10 times the show notable signs of canine distemper. The vaccine virus
maximum virus titre likely to be contained in one dose of the complies with the test if during the observation period after
vaccine. Observe the bitches at least dai1y until one day after challenge, all the vaccinated dogs survive and show no signs
whelping. of disease.
The vaccine virus complies with the test if no bitch shows
abnormallocal or systemic reactions, signs of disease or dies
Veterinary Vaccines 223
Carry out the test for safety (section 2-3-1) using unpassaged 3-6 Virus titre
vaccine virus and the maximally passaged virus that has been Titrate the vaccine virus in suitable cell cultures. The vaccirie
recovered. complies with the test if one dose contains not less than the
The vaccine virus complies with the test if no indication of minimum virus titre stated on the label.
increased virulence of the maximally passaged virus 3-7 Potency
compared with the unpassaged virus is observed. If virus is The vaccine complies with the requirements of the test
not recovered at any passage level in the first and second prescribed under Irnmunogenicity (section 2-3-3) when
series of passages, the vaccine virus also complies with the administered by a recommended route and method. It is not
test. necessary to carry out the potency test for each batch of the
2-3-3 Immunogenicity vaccine if it has been carried out on a representative batch
A test is carried out for each route and method of using a vaccinating dose containing not more than the
administration to be recommended for vaccination, using in minimum virus titre stated on the label.
each case dogs of the minimum age to be recommended. _____________________________________________ PhE~
the faeces is less than 1/100 of the geometric mean of the 3-4-2 Test in dogs for virus-neutralising antibodies Use for the
maximum titres found in the controls. test not fewer than 2 healthy dogs, 8-12 weeks old, that have
2-4 MANUFACTURER'S TESTS antibody titres less than 4 ND so per 0.1 mL of serum,
measured by the method described below. Vaccinate each
2-4-1 Residual Uve virus dog according to the schedule to be recommended. 14 days
A test for residuallive virus is carried out on the bulk harvest after vaccination, examine the serum of each dog as follows.
of each batch to confirm inactivation of the canine Heat the serum at 56 oC for 30 min and prepare serial
parvovirus. The quantity of inactivated virus harvest used in dilutions using a medium suitable for canine cells. Add to
the test is equivalent to not less than 100 doses of the each dilution an equal volume of a virus suspension
vaccine. The inactivated viral harvest is inoculated into containing an amount of virus such that when the volume of
suitable non-confluent cells; after incubation for 8 days, a serum-virus mixture appropriate for the assay system is
sub culture is made using trypsinised cells. After inc\lbation inoculated into cell cultures, each culture receives
for a further 8 days, the cultures are examined for residual approximately 104 CCID so . Incubate the mixtures at 37 oC
live parvovirus by an immunofluorescence test. for 1 h and inoculate 4 canine cell cultures with a suitable
The immunofluorescence test may be supplemented by a volume of each mixture. Incubate the cell cultures at 37 oC
haemagglutination test or other suitable tests on the for 7 days, passage and incubate for a further 7 days.
supernatant of the cell cultures. The inactivated viral harvest Examine the cultures for evidence of specific cytopathic
complies with the-test if no live virus is detected. effects and calculate the antibody titre. The vaccine complies
3 BATCH TESTS with the test if the mean titre is not less than 32 ND sO per
3-1 Identification 0.1 mL of serum. If one dog fails to respond, repeat the test
When injected into animals that do not have antibodies using 2 more dogs and calculate the result as the mean of the
against canine parvovirus, the vacc1ne stimulates the titres obtained from all of the 3 dogs that have responded.
production of such antibodies. ___________________________________________ PhE~
one dose of the vaccine. Observe the dogs at least daily for the label and the virus is at the most attenuated passage level
21 days. A count of white blood cells in circulating blood is that will be present in a batch of vaccine.
made on days 3, 5, 7 and 10 after the injection. U se for the test not fewer than 7 dogs that do not have
The test is invalid if a diminution in the number of haemagglutination-inhibiting antibodies against canine
circulating white blood cells greater than 50 per cent of the parvovirus. Vaccinate not fewer than 5 dogs. Maintain not
initial number - deterrnined as the average of the 3 values fewer than 2 dogs as controls. Challenge each dog after
found before injection of the vaccine strain - is noted. 20-22 days by the oronasal route with a sufficient quantity of
The vaccine virus complies with the test if no dog shows a suspension of virulent canine parvovirus. Observe the dogs
abnorrnal local or systemic reactions, notable signs of disease at least daily for 14 days after challenge. At the end of the
or die s from causes attributable to the vaccine virus. observation period, carry out a haemagglutination test for
2-3-1-2 Effects on the thymus For each test, use not fewer and titration of the virus in the faeces.
than 10 dogs that do not have haemagglutination-inhibiting The test is invalid if fewer than 100 per cent of the control
antibodies against canine parvovirus. Adrninister to each of dogs show typical signs of the disease andlor leucopenia and
not fewer than 5 dogs a quantity of the vaccine virus excretion of the virus. The vaccine virus complies with the
equivalent to not less than 10 times the maximurn virus titre test if all the vaccinated dogs survive and show no sign of
likely to be contained in one dos e of the vaccine. Maintain disease nor leucopenia and if the maximum titre of virus
not fewer than 5 dogs as controls. Observe the dogs at least excreted in the faeces is less than 1/100 of the geometric
daily. After 14 days, euthanise 2 dogs from each group and mean of the maximurn titres found in the controls.
after 21 days, the remaining dogs from each group. Carry out 3 BATCH TESTS
histological examination of the thyrnus of each dogo
3-1 Identification
The vaccine virus complies with the test if there is no more The vaccine is grown in a susceptible cellline in a substrate
than slight hypoplasia of the thyrnus after 14 days and no suitable for presenting for fluorescent antibody or
damage is evident after 21 days. irnrnunoperoxidase tests. Suitable controls are included.
2-3-2 Increase in virulence A proportion of the cells is tested with a monoclonal
The test for increase in virulence consists of the antibody specific for canine parvovirus and a proportion of
administration of the vaccine virus at the least attenuated the cells tested with a monoclonal antibody specific for feline
passage level that will be present between the master seed lot parvovirus. Canine parvovirus antigen is detected but no
and a batch of the vaccine to 2 dogs of the minimum age to feline parvovirus is detected in the cells inoculated with the
be recommended for vaccination, that do not have vaccine.
haemagglutination-inhibiting antibodies against canine 3-2 Bacteria and fungi
parvovirus, sequential passages, 5 times where possible, to The vaccine and, where applicable, the liquid supplied with it
further similar groups and testing of the final recovered virus comply with the test for sterility prescribed in the monograph
for increase in virulence. If the properties of the vaccine virus Vaccines for veterinary use (0062).
allow sequential passage to 5 groups via natural spreading,
3-3 Mycoplasmas (2.6. 7)
this method may be used, otherwise passage as described
The vaccine complies with the test for mycoplasmas.
below is carried out and the maximally passaged virus that
has been recovered is tested for increase in virulence. Care 3-4 Extraneous agents
must be taken to avoid contamination by virus from previous Neutralise the vaccine virus with a suitable monospecific
passages. antiserum against canine parvovirus and inoculate into cell
Adrninister to each dog by a route to be recommended a cultures known for their susceptibility to virus es pathogenic
quantity of the vaccine virus that will allow recovery of virus for the dogo The vaccine complies with the test if no
for the passages described below. Collect the faeces from cytopathic effect develops and there is no sign of
each dog from the 2nd to the 10th day after administration of haemagglutinating or haemadsorbing agents and no other
the virus, check them for the presence of the virus and pool sign of the presence of extraneous viruses.
the faeces containing virus. Adrninister 1 mL of the 3-5 Safety
suspension of pooled faeces by the oronasal route to each of U se 2 dogs of the minimum age recommended for
2 other dogs of the same age. Carry out this passage vaccination and that do not have haemagglutination-
operation not fewer than 5 times; verify the presence of the inhibiting antibodies against canine parvovirus. Adrninister to
virus at each passage. If the virus is not found at a passage each dog by a recommended route 10 doses of the vaccirie.
level, carry out a second series of passages. Observe the dogs at least daily for 14 days.
The vaccine virus complies with the test if no indication of The vaccine complies with the test if no dQg shows notable
increased virulence of the maximally passaged virus signs of disease or dies from causes attributable to the
compared with the unpassaged virus is observed; account is vaccine.
taken, notably, of the count of white blood cells, of results of 3-6 Virus titre
histological examination of the thyrnus and of the titre of Titrate the vaccine virus by inoculation into suitable cell
excreted virus. If virus is not recovered at any passage level in cultures. The vaccine complies with the test if one dose
the first and second series of passages, the vaccine virus also contains mot less than the minimum virus titre stated on the
complies with the test. labe!'
2-3-3 Irnmunogenicity 3-7 Potency
A test is carried out for each route and method of The va~cine complies with the requirements of the test
administration to be recommended using in each case dogs prescribed under Irnrnunogenicity (section 2-3-3) when
of the minimurn age to be recommended for vaccination. adrninistered by a recommended route and method. It is not
The quantity of vaccine virus to be adrninistered to each dog necessary to carry out the potency test for each batch of the
is not greater than the minimum virus titre to be stated bn vaccine if it has been carried out on a representative batch
Veterinary Vaccines 227
using a vaccinating dose containing not more than the route, twice the maximum recommended dose. Observe the
minimum virus titre stated on the labe!' animals at least daily for 7 days.
___________________________________________ ~E~ The vaccine complies with the test if no animal shows
notable signs of disease or dies from causes attributable to
the vaccine.
3-4 Residual toxicity
Inject 0.5 mL of the vaccine by the subcutaneous route into
Clostridium Botulinum Vaccine each of 5 mice, each weighing 17-22 g. Observe the animals
Botulinum Vaccine at least daily for 7 days.
(Clostridium Botulinum Vaccine for Veterinary UseJ The vaccine complies with the test if no animal shows
Ph Bur monograph 0360) notable signs of disease or dies from causes attributable to
When Clostridium Botulinum Vaccine or Botulinum Vaccine the vaccine.
is prescribed or demanded and the types to be present are 3-5 Potency.
not stated, Clostridium Botulinum Vaccine prepared from U se for the test healthy white mice from a uniform stock,
types C and D shall be dispensed or supplied. each weighing 18-20 g. Use as challenge dose a quantity of a
PhE~ ___________________________________________ toxin of C. botulinum of the same type as that used in the
preparation of the vaccine corresponding to 25 times the
1 DEFINITION paralytic dose 50 per cent, a paralytic dose 50 per cent being
Clo~tridium botulinum vaccine for veterinary use is prepared the quantity of toxin which, when injected by the
from liquid cultures of suitable strains of Clostridium intraperitoneal route into mice, causes paralysis in
botulinum type C or type D or a mixture of these types. 50 per cent of the animals within an observation period of
The whole culture or its filtra te or a mixture of the two is 7 days. If 2 types of C. botulinum have been used in the
inactivated to eliminate its toxicity while maintaining preparation of the vaccine, carry out the potency
adequate irnmunogenic properties. This monograph applies determination for each. Dilute the vaccine to be examined
to vaccines intended for active irnmunisation of animal s 1 in 8 using a 9 gIL solution of sodium chloride R. Inject
against botulismo 0.2 mL of the dilution subcutaneously into each of 20 mice.
2 PRODUCTION After 21 days, inject the challenge dos e by the intraperitoneal
2-1 PREPARATION OF THE VACCINE route into each of the vaccinated mice and into each of
C. botulinum used for production is grown in an appropriate 10 control mice. Observe the mice for 7 days and record the
liquid medium. number of animals which show signs of botulismo The test is
not valid unless all the control mice show signs of botulism
The preparation may be adsorbed, precipitated or
during the observation periodo The vaccine complies with the
concentrated. It may be treated with a suitable adjuvant and
test if not fewer than 80 per cent of the vaccinated mice are
may be freeze-dried.
protected.
2-2 CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfory with respect to safety 4 LABELLING
(5.2.6) and efficacy (5.2.7) for the animals for which it is The label states:
intended. -- the type or types of C. botulinum from which the vaccine
has been prepared,
2-3 MANUFACTURER'S TESTS -- whether the preparation is a toxoid or a vaccine prepared
2-3-1 Batch potency test from a whole inactivated culture or a mixture of the two.
It is not necessary to carry out the Potency test (section 3-5) ___________________________________________ PhE~
for each batch of the vaccine if it has been carried out using
a batch of vaccine with a minimum potency. Where the test
is not carried out, an altemative validated method is used,
the criteria for acceptance being set with reference to a batch
of vaccine that has given satisfactory results in the test Clostridium Chauvoei Vaccine
described under Potency.
Blackleg Vaccine
3 BATCH TESTS
The identification, the tests and the determination of potency (Clostridium Chauvoei Vaccine for Veterinary UseJ
apply to the liquid preparation and to the freeze-dried Ph Bur monograph 0361)
preparation reconstituted as stated on the labe!' ~E~ ___________________________________________
2-2 CHOICE OF VACCINE COMPOSITION The whole culture or its filtrate or a mixture of the 2 is
The vaccine is shown to be satisfactory with respect to safety inactivated to eliminate its toxicity while maintaining
(5.2.6) and efficacy (5.2.7) for the animals for which it is adequate irnmunogenic properties. This monograph applies
intended. to vaccines intended for active immunisation of animals
3 BATCH TESTS and/or to protect passively their progeny against disease
caused by C. novyi (type B).
3-1 Identification
The vaccine protects susceptible animals against infection 2 PRODUCTION
with C. chauvoei. The potency test may also serve for 2-1 PREPARATION OF THE VACCINE
identification. C. novyi (type B) used for production is grown in an
3-2 Bacteria and fungi appropriate liquid medium. Toxoids and/or inactivated
The vaccine and, where applicable, the liquid supplied with it cultures may be treated with a suitable adjuvant, after
comply with the test for sterility prescribed in the monograph concentration if necessary.
on Vaccines for veterinary use (0062). 2-2 CHOICE OF VACCINE COMPOSITION
3-3 Safety The vaccine is shown to be satisfactory with respect to safety
U se 2 animals of one of the species for which the vaccine is (5.2.6) and efficacy (5.2.7) for the animal s for which it is
intended and that have not been vaccinated against C. intended. For the latter, it shall be demonstrated that for
chauvoei. Administer to each animal at a single site, by a each target species the vaccine, when administered according
recommended route, twice the maximum recommended to the recommended schedule, stimulates an immune
dose. Observe the animals at least daily for 7 days. response (for example, induction of antibodies) consistent
with the c1aims made for the producto
The vaccine complies with the test if no animal shows
notable signs of disease or dies from causes attributable to 2-3 MANUFACTURER'S TESTS
the vaccine. 2-3-1 Residual toxicity
3-4 Potency A test for detoxification is carried out immediately after the
U se for the test not fewer than 10 healthy guinea-pigs, each detoxification process and, when there is risk of reversion, a
nd
weighing 350-450 g. Administer to each guinea-pig by the 2 test is carried out at as late a stage as possible during the
subcutaneous route a quantity of the vaccine not greater than production process. The test for residual toxicity (section
the minimum dose stated on the label as the first dose. After 3-4) may be omitted by the manufacturero
28 days, administer into the same animal s a quantity of the 2-3-2 Batch potency test
vaccine not greater than the minimum dos e stated on the It is not necessary to carry out the Potency test (section 3-5)
label as the second dose. 14 days after the second for each batch of vaccine if it has been carried out using a
vaccination, inoculate by the intramuscular route into each of batch of vaccine with a minimum potency.
the vaccinated guinea-pigs and into each of 5 control animals Where the test is not carried out, an alternative validated
a suitable quantity of a virulent culture, or of a spore method is used, the criteria for acceptance being set with
suspension, of C. chauvoei, activated if necessary with an reference to a batch of vaccine that has given satisfactory
activating agent such as calcium chloride. results in the test described under Potency and that has been
The vaccine complies with the test if not more than shown to be satisfactory with respect to irnmunogenicity in
10 per cent of the vaccinated guinea-pigsdie from C. the target species. The following test may be used after a
chauvoei infection within 5 days and all the control animals satisfactory correlation with the test under Potency (section
die from C. chauvoei infection within 48 h of challenge or 3':'5) has been established.
within 72 h if a spore suspension was used for the challenge. Vaccinate rabbits as described under Potency and prepare
If more than 10 per cent but not more than 20 per cent of sera. Determine the level of antibodies against the alpha toxin
the vaccinated animal s die, repeat the test. The vaccine of C. novyi in the individual sera by a suitable method such
complies with the test if not more than 10 per cent of the asan irnmunochemical method (2.7.1) or neutralisation in
second group of vaccinated animals die within 5 days and all cell cultures. Use a homologous reference serum calibrated in
of the second group of control animals die within 48 h of International Units of C. novyi alpha antitoxin. Clostridia
challenge or within 72 h if a spore suspension was used for (multicomponent) rabbit antiserum BRP is suitable for use as a
the challenge. To avoid unnecessary suffering following reference serum.
virulent challenge, moribund animals are euthanised and are
The vaccine complies with the test if the leve! of antibodies is
then considered to have died from C. chauvoei infection.
not less than that found for a batch of vaccine that has given
___________________________________________ PhE~
3-3 Safety mixtures to stand at room temperature for 60 min. Using not
U se 2 animals of 1 of the species for which the vaccine is fewer than 2 mice, each weighing 17-22 g, for each mixture,
intended and that have not been vaccinated against C. novyi inject adose of 0.2 mL by the intramuscular or the
(type B). Administer to each animal, by a recommended subcutaneous route into each mouse. Observe the mice for
route, twice the maximum recommended dose of the vaccine. 72 h. If all the mice die, the amount of toxin present in
Observe the animals at least daily for not less than 14 days. 0.2 mL of the mixture is in excess of the test dose. If none of
The vaccine complies with the test if no animal shows the mice die, the amount of toxin present in 0.2 mL of the
notable signs of disease or dies from causes attributable to mixture is less than the test dose. Prepare fresh mixtures
the vaccine. such that 2.0 mL of each mixture contains 1.0 mL of the
solution of the reference preparation (1 IU) and 1 of a series
3-4 Residual t o x i c i t y ,
of graded volumes of the solution of the test toxin separated
Administer 0.5 mL of the vaccine by the subcutaneous route
from each other by steps of not more than 20 per cent and
into each of 5 mice, each weighing 17-22 g. Observe the
covering the expected end-point. Allow the mixtures to stand
animals at least daily for 7 days.
at room temperature for 60 mino Using not fewer than 2
The vaccine complies with the test if no animal shows mice for each mixture, inject adose of 0.2 mL by the
notable signs of disease or dies from causes attributable to intramuscular or the subcutaneous route into each mouse.
the vaccine. Observe the mice for 72 h. Repeat the determination at least
3-5 Potency once and combine the results of the separate tests that have
Use for the test not fewer than 10 healthy rabbits, been made with mixtures of the same composition so that a
3-6 months old. Administer to each rabbit by the series of total s is obtained, each total representing the
subcutaneous route a quantity of vaccine not greater than the mortality due to a mixture of a given composition.
minimum dos e stated on the label as the first dose. After The test dos e of toxin is the amount present in 0.2 mL of
21-28 days, administer to the same animals a quantity of the that mixture which causes the death of one half of the total
vaccine not greater than the minimum dose stated on the number of mice injected with it.
label as the 2nd dose. 10-14 days after the 2nd injection, bleed 3-5-3 Determination of the potency of the serum obtained from
the rabbits and pool the sera.
rabbits Preliminary test Dissolve a quantity of the test toxin
The vaccine complies with the test if the potency of the in a suitable liquid so that 1.0 mL contains 10 times the test
pooled sera is not less than 3.5 IU/mL. dose (solution of the test toxin). Prepare a series of mixtures
The International Unit is the specific neutralising activity for of the solution of the test toxin and of the serum to be
C. novyi alpha toxin contained in a stated amount of the examined such that each mixture contains 1.0 mL of the
International Standard, which consists of a quantity of dried solution of the test toxin, 1 of a series of graded volumes of
immune horse serum. The equivalence in International Units the serum to be examined and sufficient of a suitable liquid
of the International Standard is stated by the World Health to bring the final volume to 2.0 mL. Allow the mixtures to
Organisation. stand at room tempel'ature for 60 min. U sing not fewer than
The potency of the pooled sera obtained from the rabbits is 2 mice for each mixture, inject adose of 0.2 mL by the
determined by comparing the quantity necessary to protect intramuscular or the subcutaneous route into each mouse.
mice or other suitable animals against the toxic effects of a Observe the mice for 72 h. If none of the mice dies, 0.2 mL
fixed dose of C. novyi alpha toxin with the quantity of a of the mixture contains more than 0.1 IU. If all the mice die,
reference preparation of Clostridium novyi alpha antitoxin, 0.2 mL ofthe mixture contains less than 0.1 IU.
calibrated in International Units, necessary to give the same Final test Prepare a series of mixtures of the solution of the
protection. For this comparison, a suitable preparation of C. test toxin and the serum to be examined such that 2.0 mL of
novyi alpha toxin for use as a test toxin is required. The dose each mixture contains 1.0 mL of the solution of the test
of the test toxin is determined in relation to the reference toxin and 1 of a series of graded volumes of the serum to be
preparation; the potency of the serum to be examined is examined, separated from each other by steps of not more
determined in relation to the reference preparation using the than 20 per cent and covering the expected end-point as
test toxin. determined by the preliminary test. Prepare further mixtures
Clostridia (multicomponent) rabbit antiserum BRP is suitable for of the solution of the test toxin and of the solution of the
use as a reference serum. reference preparation such that 2.0 mL of each mixture
3-5-1 Preparation of test toxin Prepare the test toxin from a
contains 1.0 mL of the solution of the test toxin and 1 of a
sterile filtrate of an approximately 5-day culture in liquid series of graded volumes of the solution of the reference
preparation, in order to confirm the test dose of the toxin.
medium of C. novyi type B and dry by a suitable method.
Allow the mixtures to stand at room temperature for 60 min.
Select the test toxin by determining for mice the L+/10 dose
and the LD so, the observation period being 72 h. U sing not fewer than 2 mice for each mixture, proceed as
described in the preliminary test.
A suitable alpha toxin contains not less than one L+/10 dose
in 0.05 mg and not less than 10 LDso in each L+/10 dose. The test mixture which contains 0.1 IV in 0.2 mL is that
mixture which IdUs the same or almost the same number of
3-5-2 Determination of test dose of toxin Prepare a solution of mice as the reference mixture containing 0.1 IV in 0.2 mL.
the reference preparation in a suitable liquid so that it Repeat the determination at least once and calculate the
contains 1 IU/mL. Prepare a solution of the test toxin in a average of all valid estimates. The test is valid only if the
suitable liquid so that 1 mL contains a precisely known reference preparation gives a result within 20 per cent of the
amount such as 1 mg. Prepare mixtures of the solution of the expected value.
reference preparation and the solution of the test toxin such
The confidence limits (P = 0.95) have been estimated to be:
that each mixture contains 1.0 mL of the solution of the
- 85 per cent and 114 per cent when 2 animals per dose
reference preparation (1 IU), 1 of a series of graded volumes
of the solution of the test toxin and sufficient of a suitable are used,
liquid to bring the total volume to 2.0 mL. Allow the
230 Veterinary Vaccines
-- 91.5 per cent and 109 per cent when 4 animals per dose Where the test is not carried out, an alternative validated
are used, method is used, the criteria for acceptance being set with
-- 93 per cent and 108 per cent when 6 animals per dose reference to a batch of vaccine that has given satisfactory
are used. results in the test described under Potency and that has been
shown to be satisfactory with respect to immunogenicity in
4 LABELLING
the target species. The following test may be used after a
The label states:
satisfactory correlation with the test under Potency (section
-- whether the product is a lOxoid, a vaccine prepared from
3-5) has been established.
a whole inactivated culture or a mixture of the 2,
-- for each target species, the immunising effect produced Vaccinate rabbits as described under Potency and prepare
(for example, antibody production, protection against sera. Determine the level of antibodies against the beta
signs of disease or infection). and/or epsilon toxins of C. perfringens in the individual sera
___________________________________________ ~Ew
by a suitable method such as an immunochemical method
(2.7.1) or neutralisation in cell cultures. Use a homologous
reference serum calibrated in International Units of C.
perfringens beta and/or epsilon antitoxin. Clostridia
(multicomponent) rabbit antiserum BRP is suitable for use as a
reference serum.
Clostridium Perfringens Vaccines The vaccine complies with the test if the level or levels of
(Clostridium Perfringens Vaccine for Veterinary Use~ antibodies are not les s than that found for a batch of vaccine
Ph Eur monograph 0363) that has given satisfactory results in the test described under
The following titles may be used for appropriate vaccines: Potency and that has been shown to be satisfactory with
Clostridium Perfringens Type B Vaccine; Lamb Dysentery respect to immunogenicity in the target species.
Vaccine; 3 BATCH TESTS
Clostridium Perfringens Type C Vaccine; Struck Vaccine; 3-1 Identification
Clostridium Perfringens Type D Vaccine; Pulpy Kidney Type B When injected into animals that do not have beta
Vaccine and epsilon antitoxins, the vaccine stimulates the formation
~Ew ____________ ~ _____________________________
of such antitoxins.
Type C When injected into animals that do not have beta
1 DEFINITION antitoxin, the vaccine stimulates the formation of such
Clostridium perfringens vaccine for veterinary use is prepared antitoxin.
from liquid cultures of suitable strains of Clostridium Type D When injected into animals that do not have
perfringens type B, C. perfringens type C or C. perfringens type epsilon antitoxin, the vaccine stimulates the formation of
D or a mixture of these types. such antitoxin.
The whole cultures or their filtrates or a mixture of the two
3-2 Bacteria and fungi
are inactivated to eliminate their toxicity while maintaining
The vaccine and, where applicable, the liquid supplied with it
adequate immunogenic properties. This monograph applies
comply with the test for sterility prescribed in the monograph
lO vaccines intended for active immunisation of animal s
on Vaccines for veterinary use (0062).
andlor to protect passively their progeny against disease
caused by C. perfringens. 3-3 Safety
U se 2 animals of one of the species for which the vaccine is
2 PRODUCTION intended and that have not been vaccinated against C.
2-1 PREPARATION OF THE VACCINE perfringens. Administer by a recommended route to each
C. perfringens used for production is grown in an appropriate animal, twice the maximum recommended dose. Observe the
liquid medium. Toxoids andlor inactivated cultures may be animals at least daily for 14 days.
treated with a suitable adjuvant.
The vaccine complies with the test if no animal shows
2-2 CHOICE OF VACCINE COMPOSITION notable signs of disease or dies from causes attributable to
The vaccine is shown to be satisfaclOry with respect to safety the vaccine.
(5.2.6) and efficacy (5.2.7) for the animals for which it is
3-4 Residual toxicity
intended. For the latter, it shall be demonstrated that for
Administer 0.5 mL of the vaccine by the subcutaneous route
each target species the vaccine, when administered according
into each of 5 mice, each weighing 17-22 g. Observe the
to the recommended schedule, stimulates an immune mice at least daily for 7 days. ..-.
response (for example, induction of antibodies) consistent
with the claims made for the producto The vaccine complies with the test if no animal shows
notable signs of disease or dies from causes attributable to
2-3 MANUFACTURER'S TESTS
the vaccine.
2-3-1 Residual toxicity 3-5 Potency
A test for detoxification is carried out immediately after the
U se for ili~ test not fewer than 10 healthy rabbits,
detoxification process and, when there is risk of reversion, a
3-6 months old. Administer to each rabbit by the
second test is carried out at as late a stage as possible during
subcutaneous route a quantity of vaccine not greater than the
the production process. The test for residual toxicity (section
minimúm dose stated on the label as the first dose. After
3-4) may be omitted by the manufacturero
21 to 28 days, administer to the same animals a quantity of
2-3-2 Batch potency test the vaccine not greater than the minimum dose stated on the
It is not necessary to carry out the Potency test (section 3-5) label as the second dose. lOto 14 days after the second
for each batch of vaccine if it has been carried out using a injection, bleed the rabbits and pool the sera.
batch of vaccine with a minimum potency.
Veterinary Vaccines 231
Type B The vaccine complies with the test if the potency of fewer than 2 mice, each weighing 17-22 g, for each mixture,
the pooled sera is not les s than 10 IU of beta antitoxin and inject adose of 0.5 mL by the intravenous or the
not les s than 5 IU of epsilon antitoxin per millilitre. intraperitoneal route to each mouse. Observe the mice for
Type C The vaccine complies with the test if the potency of 72 h. If all the mice die, the amount of toxin present in
the pooled sera is not less than 10 IU of beta antitoxin per 0.5 mL of the mixture is in excess of the test dose. If none of
millilitre. the mice dies the amount of toxin present in 0.5 mL of the
Type D The vaccine complies with the test if the potency of mixture is less than the test dose. Prepare fresh mixtures
the pooled sera is not les s than 5 IU of epsilon antitóxin per such that 5.0 mL of each mixture contains 2.0 mL ofthe
millilitre. solution of the reference preparation and one of a series of
graded volumes of the solution of the test toxin separated
3-5-1 International standard for Clostridium peifringm~ beta
from each other by steps of not more than 20 per cent and
antitoxin The International Unit is the specific neD.tralising
covering the expected end-point. Allow the mixtures to stand
activity for C. peifringens beta toxin contained in a stated
at room temperature for 30 mino Using not fewer than 2
amount of the International Standard which consists of a
mice for each mixture, inject adose of 0.5 mL by the
quantity of dried immune horse serum. The equivalence in
intravenous or the intraperitoneal route to each mouse.
International Units of the Intemational Standard is stated by
Observe the mice for 72 h. Repeat the determination at least
the W orld HealthOrganisation.
once and add together the results of the separate tests that
3-5-2 International standard for Clostridium peifringens epsilon have been made with mixtures of the same composition so
antitoxin The International Unit is the specific neutralising that a series of totals is obtained, each total representing the
activity for C. peifringens epsilon toxin contained in a stated mortality due to a mixture of given composition.
amount of the Intemational Standard which consists of a
The test dos e of toxin is the amount present in 0.5 mL of
quantity of dried immune horse serum. The equivalence in
that mixture which causes the death of one half of the total
Intemational Units of the Intemational Standard is stated by
number of mice injected with it.
the World Health Organisation.
3-5-5 Determination of the potency of the serum obtained from
The potency of the pooled sera obtained from the rabbits is rabbits Preliminary test Dissolve a quantity of the test toxin
determined by comparing the quantity necessary to protect in a suitable liquid so that 2.0 mL contains 10 times the test
mice or other suitable animals. against the toxic effects of a
dos e (solution of the test toxin). Prepare a series of mixtures
fixed dose of C. peifringens beta toxin or C. peifringens epsilon
of the solution of the test toxin and of the serum to be
toxin with the quantity of a reference preparation of
examined such that each contains 2.0 mL of the solution of
Clostridium perfringens beta antitoxin or Clostridium the test toxin, one of a series of graded volumes of the serum
perfringens epsilon antitoxin, as appropriate, calibrated in
to be examined and sufficient of a suitable liquid to bring the
Intemational Units, necessary to give the same protection.
final volume to 5.0 mL. Allow the mixtures to stand at room
For this comparison, a suitable preparation of C. peifringens temperature for 30 mino Using not fewer than 2 mice for
beta or epsilon toxin for use as a test toxin is required. each mixture, inject adose of 0.5 mL by the intravenous or
The dos e of the test toxin is determined in relation to the
the intraperitoneal route into each mouse. Observe the mice
appropriate reference preparation; the potency of the serum for 72 h. If none of the mice die, 0.5 mL of the mixture
to be examined is determined in relation to the appropriate contains more than 1 IU of beta antitoxin or 0.1 IU of
reference preparation using the appropriate test toxin.
epsilon antitoxin. If all the mice die, 0.5 mL of the mixture
Clostridia (multicomponent) rabbit antiserum BRP is suitable for contains less than 1 IU of beta antitoxin or 0.1 IU of epsilon
use as a reference serum. antitoxin.
3-5-3 Preparation of test toxin Prepare the test toxin from a Final test Prepare a series of mixtures of the solution of the
sterile filtrate of an early culture in liquid medium of C. test toxin and the serum to be examined such that 5.0 mL of
peifringens type B, type C or type D as appropriate and dry each mixture contains 2.0 mL of the solution of the test
by a suitable method. Use a beta or epsilon toxin as toxin and one of a series of graded volumes of the serum to
appropriate. Select the test toxin by determining for mice the be examined separated from each other by steps of not more
L+ and the LDso for the beta toxin and the L+/IO dos e and than 20 per cent and covering the expected end-point as
the LDso for the epsilon toxin, the observation period being determined by the preliminary test. Prepare further mixtures
72 h. of the solution of the test toxin and of the solution of the
A suitable beta toxin contains not les s than one L+ in reference preparation such that 5.0 mL of each mixture
0.2 mg and not less than 25 LDso in one L+ dose. contains 2.0 mL of the solution of the test toxin and one of a
A suitable epsilon toxin contains not less than one L+/IO series of graded volumes of the solution of the reference
dos e in 0.005 mg and not less than 20 LDso in one L+/10 preparation, in order to confirm the test dose of the toxin.
dose. Allow the mixtures to stand at room temperature for 30 mino
3-5-4 Determination of test dose of toxin Prepare a solution of U sing not fewer than 2 mice for each mixture proceed as
the reference preparation in a suitable liquid so that it described in the preliminary test.
contains 5 IU/mL for Clostridium perfringens beta antitoxin Beta antitoxin The test mixture which contains 1 IU in
and 0.5 IU/rnL for Clostridium perfringens epsilon antitoxin. 0.5 mL is that mixture which kills the same or almost the
Prepare a solution of the test toxin in a suitable liquid so that same number of mice as the reference mixture containing
1 mL contains a precisely known amount such as 10 mg for 1 IU in 0.5 mL.
beta toxin and 1 mg for epsilon toxin. Prepare mixtures of Epsilon antitoxin The test mixture which contains 0.1 IU in
the solution of the reference preparation and the solution of 0.5 mL is that mixture which kills the same or almost the
the test toxin such that each contains 2.0 mL of the solution same number of mice as the reference mixture containing
of the reference preparation, one of a series of graded 0.1 IU in 0.5 mL. Repeat the determination at least once
volumes of the solution of the test toxin and sufficient of a and calculate the average of all valid estimates. The test is
suitable liquid to bring the total volume to 5.0 mL. Allow the
mixtures to stand at room temperature for 30 mino Using not
232 Veterinary Vaccines
valid only if the reference preparation gives a result within reference to a batch of vaccine that has given satisfactory
20 per cent of the expected value. results in the test described under Potency and that has been
The confidence limits (P = 0.95) have been estimated to be: shown to be satisfactory with respect to immunogenicity in
-- 85 per cent and 114 per cent when 2 animals per dose the target species. The following test may be used after a
are used, satisfactory correlation with the test under Potency (section
-- 91.5 per cent and 109 per cent when 4 animals per dose 3-5) has been established.
are used, Vaccinate rabbits as described under Potency and prepare
-- 93 per cent and 108 per cent when 6 animals per dose sera. Determine the level of antibodies against the toxin of
are used. C. septicum in the individual sera by a suitable method such
as an immunochemical method (2.7.1) or neutralisation in
4 LABELLING
cell cultures. Use a homologous reference serum calibrated in
The label states:
International Units of C. septicum antitoxin. Clostridia
-- whether the preparation is a toxoid or a vaccine prepared
(multicomponent) rabbit antiserum BRP is suitable for use as a
from a whole inactivated culture or a mixture of the two,
reference serum. The vaccine complies with the test if the
-- for each target species, the immunising effect produced
level of antibodies is not less than that found for a batch of
(for example, antibody production, protection against
vaccine that has given satisfactory results in the test described
signs of disease or infection).
under Potency and that has been shown to be satisfactory
_____________________________________________ PhE~
preparation of Clostridium septicum antitoxin, calibrated in contains more than 0.2 IV. If all the mice die, 0.5 mL of the
Intemational Units, necessary to give the same protection. mixture contains les s than 0.2 IU.
For this comparison, a suitable preparation of C. septicum Final test Prepare a series of mixtures of the solution of the
toxin for use as a test toxin is required. The dose of the test test toxin and of the serum to be examined such that 5.0 mL
toxin is determined in relation to the reference preparation; of each mixture contains 2.0 mL of the solution of the test
the potency of the serum to be examined is determined in toxin and one of a series of graded volumes of the serum to
relation to the reference preparation using the test toxin. be examined, separated from each other by steps of not more
Clostridia (multicomponent) rabbit antiserum BRP is suitable for than 20 per cent and covering the expected end-point as
use as a reference serum. determined by the preliminary test. Prepare further mixtures
3-5-1 Preparation of test toxin Prepare the test toxin ,from a of the solution of the test toxin and of the solution of the
sterile filtra te of a 1- to 3-day culture of C. septicum in liquid reference preparation such that 5.0 mL of each mixture
medium and dry by a suitable method. Select the test toxin contains 2.0 mL of the solution of the test toxin and one of a
by determining for mice the L+/5 dose and the LDso the series of graded volumes of the solution of the reference
observation period being 72 h. preparation to confirm the test dose of the toxin. Allow the
mixtures to stand at room temperature for 60 mino Using not
A suitable toxin contains not less than one L+/5 dos e in
fewer than 2 mice for each mixture proceed as described in
1.0 mg and not le-ssthan 10 LDso in each L+/5 dose.
the preliminary test. The test mixture which contains 0.2 IU
3-5-2 Determination of test dose of toxin Prepare a solution of in 0.5 mL is that mixture which kills the same or almost the
the reference preparation in a suitable liquid so that it same number of mice as the reference mixture containing
contains 1.0 IU/rnL. Prepare a solution of the test toxin in a 0.2 IU in 0.5 mL. Repeat the determination at least once
suitable liquid so that 1 mL contains a precisely known and calculate the average of all valid estimates. The test is
amount, such as 4 mg. Prepare mixtures of the solution of valid only if the reference preparation gives a result within
the reference preparation and the solution of the test toxin 20 per cent of the expected value.
such that each mixture contains 2.0 mL of the solution of the
The confidence limits (P = 0.95) have been estimated to be:
reference preparation (2 IU), one of a series of graded
-- 85 per cent and 114 per cent when 2 animals per dose
volumes of the solution of the test toxin and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. Allow the are used,
-- 91.5 per cent and 109 per cent when 4 animals per dose
mixtures to stand at room temperature for 60 mino Using not
fewer than 2 mice, each weighing 17-22 g, for each mixture, are used,
-- 93 per cent and 108 per cent when 6 animals per dose
inject adose ofO.5 mL by the intravenous or the
are used.
intraperitoneal route into each mouse. Observe the mice for
72 h. If all the mice die, the amount of toxin present in 4 LABELLING
0.5 mL of the mixture is in excess of the test dose. If none of The label states:
the mice die, the amount oftoxin present in 0.5 mL of the -- whether the preparation is a toxoid or a vaccine prepared
mixture is less than the test dose. Prepare fresh mixtures from a whole inactivated culture, or a mixture of the two,
such that 5.0 mL of each mixture contains 2.0 mL of the -- for each target species, the immunising effect produced
reference preparation (2 IV) and one of a series of graded (for example, antibody production, protection against
volumes of the solution of the test toxin separated from each signs of disease or infection).
other by steps of not more than 20 per cent and covering the _____________________________________________ PhE~
protein and shown to be not less than the value approved for Tetanus vaccine intended for use in animals other than
the particular producto horses complies with the test if the average antibody titre is
2-2 CHOICE OF VACCINE COMPOSITION not less than 7.5 IU/rnL.
The vaccine is shown to be satisfactory with respect to safety Tetanus vaccine intended for use in horses complies with the
(5.2.6) and efficacy (5.2.7) for the animals for which it is test if the average antibody titre is not less than 30 IU/mL.
intended. The following tests for Production of antigens For tetanus vaccine presented as a combined vaccine for use
(section 2-2-1), Safety (section 2-2-2) and Immunogenicity in animals other than horses, the aboye test may be carried
(section 2-2-3) may be used during demonstration of safety out in susceptible rabbits instead of guinea-pigs. The vaccine
and efficacy. complies with the test if the average antibody titre of the sera
The C. tetani strain used in the preparation of the vaccine is of the vaccinated rabbits is not less than 2.5 IU/mL.
shown to be satisfactory with respect to the production of the Clostridia (multicomponent) rabbit antiserum BRP and
neurotoxin. Clostridium tetani rabbit antiserum BRP are suitable as
2-2-1 Production of antigens reference serum.
The production of the neurotoxin of C. tetani is verified by a 2-3 MANUFACTURER'S TESTS
suitable irnmunochemical method (2.7.1) carried out on the 2-3-1 Absence of toxin and irreversibility of toxoid
neurotoxin obtained from the vaccine strain under the Carry out a test for reversion to toxicity on the detoxified
conditions used for the production of the vaccine. harvest using 2 groups of 5 guinea-pigs, each weighing
2-2-2 Safety 350-450 g; if the vaccine is adsorbed, carry out the test with
Carry out the test for each route and method of the shortest practical time interval before adsorption. Prepare
administration to be recommended and for each species of a dilution of the detoxified harvest so that the guinea-pigs
animal for which the vaccine is intended; use animals of the each receive 10 times the amount of toxoid (measured in Lf
minimum age recommended for vaccination and of the most units) that will be present in adose of vaccine. Divide the
sensitive category for the species. Use a batch of vaccine dilution into 2 equal parts. Keep one part at 5 ± 3 oC and
containing not less than the maximum potency that may be the other at 37 oC for 6 weeks. Attribute each dilution to a
expected in a batch of vaccine. separate group of guinea-pigs and inject into each guinea-pig
2-2-2-1 General safety For each test use not fewer than the dilution attributed to its group. Observe the animals at
15 animals, free from antitoxic antibodies. Administer to least daily for 21 days. The toxoid complies with the test if
each animal a double dose of vaccine. Administer to each no guinea-pig shows signs of disease or dies from causes
animal a single dos e of vaccine after the interval to be attributable to the neurotoxin of C. tetani.
recommended. Observe the animals at least daily until 2-3-2 Batch potency test
14 days after the last administration. The vaccine complies It is not necessary to carry out the Potency test (section 3-4)
with the test if no animal shows notable signs of disease or for each batch of vaccine if it has been carried out using a
die s from causes attributable to the vaccine. batch of vaccine with a minimum potency.
2-2-2-2 Safety in pregnant animals If the vaccine is intended Where the test described under Potency is used as the batch
for use in pregnant animals, vaccinate the animals at the potency test, the vaccine complies with the test if the
stage of pregnancy and according to the scheme stated on the antibody titre in International Units is not less than that
labe1 and prolong the observation period required in the found for a batch of vaccine shown to be satisfactory with
general safety test until 1 day after parturition. respect to immunogenicity in the target species.
The vaccine complies with the test if no animal shows 3 BATCH TESTS
notable signs of disease or dies from causes attributable to
3-1IDENTIFICATION
the vaccine and if no adverse effects on the pregnancy or the
1f the nature of the adjuvant allows it, carry out test A. Otherwise
offspring are noted.
carry out test B.
2-2-3 Innnunogenicity A. Dissolve in the vaccine sufficient sodium citrate R to give a
2-2-3-1 1mmunogenicity test in the target species It shall be 100 gIL solution. Maintain the solution at 37 oC for about
demonstrated for each target species that the vaccine, when 16 h and centrifuge until a c1ear supernatant liquid is
administered according to the recommended schedule and by obtained. The supernatant reacts with a suitable tetanus
the recommende route, stimulates an irnmune response (for antitoxin, giving a precipitate.
example, induction of antitoxic antibodies or induction of
protective levels of antitoxic antibodies) consistent with the
B. When injected into animal s that do not have antibodies
against fue neurotoxin of C. tetani, the vaccine stimulates the
c1aims made for the producto
production of such antibodies.--
2-2-3-2 1mmunogenicity test in guinea-pigs Administer to each
of at least 5 guinea-pigs that do not have antibodies against 3-2 BACTERIA AND FUNGI
the neurotoxin of C. tetani by the subcutaneous route 1 dos e The vaccine and, where applicable, the liquid supplied with it
of vaccine. After 28 days, administer again to each guinea-pig comply with the test for sterility prescribed in the monograph
1 dose by the subcutaneous route. 14 days after the second on Vaccines for veterinary use (0062).
dose, collect blood from each guinea-pig and prepare serum 3-3 SAFEiv
samples. Determine for each serum the titre of antibodies Administer 5 mL of the vaccine by the subcutaneous route as
against the neurotoxin of C. tetani using a suitable 2 equal divided dos es, at separate sites into each of 5 healthy
irnmunochemical method (2.7.1) such as a toxin-binding- guinea-pigs, each weighing 350-450 g, that have not
inhibition test (ToBI test) and a homologous reference previously been treated with any material that will interfere
serum. Determine the average antibody titre of the serum with the test. The vaccine complies witl:i the test if no animal
samples. shows notable signs of disease or die s from causes
Clostridium tetani guinea-pig antiserum for vaccines for veterinary attributable to the vaccine. If within 21 days of the injection
use BRP is suitable as reference sera. any of the animals shows signs of or die s from tetanus, the
Vaccines 235
vaccine does not comply with the test. If more than 1 animal 2-3 CHOICE OF VACCINE COMPOSITION
dies from non-specific causes, repeat the test. If any animal Only coccidial lines shown to be satisfactory with respect to
dies in the second test, the vaccine does not comply with the residual pathogenicity and increase in virulence may be used
test. in the preparation of the vaccine, and the tests described
3-4POTENCY below (sections 2-3-2 and 2-3-3) may be used to
The vaccine complies -with the requirements of the test demonstrate this. The vaccine shall be shown to be
mentioned under Immunogenicity (section 2-2-3-2). satisfactory with respect to safety (5.2.6) and efficacy (5.2.7)
___________________________________________ ~E~
for the chickens for which it is intended. The following tests
under Specific test for the safety of the vaccine composition
(section 2-3-1) and Immunogenicity (section 2-3-4) may be
used during the demonstration of safety and efficacy.
2-3-1 Specific test for the safety of the vaccine
Coccidiosis Vaccine (Live) for composition
Carry out the test with a preparation containing oocysts of
Chickens each species at the least attenuated passage level that will be
(Ph Eur monograph2326) present in a batch of vaccine. Use not fewer than 20 chickens
PhE~ ___________________________________________ from an SPF flock (5.2.2). The chickens must be hatched
and reared as described in section 2-1 and must not have
1 DEFINITION been treated with coccidiostats. Use chickens of the category
Coccidiosis vaccine (live) for chickens is a preparation of that is expected to be the most sensitive, i.e. 14-day-old
sporulated oocysts of a suitable line or lines of species of chickens. During the test, chickens are housed in suitable
coccidial parasites (Eimeria spp.). This monograph applies to conditions with the use of floor pens or cages with solid
vaccines intended for administration to chickens for active floors to favour reinfection with oocysts. Administer by
immunisation. gavage or another suitable route to each chicken a quantity of
2 PRODUCTION vaccinal oocysts consisting of the equivalent of not less than
2-1 PREPARATION OF THE VACCINE 10 times the maximum quantity of oocysts of each coccidial
Oocysts are produced in chickens from a flock free from species likely to be contained in 1 dose of the vaccine.
specified pathogens (SPF) (5.2.2) or in embryonated hens' Observe the chickens at least daily for 21 days. The test is
eggs from an SPF flock (5.2.2). The eggs must be subject to not valid if more than 10 per cent of the vaccinated chickens
disinfection and/or incubation conditions validated to ensure die from causes not attributable to the vaccinal oocysts.
the inactivation of any Eimeria that may be on the shells. The vaccine complies with the test if no vaccinated chicken
The hatched chickens must then be reared in disinfected shows notable clinical signs of coccidiosis or die s from causes
premises, in isolation conditions that ensure no infection with attributable to the vaccine.
Eimeria. The chickens must not have been treated with 2-3-2 Test for residual pathogenicity
coccidiostats. Oocysts are collected from faeces or contents of Carry out a separate test with each coccidial species and line
the intestinal tract of infected chickens during the patent to be included in the vaccine. Use in each case a prepáration
periodo Oocysts of different Eimeria lines are produced containing oocysts at the least attenuated passage level that
separately. Oocysts are isolated, purified, disinfected, will be present between the master seed lot and a batch of
sporulated and counted. The vaccine is produced by the vaccine. For each test use not fewer than 20 chickens
blending defined numbers of sporulated oocysts of each line from an SPF flock (5.2.2). The chickens must be hatched
in a suitable medium. and reared as described in section 2-1 and must not have
2-2 SEED LOTS been treated with coccidiostats. Use chickens of the category
that is expected to be the most sensitive, i.e. 14-day-old
2-2-1 Identification chickens. During the test, the chickens are placed in cages
The identity of each Eimeria master seed is established from (or any other suitable accomodation that prevents reinfection
the characteristics of the coccidia produced from it, based on
and allows collection of faeces). Administer by gavage or
an appropriate selection of the following characteristics: size another suitable route to each chicken the equivalent of not
and shape of the oocyst; localisation of the developmental less than 10 times the maximum quantity of the vaccinal
stages in the chicken intestine; pathognomonic lesions
oocysts likely to be contained in 1 dos e of the vaccine.
(E. ten ella, E. acervulina, E. necatrix, E. maxima and
Observe the chickens at least daily for 14 days. The test is
E. brunettt) and lack of macroscopic lesions (E. praecox and
not valid if more than 10 per cent of the chickens die from
E. mitis); size of schizonts in the intestinal mucosa; size of
causes not attributable to the vaccinal oocysts. Collect faeces
gametocytes in the mucosa; differences in the electrophoretic and determine oocyst production daily from day 3 until day
mobilities of certain isoenzymes, e.g. lactate dehydrogenase 14. On one day between days 4 and 8, depending on the
and glucose phosphate isomerase; and by the use of length of the pre-patent period, when lesions are expected to
molecular biology techniques. Artificially attenuated lines be maximal, and on day 14, euthanise not fewer than 9
may be distinguished from the parent strains by studying chickens and examine the intestinal tract for specific lesions
parameters appropriate to the method of attenuation. indicative of infection with the coccidial species or, for
2-2-2 Extraneous agents species known not to induce macroscopic lesions (E. mitis
Carry out tests 1-6 of chapter 2.6.24. Avian viral vaccines: and E. praecox), microscopic evidence of infection such as
tests for extraneous agents in seed lots. General provisions a-d, f demonstration of oocysts or developing oocysts in the
and h and section 7 of chapter 2.6.24 are also applicable. intestinal contents or scrapings of the intenstinal wall.
In these tests on the master seed lot, use organisms that are For species that have the potential to produce relevant
not more than 5 passages from the master seed lot at the macroscopic pathological changes if not attenuated, a scoring
start of the tests. Each master seed lot complies with the system with a scale from O to 4 is used to record the species-
requirements of each test. specific lesions visible in the intestine as follows.
236 Vaccines
administration of passaged and unpassaged oocysts. The line - for challenges with E. mitis or E. praecox, fewer than
complies with the test if no indication of an increase in 80 per cent of the control chickens euthanised between
virulence of the maximally passaged oocysts compared with days 4 and 8 are infected.
the unpassaged oocysts is observed. The test is invalid if The vaccine complies with the test if:
oocysts are not recovered at any passage level. - for all the Eimeria challenge species, the production of the
2-3-4 Immunogenicity oocysts is significantly decreased in vaccinates compared
The efficacy of each coccidial species and line inc1uded in the with controls;
vaccine is determined in a separate study with an apP!"opriate - for all the Eimeria challenge species, no vaccinated
challenge strain. For each component, a test is carried out chicken die s due to the challenge infection;
with vaccine administered by each route and method of - for challenge with E. ten ella, E. acervulina, E. necatrix,
administration to be recommended, using in each case E. maxima or E. brunetti, at least 80 per cent of the
chickens not older than the youngest age to be reco~mended vaccinates show no more than mild signs of disease and
for vaccination. The quantity of each of the components in these are les s marked than those in the controls;
the batch of vaccine administered to each chicken is not - for challenges with E. ten ella, E. acervulina, E. necatrix,
greater than the minimum number of oocysts to be stated on E. maxima or E. brunetti, at least 80 per cent of the
the label and the oocysts are at the most attenuated passage vaccinates have no or minimallesions in the intestine
level that will be present in a batch of vaccine. Use for the (e.g. mean lesion scores not greater than 1) and no bird
test not fewer than 40 chickens from an SPF flock (5.2.2). has a lesion score of 4;
The chickens must be hatched and reared as described in - for challenges with E. ten ella, E. acervulina, E. necatrix,
section 2-1 and must not have been treated with E. maxima, E. brunetti, E. mitis, or E. praecox, the growth
coccidiostats. Vaccinate not fewer than 20 chickens and rate in the vaccinates is significantly greater than in the
maintain not fewer than 20 chickens as controls. For the controls.
evaluation of weight gain with Eimeria strains showing a low 2-4 MANUFACTURER'S TESTS
pathogenicity, the number of chickens used may be higher 1
2-4-1 In-process test for sporulation rate and oocyst
The test may require different challenge doses for differen!
count
test parameters and so may be assessed as separate challenge
A sample of each oocyst bulk is examined microscopically
groups. For example, a lower challenge dose may be needed
after the sporulation step and before blending to determine
to determine the effect on oocyst output than the dose
the percentage of sporulated oocysts and the oocyst count.
needed to determine the effect on weight gain and lesion
The values obtained are within the limits shown to allow
scoring. After vaccination, the chickens are housed in suitable
preparation of a satisfactory vaccine.
conditions with the use of floor pens or cages with solid
floors to favour reinfection with oocysts. On a suitable day 2-4-2 Batch potency test for each Eimeria species in
between days 14 and 21 after vaccination, weigh each the vaccine
chicken, move them to cages (or any other suitable It is not necessary to carry out the potency test (section 3-7)
accomodation that prevents reinfection and allows collection for each batch of the vaccine if it has been carried out using
of faeces) and challenge each chicken by gavage or another a batch or batches of vaccine with minimum potency and
suitable route with a sufficient quantity of virulent coccidia to sporulated oocyst contento Where the test is not carried out,
induce in the unvaccinated control s signs of disease an altemative validated method is used, the criteria for
characteristic of the Eimeria challenge species. Observe the acceptance for each component being set with reference to a
chickens at least daily until the end of the test. Record deaths batch of vaccine that has given satisfactory results in the test
and the number of surviving chickens that show c1inical signs described under Potency.
of disease. Collect faeces and determine oocyst production 2-4-3 Freedom from extraneous agents
from day 3 after challenge until the end of the test. On an The disinfection method applied during the preparation of
appropriate day between days 4 and 8 after challenge, the final product from the harvested oocysts may be validated
depending on the length of the pre-patent period of the to show effective inactivation of certain potential extraneous
challenge species, weigh each chicken. Euthanise 10 chickens agents. Where relevant validation data is available and where
from each group and examine them for lesions in the justified and authorised, sorne or all of the tests indicated
intestinal tracto Where appropriate, record the specific lesions under Extraneous agents (section 3-4) may be omitted as
indicative of the coccidial challenge species (using the scoring routine tests on each batch.
system described in section 2-3-2). For species known not to
3 BATCH TESTS
induce macroscopic lesions (E. mitis and E. praecox),
3-1 Identification
examine the chickens for microscopic evidence of infection
3-1-1 Microscopical examination is used to confirm the
such as demonstration of oocysts or developing oocysts in the
presence of coccidial oocysts in the batch of vaccine.
intestinal contents or scrapings of the intestinal wal1. On day
14 after challenge, weigh each of the remaining chickens. 3-1-2 The potency test (or batch potency test) is used to
confirm the presence of oocysts of each of the Eimeria species
The test is invalid if:
stated on the label.
- during the period between vaccination and challenge,
more than 10 per cent of the vaccinated or control 3-2 Bacteria and fungi
chickens show abnormal c1inical signs or die from causes The vaccine and where applicable the liquid supplied with it
not attributable to the vaccine; comply with the test for sterility prescribed in the monograph
- for challenges with E. tenella, E. acervulina, E. necatrix, Vaccines for veterinary use (0062) and comply with the test
E. maxima or E. brunetti, fewer than 80 per cent of the with a medium selective for Campylobacter spp.
control chickens euthanised between days 4 and 8 have 3-3 Mycoplasmas
marked characteristic lesions of the challenge infection in The vaccine complies with the test for mycoplasmas (2.6.7).
the intestine at post-mortem examination (e.g. lesion
scores not less than 2);
238 Veterinary Vaccines
to measure the virus contento The batch complies with the 2-2-2 Cell cultures
tests if the titre of the batch is not less than the minimum If the vaccine virus is grown in cell cultures, they comply
titre stated on the label. with the requirements for cell cultures for production of
The vaccine complies with the requirements stated under veterinary vaccines (5.2.4).
Veterinary Vaccines with the following modifications. 2-3 SEED LOTS
IDENTIFICATION 2-3-1 Extraneous agents
Produces the characteristic lesions of contagious pustular The master seed lot complies with the test for extraneous
dermatitis when applied to a scarified area of the skin/of agents in seed lots (2.6.24). In these tests on the master seed
lambs. lot, the organisms used are not more than 5 passages from
the master seed lot at the start of the tests.
TESTS
Extraneous bacteria and fungi 2-4 CHOICE OF VACCINE VIRUS
The vaccine is shown by appropriate methods to be free from The vaccine virus shall be shown to be satisfactory with
pathogenic organisms. respect to safety (5.2.6) and efficacy (5.2.7) for the ducks for
which the vaccine is intended.
Mycoplasmas
Complies with the test for absence of mycoplasmas, The following tests for safety (section 2-4-1), increase in
Appendix XVI R(Vet) 3. virulence (section 2-4-2) and irnrnunogenicity (section 2-4-3)
may be used during demonstration of safety and
Safety irnrnunogenicity.
Adrninister by skin scarification of one site or a small number
of contiguous sites, ten doses of the vaccine to each of two 2-4-1 Safety
lambs of the minimum age to be recommended for Carry out the test for each route and method of
vaccination and that are free from antibodies to orf virus. adrninistration to be recommended for vaccination, using in
Observe the lambs for two weeks. No systemic or local each case ducks from a species considered to be the most
reactions occur except for mild locallesions of orfo susceptible among the species to be recommended for
vaccination and not older than the youngest age to be
Extraneous viruses recommended for vaccination. Use vaccine virus at the least
Neutralise the vaccine if necessary. Inoculate the vaccine attenuated passage level that will be present in a batch of
onto suitable cell cultures that are susceptible to ovine vaccine. For each test, use not fewer than 20 susceptible
viruses, make at least one passage and maintain the cultures ducks that do not have antibodies against duck plague virus.
for at least 14 days. No cytopathic effect deve1ops. Conduct a Adrninister to each duck a quantity of vaccine virus
test for freedoin from haemadsorbing agents. The cell equivalent to not less than 10 times the maximum virus titte
cultures show no signs of viral contamination. likely to be contained in 1 dose of the vaccine. Observe the
POTENCY ducks at least daily for 21 days. The test is not valid if more
Vaccinate each of no fewer than two healthy susceptible than 10 per cent of the ducks die from causes not
sheep, 6 to 12 months old, with serial dilutions of the attributable to the vaccine virus. The vaccine virus complies
vaccine applied to the scarified skin. Characteristic lesions of with the test if no duck shows notable signs of duck plague
contagious pustular dermatitis appearing on the fourth to or die s from causes attributable to the vaccine virus.
eighth day after inoculation are recorded. The vaccine 2-4-2 Increase in virulence
contains not les s than 100 MID in the dose stated on the The test for increase in virulence consists of the
label. administration of the vaccine virus, at the least attenuated
STORAGE passage leve1 that will be present between the master seed lot
When stored in the prescribed conditions the vaccine may be and a batch of vaccine, 10 a group of 5 domestic ducks that
expected to retain its potency for at least ayear. do not have antibodies against duck plague virus and of an
age suitable for the multiplication of the virus then its
sequential passage, 5 times where possible, to further similar
groups of ducks of an age suitable for the multiplication of
the virus and testing of the final recovered virus for increase
Duck Plague Vaccine (Live) in virulence. If the properties of the vaccine virus allow
sequential passage to 5 groups via natural spreading, this
(Ph Eur monograph 1938)
method may be used, otherwise passage as described be10w is
~E~ ___________________________________________
carried out and the maximally passaged virus that has been
1 DEFINITION recovered is tested for increase in virulence. Care must be
Duck plague vaccine (live) is a preparation of a suitable taken to avoid contamination by virus from previous
strain of duck plague virus (anatid herpesvirus 1). This passages. Administer by a recommended route a quantity of
monograph applies to vaccines intended for the active the vaccine virus that will allow recovery of virus for the
immunisation of ducks. passages described below. 2 to 4 days later, take samples of
liver and spleen from each duck and pool all samples.
2 PRODUCTION Adrninister 0.1 mL of the pooled suspension by the oro-nasal
2-1 PREPARATION OF THE VACCINE or a parenteral route to each of 5 other domestic ducks of
The vaccine virus is grown in embryonated hens' eggs or in the same age that do not have antibodies against duck plague
cell cultures. The vaccine may be freeze-dried. virus. Carry out this passage operation not fewer than
2-2 SUBSTRATE FOR VIRUS PROPAGATION 5 times; veri:fy the presence of the virus at each passage.
2-2-1 Embryonated hens' eggs If the virus is not found at a passage level, carry out a second
If the vaccine virus is grown in embryonated hens' eggs, they series of passages. Carry out the test for safety (section 2-4-1)
are obtained from flocks free from specified pathogens (SPF) using the unpassaged vaccine virus and the maximally
(5.2.2). passaged vaccine virus that has been recovered. The vaccine
240 Veterinary Vaccines
virus complies with the test if no indication of increase in contains not less than the minimum virus titre stated on the
virulence of the maximally passaged virus compared with the label.
unpassaged virus is observed. If virus is not recovered at any 3-7 Potency
passage level in the first and second series of passages, the The vaccine complies with the test prescribed under
vaccine virus also complies with the test. immunogenicity (section 2-4-3), when administered by a
2-4-3 Immunogenicity recommended route and method. It is not necessary to carry
A test is carried out for each route and method of out the potency test for each batch of the vaccine if it has
administration to be recommended for vaccination, using in been carried out on a representative batch using a vaccinating
each case domes tic ducks not older than the youngest age to dose containing not more than the minimum virus titre
be recommended for vaccination. The quantity of the vaccine stated on the label.
virus administered to each duck is not greater than the ___________________________________________ ~E~
of vaccine virus equivalent to not les s than 10 times the - during the observation period after challenge fewer than
maximum virus titre likely to be contained in 1 dose of 70 per cent of the challenged ducklings from the control
vaccine. Observe the ducklings at least daily for 21 days. ducks die or show typical signs of the disease,
The test is not valid if more than 10 per cent of the and/or during the period between vaccination and
ducklings die from causes not attributable to the vaccine collection of the eggs more than 10 per cent of the
virus. The vaccine virus complies with the test if no duckling control or vaccinated ducks show abnormal clinical signs
shows notable signs of duck viral hepatitis or die s from or die from causes not attributable to the vaccine.
causes attributable to the vaccine virus. The vaccine virus complies with the test if during the
2-4-2 Increase in virulence observation period after challenge the percentage relative
The test for increase in virulence consists of the protection calculated using the following expression is not
administration of the vaccine virus at the least atten~ated less than 80 per cent:
passage level that will be present between the master seed lot
and a batch of vaccine to a group of five 1-day-old domestic
v-c
ducklings that do not have antibodies against duck hepatitis e
100 _ x 100
virus type I, sequential passages, 5 times where possible, to
further similar groups of 1-day-old ducklings and testing of
the final recovered~virus for increase in virulence. If the V percentage of challenged ducklings from vaccinated
properties of the vaccine virus al10w sequential passage to ducks that survive to the end of the observation period
5 groups via natural spreading, this method may be used, without clinical signs of the disease.
otherwise passage as described below is carried out and the e percentage of challenged ducldings from unvaccinated
maximally passaged virus that has been recovered is tested control ducks that survive to the end of the observation
for increase in virulence. Care must be taken to avoid period without clinical signs of the disease.
contamination by virus from previous passages. Administer 2-4-3-2 Vaccines far active immunisatian af ducklings Use for
by the oro-nasal route a quantity of vaccine virus that will , the test not fewer than 30 ducklings of the same origin and
allow recovery of virus for the passages described below. 2 lo that do not have antibodies against duck hepatitis virus type
4 days later, take samples of liver from each duckling and I. Vaccinate by a recommended route not fewer than 20
pool the samples. Administer 1 mL of the pooled liver ducklings. Maintain not fewer than 10 ducklings as controls.
suspension by the oro-nasal route to each of five 1-day-old Challenge each duckling after at least 5 days by the oro-nasal
seronegative domestic ducklings. Carry out this operation route with a sufficient quantity of virulent duck hepatitis
5 times. Verify the presence of the virus at each passage. virus type I. Observe the ducklings at least daily for 14 days
If the virus is not found at a passage level, carry out a second after challenge. Record the deaths and the number of
series of passages. Observe the ducklings given the last surviving ducklings that show clinical signs of disease.
passage at least daily for 21 days. Compare the results The test is not valid if:
obtained with unpassaged virus in the safety studies and - during the observation period after challenge fewer than
those obtained with maximally passaged virus to assess the 70 per cent of the control ducklings die or show typical
degree of increase in virulence. If the virus is not recovered at signs of the disease,
any passage level in the first and second series of passages, it - and/or during the period between vaccination and
is considered not to show increase in virulence. challenge more than 10 per cent of the control or
2-4-3 Immunogenicity vaccinated ducklings show abnormal clinical signs or die
A test is carried out for each route and method of from causes not attributable to the vaccine.
administration to be recommended, using in each case The vaccine virus complies with the test if during the
domestic ducks not older than the youngest age to be observation period after challenge the percentage relative
recommended for vaccination. The quantity of the vaccine protection ca1culated using the following expression is not
virus administered to each bird is not greater than the less than 80 per cent:
minimum virus titre to be stated on the label and the virus is
at the most attenuated passage level that will be present in a
batch of the vaccine. v- c x 100
2-4-3-1 Vaccines far passive immunisatian af ducklings Use for 100- e
the test not fewer than 15 laying ducks or ducks intended for
laying, as appropriate, of the same origin and that do not v = percentage of challenged vaccinated ducklings that
have antibodies against duck hepatitis virus type I. Vaccinate survive to the end of the observation period without
by a recommended route not fewer than 10 ducks using the clinical signs of the disease.
schedule to be recommended. Maintain not fewer than 5 e percentage of challenged unvaccinated control
ducks as controls. Starting from 4 weeks after onset of lay, ducklings that survive to the end of the observation
collect embryonated eggs from vaccinated and control ducks period without clinical signs of the disease.
and incubate them. Challenge not fewer than twenty 1-week-
old ducklings representative of the vaccinated group and not 3 BATCH TESTS
fewer than 10 from the control group by the oro-nasal route 3-1 Identification
with a sufficient quantity of virulent duck hepatitis virus type The vaccine, diluted if necessary and mixed with a
I. Observe the ducklings at least daily for 14 days after monospecific duck hepatitis virus type I antiserum, no longer
challenge. Record the deaths and the number of surviving infects embryonated hens' eggs from an SPF flock (5.2.2) or
ducklings that show clinical signs of disease. susceptible cell cultures (5.2.4) into which it is inoculated.
The test is not valid if: 3-2 Bacteria and fungi
Vaccines intended for administration by injection comply
with the test for sterility prescribed in the monograph
Vaccines far veterinary use (0062).
242 Veterinary Vaccines
When the second group of vaccinated birds and the group of more sensitive test system, in hens' eggs from an SPF flock
30 control birds are nearing the end of lay, challenge these (5.2.2). Inject 2/5 th of adose into the allantoic cavity of each
birds, as before. The test is invalid unless there is a well of ten 10- to 14-day-old embryonated eggs that are free from
marked drop in egg production and/or quality in the control parental antibodies to egg drop syndrome '76 virus. Incubate
birds. The vaccine complies with the test if the vaccinated the eggs and observe for 8 days. Pool separately the allantoic
birds show no marked drop in egg production andlor quality. fluid from eggs containing live embryos, and that from eggs
Carry out serological tests on serum samples obtained at the containing dead embryos, exc1uding those that die from non-
time of vaccination, 4 weeks later and just prior to ch~l1enge. specific causes within 24 h of the injection. Inject into the
The test is invalid if antibodies against egg drop syndrome allantoic cavity of each of ten 10- to 14-day-old embryonated
'76 virus are detected in any sample from control birds. eggs that do not have parental antibodies to egg drop
syndrome '76 virus, 0.2 mL of the pooled allantoic fluid
2-5 MANUFACTURER'S TESTS
from the live embryos and into each of 10 similar eggs,
2-5-1 Residuallive virus 0.2 mL of the pooled allantoic fluid from the dead embryos
The test for residuallive virus is carried out in embryonated and incubate for 8 days. Examine the allantoic fluid from
ducks' eggs from a flock free from egg drop syndrome '76 each egg for the presence of haemagglutinating activity using
virus infection, or in embryonated hens' eggs from an SPF chicken erythrocytes. If more than 20 per cent of the
flock (5.2.2), or illsuitable cell cultures, whichever is the embryos die at either stage, repeat that stage. The vaccine
most sensitive for the vaccine strain. The quantity of complies with the test if there is no evidence of
inactivated virus harvest used in the test is equivalent to not haemagglutinating activity and if, in any repeat test, not more
less .than 10 doses of the vaccine. The inactivated virus than 20 per cent of the embryos die from non-specific
harvest complies with the test if no live virus is detected. causes. Antibiotics may be used in the test to control
2-5-2 Batch potency test extraneous bacterial infection.
It is not necessary to carry out the Potency test (section 3-6) B. For a vaccine adapted to growth in cell cultures, inoculate
for each batch of vaccine if it has been carried out using a 10 doses into suitable cell cultures. If the vaccine contains an
batch of vaccine with a minimum potency. Where the test is oily adjuvant, eliminate it by suitable means. Incubate the
not carried out, an alternative validated method is used, the cultures at 38 ± 1 oC for 7 days. Make a passage on another
criteria for acceptance being set with reference to a batch of set of cell cultures and incubate at 38 ± 1 oC for 7 days.
vaccine that has given satisfactory results in the test described Examine the cultures regular1y and at the end of the
under Potency. The following test may be used. incubation period examine the supernatant liquid for the
Vaccinate not fewer than ten 14- to 28-day-old chickens presence of haemagglutinating activity. The vaccine complies
from an SPF flock (5.2.2) with 1 dos e ofvaccine by one of with the test if the cell cultures show no sign of infection and
the recommended routes. 4 weeks later, collect serum if there is no haemagglutinating activity in the supernatant
samples from each bird and from 5 unvaccinated control liquido
birds of the same age and from the same source. Measure 3-5 Extraneous agents
the antibody response in a haemagglutination (HA) inhibition U se the chickens from the safety test. 21 days after
test on each serum using 4 HA units of antigen and chicken administration of the double dos e of vaccine, administer one
erythrocytes. The test is invalid if there are specific antibodies dose by the same route into each chicken. Collect serum
in the sera of the unvaccinated birds. The vaccine complies samples from each chicken 2 weeks later and carry out tests
with the test if the mean titre of the vaccinated group is not for antibodies against the following agents by the methods
les s than that found previously for a batch of vaccine that has prescribed in general chapter 5.2.2. Chicken fiocks free from
given satisfactory results in the test described under Potency. specified pathogens for the production and quality control of
3 BATCH TESTS vaccines: avian encephalomyelitis virus, avian leucosis viruses,
3-1 Identification infectious bronchitis virus, infectious bursal disease virus,
When injected into chickens that do not have antibodies infectious laryngotracheitis virus, influenza A virus, Marek's
against egg. drop syndrome '76 virus, the vaccine stimulates disease virus, Newcastle disease virus and, for vaccine
the production of such antibodies. produced in duck eggs, Chlamydia (by a complement-fixation
test or agar gel precipitation test), duck hepatitis virus type 1
3-2 Bacteria and fungi (by a fluorescent-antibody test or serum-neutralisation test)
The vaccine and, where applicable, the liquid supplied with it and Derzsy's disease virus (by a serum-neutralisation test).
comply with the test for sterility prescribed in the monograph The vaccine complies with the test if it does not stimulate the
Vaccines for veterinary use (0062). formation of antibodies against these agents.
3-3 Safety 3-6 Potency
Use 10 chickens, 14 to 28 days old, from an SPF flock The vaccine complies with the requirements of the test
(5.2.2). Administer a double dos e of vaccine by a mentioned under Immunogenicity (section 2-4-1) when
recommended route into each chicken. Observe the chickens administered by a recommended route and method.
at least daily for 21 days.
The vaccine complies with the test if no chicken shows 4LABELLING
notable signs of disease or dies from causes attributable to The labe1 states whether the strain in the vaccine is duck- or
the vaccine. hen-embryo-adapted or cell-culture-adapted.
___________________________________________ PhE~
2-3-2 Irnmunogenicity
Equine Herpesvirus Vaccine, The type of immunogenicity test depends on the claims for .
Inactivated the product. For vaccines intended to protect against the
(Equine Herpesvirus Vaecine (Inaetivated) J disease of the respiratory tract, carry out test 2-3-2-1, using
Ph Eur monograph 1613) equid herpesvirus 1 and/or equid herpesvirus 4 depending on
~E~ ___________________________________________ the claims for protection. For vaccines intended to protect
against abortion carry out test 2-3-2-2.
1 DEFINITION A test is carried out for eac:h route and method of
Equine herpesvirus vaccine (inactivated) is a preparation of administration to be recommended, using in each case horses
one or more suitable strains of equid herpesvirus 1 ami/or that have not been vaccinated with an equine herpesvirus
equid herpesvirus 4, inactivated while maintaining adequate vaccine, that have at most a low antibody titre not indicative
immunogenic properties or a suspension of an inactivated of recent infection, and that do not excrete equid herpesvirus.
fraction of the virus. This monograph applies to vaccines To demonstrate that no recent infection occurs, immediately
intended for the active immunisation of horses against disease before vaccination: draw a blood sample from each horse and
caused by equid herpesvirus 1 and/or equid herpesvirus 4. test individually for antibodies against equid herpesviruses 1
2 PRODUCTION and 4; collect 10 mL of heparinised blood and test the
2-1 PREPARATION OF THE VACCINE washed leucocytes for equid herpesviruses 1 and 4; collect a
Each strain of vaccine virus is grown separately in cell nasopharyngeal swab and test for equid herpesviruses 1 and
cultures. The viral suspensions may be purified and 4. There is no indication of an active infection. Immediately
concentrated and are inactivated; they may be treated to before challenge collect a nasopharyngeal swab and test for
fragment the virus and the viral fragments may be purified equid herpesviruses 1 and 4. If there is an indication of virus
and concentrated. The vaccine may be adjuvanted. excretion remove the horse from the test. Keep the horses in
strict isolation. The vaccine administered to each horse is of
2-2 SUBSTRATE FOR VIRUS PROPAGATION
minimum potency.
2-2-1 Cell cultures 2-3-2-1 Vaecines intended for proteetion against disease of the
The cell cultures comply with the requirements for cell respiratory traet U se for the test not fewer than 10 horses,
cultures for production ofveterinary vaccines (5.2.4). not less than 6 months old. Vaccinate not fewer than 6
2-3 CHOICE OF VACCINE COMPOSITION horses according to the schedule to be recommended.
The vaccine is shown to be satisfactory with respect to safety Maintain not fewer than 4 horses as controls. At least
(5.2.6) and efficacy (5.2.7) for the horses for which it is 2 weeks after the last vaccination, challenge each horse by
intended. Where a particular breed of horse is known to be nasal instillation with a quantity of equid herpesvirus 1 or 4,
especially sensitive to the vaccine, horses from that breed are sufficient to produce in a susceptible horse characteristic
included in the test for safety. signs of the disease such as pyrexia and virus excretion (and
The following tests for safety (section 2-3-1) and possibly nasal discharge and coughing). Observe the horses at
immunogenicity (section 2-3-2) may be used during the least daily for 14 days. Collect nasopharyngeal swabs daily
demonstration of safety and efficacy. from each individual horse to isolate the virus.
2-3-1 Safety The vaccine complies with the test if the vaccinated horses
Carry out the test for each route and method of show no more than slight signs; the signs in vaccinates are
administration to be recommended for vaccination and in less severe than in controls. The average number of days on
horses of each category for which the vaccine is intended. which virus is excreted, and the respective virus titres are
U se a batch of vaccine containing not les s than the significantly lower in vaccinated horses than in controls.
maximum potency that may be expected in a batch of 2-3-2-2 Vaecines intended for proteetion against abortion Use
vaccine. not fewer than 10 pregnant horses. In addition to the testing
2-3-1-1-1 General safety Use for the test not fewer than 10 described above, 6, 4, 3, 2 and 1 month before the first
horses that have not been previously vaccinated with an vaccination draw a blood sample from each horse and test
equine herpesvirus vaccine, that have at most a low antibody individually for antibodies against equid herpesvirus 1 and
titre not indicative of recent infection and that do not excrete equid herpesvirus 4. Thereis no evidence of recent infection
equid herpesvirus. Administer to each horse a double dose of or virus excretion. Vaccinate not fewer than 6 horses
the vaccine, then one dose after 14 days. Observe the horses according to the schedule to be recommended. Maintain not
at least daily until 14 days after the last administration. fewer than 4 horses as controls. Between day 260 and 290 of
pregnanty but not earlier than 3 weeks after the last
The vaccine complies with the test if no horse shows
vaccination challenge each horse, by nasal Ínstillation, with a
abnormal local or systemic reactions or die s from causes
quantity of equid herpesvirus 1 sufficient to produce abortion
attributable to the vaccine during the 28 days of the test.
in susceptible horses. Observe the horses at least daily up to
2-3-1-1-2 Safety in pregnant horses If the vaccine is intended foaling or abortion. Collect samples of fetal lung and liver
for use in pregnant horses, use for the test not fewer than 10 tissues from aborted fetuses and carry out tests for virus in
pregnant horses at the relevant trimester or trimesters of cell cultures.
pregnancy according to the schedule to be recommended. I
The test is invalid if more than one control horse gives birth
Administer to each horse a double dose of the vaccine, then
to a healthy foal and if the challenge virus is not isolated
one dose after 14 days. Observe the horses at least daily until
foaling. from the aborted fetuses. The vaccine complies with the test
if not more than one vaccinated horse aborts.
The vaccine complies with the test if no horse shows
abnormallocal or systemic reactions or die s from causes 2-4 MANUFACTURER'S TESTS
attributable to the vaccine and if no adverse effects on the 2-4-1 Residuallive virus
pregnancy or the offspring are noted. The test for residual live virus is carried out using 2 passages
in the same type of cell culture as that used in the
Veterinary Vaccines 245
14 days. Observe the horses at least daily until 14 days after horse 7 days after the first vaccination and test individually
the last administration. for antibodies against equine influenza virus J to detect an
The vaccine complies with the test if no horse shows anamnestic sero-response. Horses showing sero-conversion at
abnormal local or systemic reactions or die s from causes this stage are excluded from the test. At least 2 weeks after
attributable to the vaccine during the 28 days of the test. the last vaccination, challenge each horse by aerosol with a
quantity of equine influenza virus sufficient to produce
2-3-1-2 Safety in pregnant horses If the vaccine is intended
characteristic signs of disease such as fever, nasal discharge
for use in pregnant horses, use for the test not fewer than
and coughing in a susceptible horse. Observe the horses at
10 pregnant horses at the stage or at different stages of
least daily for 14 days. Collect nasal swabs daily from each
pregnancy according to the schedule to be recommended.
individual horse to isolate the virus.
Administer to each horse a double dose of the vaccine, then
one dose after 14 days. Observe the horses at least daily until The vaccine complies with the test if the vaccinated horses
foaling. show no more than slight signs; the controls show
The vaccine complies with the test if no horse shows characteristic signs. The average number of days on which
virus is excreted, and the respective virus titres are
abnormal local or systemic reactions or dies from causes
significantly lower in vaccinated horses than in control
attributable to the vaccine and if no adverse effects on the
horses.
pregnancy or the offspring are noted.
2-3-2-2 Presence of antibodies after vaccination
2-3-2 Immunogenicity
The test described under 2-3-2-1 is suitable to demonstrate 2-3-2-2-1 Single radial haemolysis. Heat each serum at 56 oC
the immunogenicity of the strains present in the vaccine. for 30 min. Perform tests on each serum using respectively
the antigen or antigens prepared from the strain(s) used in
A test with virulent challenge is carried out for at least one the production of the vaccine. Mix 1 mL of sheep
vaccine strain (se e test 2-3-2-1). For other strains in the
erythrocyte suspension in barbital buffer solution (1 volume
vaccine, demonstration of immunogenicity may, where
of erythrocytes in 10 volumes of final suspension)with 1 mL
justified, be based on the serological response induced in
of a suitable dilution of the influenza virus strain in barbital
horses by the vaccine (see tests 2-3-2-2); justification for
buffer solution and incubate the mixture at 4 oC for 30 min.
protection against these strains may be based on published To 2 mL of the virus/erythrocyte mixture, add 1 mL of a
data on the correlation of the antibody titre with protection 3 giL solution of chromium(lll) trichloride hexahydrate R, mix
against antigenically related strains.
and allow to stand for 10 min. Heat the sensitised
Where serology is used, the test is carried out as described erythrocytes to 47 oC in a water-bath. Mix 15 mL of a
under 2-3-2-1 but instead of virulent challenge, a blood 10 gIL solution of agarose for electrophoresis R in barbital
sample is drawn 2 weeks after the last vaccination and the buffer solution, 0.7 mL of sensitised erythrocyte suspension
antibody titre of each serum is determined by a suitable and the appropriate amount of diluted guinea-pig
immunochemical method (2.7.1), such as the single radial complement in barbital buffer solution at 47 oC. Pour the
haemolysis test or the haemagglutination-inhibition test mixture into Petri dishes and allow the agar to seto Punch
shown below; a reference serum is used to validate the test. holes in the agar layer and place in each hole, 5 ¡.tL of the
The acceptance criteria depend on the strain and are based undiluted serum to be tested or control serum. Incubate the
on available data; for A/equine-2 virus, vaccines have usually Petri dishes at 37 oC for 18 h. Measure the di ame ter ofthe
been found satisfactory if the antibody titre of each serum is haemolysis zone and calculate its area, which express es the
not less than 85 mm2 where the single radial haemolysis test antibody titre, in square millimetres.
is used, or not less than 1:64 (before mixture with the
Equine influenza subtype 1 horse antiserum BRPJ equine
suspension of antigen and erythrocytes) where the
influenza subtype 2 American-like horse antiserum BRP and
haemagglutination-inhibition test is used.
equine influenza subtype 2 European-like horse antzserum BRP
Equine influenza subtype 1 horse antiserum BRPJ equine are suitable for use as reference sera for the single radial
influenza subtype 2 American-like horse antiserum BRP and ha~molysis test.
equine influenza subtype 2 European-like horse antiserum BRP
2-3-2-2-2 Haemagglutination-inhibition test. Inactivate each
are suitable for use as reference sera for the single radial
serum by heating at 56 oC for 30 mino To one volume of
haemolysis test.
each serum add three volumes of phosphate-buffered saline
The claims for the product reflect the type of pH 7.4 R and four volumes of a 250 giL suspension of light
immunogenicity demonstrated (protection against challenge kaolin R in the same buffer solution. Shake each mixture for
or antibody production). 10 min .. Centrifuge, collect the supernatant liquid and mix
2-3-2-1 Protection from signs of disease and reduction of virus with a concentrated suspension of chickenerythrocytes.
excretion Carry out the immunogenicity test using a Allow to stand at 37 oC for 60 min and centrifuge.
challenge strain against which the vaccine is stated to provide The dilution of the serum obtained is 1:8. Perform tests on
protection. Use where possible a recent isolate. each serum using each antigen prepared from the strains
A test is carried out for each route and method of used in the production of the vaccine. Using each diluted
administration to be recommended, using in each case horses serum, prepare a series of twofold dilutions. To 0.025 mL of
not les s than 6 months old. The vaccine administered to each of tll,e latter dilutions add 0.025 mL of a suspension of
each horse is of minimum potency. antigen treated with ether R and containing four
U se for the test not fewer than 10 horses that do not have haemagglutinating units. Allow the mixture to stand for
antibodies against equine influenza virus. Draw a blood 30 min/and add 0.05 mL of a suspension of chicken
sample from each horse and test individually for antibodies erythrocytes containing 2 x 107 erythrocytes/mL. Allow to
against equine influenza virus to determine seronegativity. stand for 1 h and note the last dilution of serum that still
Vaccinate not fewer than 6 horses according to the schedule completely inhibits haemagglutination.
to be recommended. Maintain not fewer than 4 horses as
controls. Draw a second blood sample from each vaccinated
Veterinary Vaccines 247
2-2 SUBSTRATE FOR VIRUS PROPAGATION criteria for acceptance being set with reference to a batch of
2-2-1 Cell cultures vaccine that has given satisfactory results in the test described
The cell cultures comply with the requirements for cell under Potency. The following test may be used.
cultures for production of veterinary vaccines (5.2.4). Use for the test groups of 15 seronegative mice. Administer
to each mouse half adose of the vaccine and 7 days later,
2-3 CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfactory with respect to safety repeat the administration. After 21 days following the first
(5.2.6) and efficacy (5.2.7) for the cats for which it is injection, take blood samples and determine the level of
antibodies against feline calicivirus by an
intended.
immunofiuorescence technique using pools of serum from
The following test for immunogenicity (2-3-1) may be used
groups of 3 mice. The vaccine complies with the test if the
during the demonstration of efficacy.
antibody levels are not significant1y lower than those obtained
2-3-1 Immunogenicity with a batch of vaccine that has given satisfactory results in
A test is carried out for each strain of feline calicivirus in the the test described under Potency.
vaccine and for each route and method of administration to
3 BATCH TESTS
be recommended for vaccination, using in each case cats
8-12 weeks old. The vaccine administered to each cat is of
3-1 Identification
When injected into animals that do not have specific
minimum potency.
antibodies against feline calicivirus, the vaccine stimulates the
U se for the test not fewer than 20 cats that do not have formation of such antibodies.
antibodies against feline calicivirus. Vaccinate not fewer than
10 cats, ,according to the schedule to be recommended. 3-2 Bacteria and fungi
Maintain not fewer than 10 cats as controls. Challenge each The vaccine and, where applicable, the liquid supplied with it
cat after 4 weeks by the intranasal route with a sufficient comply with the test for sterility prescribed in the monograph
quantity of a suspension of virulent feline calicivirus. Observe Vaccines for veterinary use (0062).
the cats at least daily for 14 days after challenge. Collect 3-3 Safety
nasal washings daily on days 2 to 14 to test for virus Use 2 cats, 8-12 weeks old, preferably that do not have
excretion. Note daily the body temperature and signs of antibodies against feline calicivirus or, where justified, use
disease using the scoring system shown below. cats that have a low level of such antibodies as long as they
The test is invalid if during the observation period after have not been vaccinated against feline calicivirosis and
challenge, fewer than 80 per cent of the control cats show administration of the vaccine does not cause an anamnestic
notable signs of feline calicivirosis (hyperthermia, buccal response. Administer to each cat by a recommended route a
uIcers, respiratory signs). The vaccine complies with the test double dose of the vaccine. Observe the cats at least daily for
if during the observation period after challenge, the score for 14 days.
the vaccinated cats is significant1y lower than that for the The vaccine complies with the test if no cat shows notable
controls. signs of disease or dies from causes attributable to the
Observed signs Score vaccine.
Death 10 3-4 Residuallive virus
Depressed state 2 Carry out a test for residuallive calicivirus using 10 doses of
Temperature 2': 39.5 oC 1 vaccine and 2 passages in cell cultures of the same type as
Temperature :S 37 oC 2 those used for preparation of the vaccine or in cell cultures
UIcer (nasal or oral) shown to be at least as sensitive. The vaccine complies with
- small and few in number 1 the test if no live virus is detected. If the vaccine contains an
- large and numerous 3 adjuvant that would interfere with the test, where possible
N asa! discharge separate the adjuvant from the liquid phase by a method that
- slight 1 does not inactivate or otherwise interfere with detection of
- copious 2 live virus.
Ocular discharge 1
3-5 Potency
Weight loss 2
The vaccine complies with the requirements of the test
Virus excretion (total number of days):
prescribed under Immunogenicity (section 2-3-1) when
:S 4 days
administered by a recommended route and method.
5-7 days 2
___________________________________________
> 7 days 3
~E~
2 PRODUCTION series of passages, the vaccine virus also complies with the
2-1 PREPARATION OF THE VACCINE test.
The vaccine virus is grown in cell cultures. 2-3-3 Immunogenicity
2-2 SUBSTRATE FOR VIRUS PROPAGATION A test is carried out for each strain of feline calicivirus in the
2-2-1 Cell cultures vaccine, for each route and method of administration to be
The cell cultures comply with the requirements for cell recommended for vaccination, using in each case cats
cultures for production ofveterinary vaccines (5.2.4). 8-12 weeks old. The quantity of vaccine virus to be
administered to each cat is not greater than the minimum
2-3 CHOICE OF VACCINE VIRUS virus titre to be stated on the label and the virus is at the
The vaccine virus is shown to be satisfactory with respect to most attenuated passage level that will be present in a batch
safety (inc1uding safety for pregnant queens if such u~e is not ofvaccine.
contra-indicated) (5.2.6) and efficacy (5.2.7) for the cats for
Use for the test not fewer than 20 cats that do not·have
which it is intended.
antibodies against fe1ine calicivirus. Vaccinate not fewer than
The following tests for safety (section 2-3-1), increase in 10 cats, according to the schedule to be recommended.
virulence (section 2-3-2) and immunogenicity (2-3-3) may be Maintain not fewer than 10 cats as controls. Challenge each
used during the demonstration of safety and efficacy. cat after 4 weeks by the intranasal route with a sufficient
2-3-1 Safety quantity of a suspension of virulent fe1ine calicivirus virus.
Carry out the test for each route and method of Observe the cats at least daily for 14 days after challenge.
administration to be recommended for vaccination. Collect nasal washings dai1y on days 2 to 14 to test for virus
Use vaccine virus at the least attenuated passage level that excretion. Note daily the body temperature and signs of
will be present between the master seed lot and a batch of disease using the scoring system shown be1ow.
the vaccine. The test is invalid if during the observation period after
F or each test, use not fewer than 10 cats of the minimum challenge, fewer than 80 per cent of the control cats show
age to be recommended for vaccination and that do not have notable signs of feline calicivirosis (hyperthermia, buccal
antibodies against fe1ine calicivirus. Administer to each cat a ulcers, respiratory signs). The vaccine virus complies with the
quantity of the vaccine virus equivalent to not less than test if during the observation period after challenge, the score
10 times the maximum virus titre likely to be contained in for the vaccinated cats is significantly lower than that for me
one dose of the vaccine. Observe the cats at least dai1y for controls.
21 days. Observed signs Score
The vaccine virus complies with the test if no cat shows Death 10
abnormallocal or systemic reactions, or dies from causes Depressed state 2
attributable to the vaccine virus. Temperature ~ 39.5 oC 1
Temperature :::; 37 oC 2
2-3-2 Increase in virulence
Ulcer (nasal or oral)
The test for increase in virulence consists of the
- small and few in number 1
administration of the vaccine virus at the least attenuated
- large and numerous 3
passage leve1 that will be present between the master seed lot
Nasal discharge
and a batch of the vaccine to 2 cats that do not have
- slight
antibodies against feline calicivirus, sequential passages,
- copious 2
5 times where possible, to further similar groups and testing
Ocular discharge 1
of the final recovered virus for increase in virulence. If the
Weight loss 2
properties of the vaccine virus allow sequential passage to
Virus excretion (total number of days):
5 groups via natural spreading, this method may be used,
:::; 4 days
otherwise passage as described below is carried out and the
5-7 days 2
maximally passaged virus that has been recovered is tested
for increase in virulence. Care must be taken to avoid
> 7 days 3 3
contamination by virus from previous passages. 3 BATCH TESTS
Administer to each cat by a route to be recommended a 3-1 Identification
quantity of the vaccine virus that will allow recovery of virus When neutralised by one or more monospecific antisera, the
for the passages described be1ow. Administer the virus by the vaccine no longer infects susceptible cell cultures into which
route to be recommended for vaccination most likely to lead it is inoculated.
to reversion of virulence. After 5 days, remove the nasal 3-2 Bacteria and fungi
mucus, tonsils and trachea of each cato Mix, homogenise in The vaccine and, where applicable, the liquid supplied with it
10 mL of buffered saline and decanto Administer the comply with the test for sterility prescribed in the monograph
supernatant by the intranasal route to each of 2 other cats. Vaccines far vetennary use (0062).
Carry out this passage operation 5 times; verify the presence
3-3 Mycoplasmas (2.6.7)
of the virus at each passage. If the virus is not found at a
The vaccine complies with the test for mycoplasmas.
passage level, carry out a second series of passages.
Carry out the test for safety (section 2-3-1) using unpassaged 3-4 Extraneous agents
N eutralise the vaccine virus with one or more suitable
vaccine virus and the maximally passaged virus that has been
monospecific antisera against fe1ine calicivirus and inoculate
recovered.
into cell cultures known for their susceptibility to virus es
The vaccine virus complies with the test if no indication of pathogenic for the cat. Carry out at least one passage and
increased virulence of the maximally passaged virus maintain the cultures for 14 days. The vaccine complies with
compared with the unpassaged virus is observed. If virus is the test if no cytopathic effect deve10ps and there is no sign
not recovered at any passage leve1 in the first and second of the presence of haemadsorbing agents.
250 Veterinary Vaccines
3-5 Safety cats typical signs of disease such as conjunctivitis and nasal
U se 2 cats 8-12 weeks old, that do not have antibodies discharge. Observe the cats for 28 days. Where reduction of
against feline calicivirus. Administer to each cat by a chlamydophila excretion is to be c1aimed, collect nasal
recommended route 10 doses of the vaccine. Observe the washings and/or conjunctival swabs on days 7, 14, 17, 21, 24
cats at least daily for 14 days. and 28 after challenge to test for chlamydophila excretion.
The vaccine complies with the test if no cat shows notable The duration of excretion for the vaccinated animals is
signs of disease or dies from causes attributable to the significandy lower than for the controls. Note daily the body
vaccine. temperature and signs of disease using a suitable scoring
system. The vaccine complies with the test if the score for
3-6 Virus titre
the vaccinated cats is significandy lower than that for the
Titrate the vaccine virus in suitable cell cultures at a
controls.
temperature favourable to replication of the virus.
The vaccine complies with the test if one dose contains not 2-3 MANUFACTURER'S TESTS
less than the minimum virus titre stated on the labe!' 2-3-1 Batch potency test
3-7 Potency It is not necessary to carry out the potency test (section 3-5)
The vaccine complies with the requirements of the test for each batch of the vaccine if it has been carried out using
prescribed under Irnmunogenicity (section 2-3-3) when a batch of vaccine with a minimum potency. Where the test
administered by a recommended route and method. It is not is not carried out on a batch, an alternative validated method
necessary to carry out the potency test for each batch of the is used, the criteria for acceptance being set with reference to
vaccine if it has been carried out on a representative batch a batch of vaccine that has given satisfactory results in the
using a vaccinating dose containing not more than the potency test (section 3-5). The following test may be used.
minimum virus titre stated on the labe!' Inject a suitable dose by a suitable route into each of 5
___________________________________________ ~E~ seronegative cats or another suitable species. Where the
schedule stated on the label requires a booster injection to be
given, a booster vaccination may also be given in this test
provided it has been demonstrated that this will still provide
a suitably sensitive test system. Before the vaccination and at
Feline Chlamydiosis Vaccine a given interval usually within the range of 14-21 days after
the last injection, collect blood from each animal and prepare
(Inactivated) serum samples. Determine individually for each serum the
(Feline Chlamydiosis Vaccine (Inactivated), titre of antibodies against each strain stated on the label,
Ph Bur monograph 2324) using a suitable test such as enzyme-linked irnmunosorbent
PhE~ ___________________________________________ assay (2.7.1). The vaccine complies with the test if the
antibody levels are not significantly lower than those obtained
1 DEFINITION
for a batch that has given satisfactory results in the potency
Feline chlamydiosis vaccine (inactivated) is a preparation of
test (section 3-5).
one or more suitable strains of Chlamydophila felis, which
have been inactivated by a suitable method. This monograph 2-3-2 Bacteria! endotoxins
applies to vaccines intended for administration to cats for A test for bacterial endotoxins (2.6.14) is carried out on the
active irnmunisation. final lot or, where the nature of the adjuvant prevents
performance of a satisfactory test, on the bulk antigen or the
2 PRODUCTION mixture of bulk antigens immediately before addition of the
2-1 PREPARATION OF THE VACCINE adjuvant. The maximum acceptable amount of bacterial
The seed material is cultured in embryonated hens' eggs endotoxins is that found for a batch of vaccine that has been
from a healthy flock or in suitable cell cultures (5.2.4). Ifthe sh,own to be satisfactory in the safety test (section 3-4).
vaccine contains more than one strain of bacterium, the The method chosen for determining the maximum
different strains are grown and harvested separately. acceptable amount of bacterial endotoxins is subsequendy
The bacterial harvests are inactivated using suitable and used for the testing of each batch.
validated methods. The suspensions may be treated to
fragment the micro-organisms and the fragments may be 3 BATCH TESTS
purified and concentrated. The vaccine may contain 3-1IDENTIFICATION
adjuvants. When injected into seronegative animals, the vaccine
stimulates the production of antibodies against each of the
2-2 CHOICE OF VACCINE COMPOSITION
strains of C. felis present in the vaccine. ---
The vaccine is shown to be satisfactory with respect to safety
(5.2.6) and efficacy (5.2.7) in cats for which it is intended. 3-2 RESIDUAL UVE CHLAMYDOPHILA
The following test for immunogenicity (section 2-2-1) may The vaccine complies with a suitable test for residual live
chlamydophila.
be used during the demonstration of efficacy.
3-3 BACTERIA AND FUNGI
2-2-1 Immunogenicity
The vacdine complies with the test for sterility prescribed in
Carry out the test for each route and method of
administration to be recommended for vaccination, using in the monograph Vaccines for veterinary use (0062).
each case cats not older than the minimum age 3-4 SAI'"ETY
recommended for vaccination and which do not have U se 2 cats not older than the minimum age recommended
antibodies against C. felis. Vaccinate 10 cats according to the for vaccination and free from antibodies against C. felis.
instructions for use and keep 10 cats as controls. Not later Administer to each cat by a recommended route a double
than 4 weeks after the last administration of vaccine, dos e of the vaccine. Observe the cats at least daily for
administer by a suitable route to each cat a quantity of a 2 weeks. The vaccine complies with the test if the cats
virulent strain of C. felis sufficient to produce in susceptible
Veterinary Vaccines 251
remain in good health and no abnormal local or systemic leucocytes does not exceed, in any of the 4 counts,
reactions occur. 50 per cent of the initial value.
3-5 POTENCY 2-4 MANUFACTURER'S TESTS
The vaccine complies with the test for immunogenicity 2-4-1· Residua1live virus
(section 2-2-1). The test for residual live virus is carried out using a quantity
___________________________________________ ~E~
of inactivated virus harvest equivalent to not less than 100
doses of the vaccine by a validated method such as the
following: inoculate into suitable non-confluent cells and after
incubation for 8 days, make a sub culture using trypsinised
Feline Infectious Enteritis Vaccine, \ :***~ cells. After incubation for a further 8 days, examine the
cultures for residual live parvovirus by an
Inactivated ***** immunofluorescence test. The immunofluorescence test may
Feline Panleucopenia Vaccine, Inactivated be supplemented by a haemagglutination test or other
(Feline Infectious Enteritis (Feline Panleucopenia) Vaccine
suitable tests on the supernatant of the cell cultures.
(Inactivated)J Ph Eur monograph 0794)
The inactivated viral harvest complies with the test if no live
~E~ ___________________________________________ virus is detected.
2-4-2 Batch potency test
1 DEFINITION For routine testing of batches of vaccine, a test based on
Feline infectious enteritis (feline panleucopenia) vaccine production of haemagglutination-inhibiting antibodies in
(inactivated) is a preparation of a suitable strain of feline guinea-pigs may be used instead of test 3-4-1 or 3-4-2
panleucopenia virus or canine parvovirus inactivated while described under Potency if a satisfactory correlation with the
maintaining adequate immunogenic properties. This test for immunogenicity has been established.
monograph applies to vaccines intended for the active
3 BATCH TESTS
immunisation of cats against feline infectious enteritis (feline
panleucopenia) . 3-1IDENTIFICATION
When injected into animals, the vaccine stimulates the
2 PRODUCTION production of antibodies against the parvovirus present in the
2-1 PREPARATION OF THE VACCINE vaccine.
The vaccine virus is grown in cell cultures. The virus harvest
3-2 BACTERIA AND FUNGI
is inactivated. The vaccine may be adjuvanted. The vaccine and, where applicable, the liquid supplied with it
2-2 SUBSTRATE FOR VIRUS PROPAGATION comply with the test for sterility prescribed in the monograph
2-2-1 Cell cultures Vaccines for veterinary use (0062).
The cell cultures comply with the requirements for cell 3-3 SAFETY
cultures for production of veterinary vaccines (5.2.4). U se 2 cats of the minimum age recommended for
2-3 CHOICE OF VACCINE COMPOSITION vaccination and preferably that do not have antibodies
The vaccine is shown to be satisfactory with respect to safety against feline panleucopenia virus or against canine
(5.2.6) and efficacy (5.2.7) for the cats for which it is parvovirus or, where justified, use cats that have a low level
intended. of such antibodies as long as they have not been vaccinated
The following test for immunogenicity (2-3-1) may be used against feline panleucopenia virus or against canine
during the demonstration of efficacy. parvovirus, and administration of the vaccine does not cause
an anamnestic response. Administer to each cat by a
2-3-1 Immunogenicity recommended route a double dose of the vaccine. Observe
A test is carried out for each route and method of the cats at least daily for 14 days.
administration to be recommended for vaccination, using in
The vaccine complies with the test if no cat shows notable
each case cats 8-12 weeks old. The vaccine administered to
signs of disease or dies from causes attributable to the
each cat is of minimum potency.
vaccine.
U se for the test not fewer than 10 cats that do not have
antibodies against feline panleucopenia virus and canine 3-4 POTENCY
parvovirus. Vaccinate not fewer than 5 cats, according to the Carry out test 3-4-1 or test 3-4-2.
schedule to be recommended. Maintain not fewer than 5 cats 3-4-1 Test in cats for haemagglutination-inhibiting
as controls. Carry out leucocyte counts 8 days and 4 days antibodies U se for the test not fewer than 4 cats, 8-12 weeks
before challenge and calculate the mean of the 2 counts to old, that do not have antibodies against feline panleucopenia
serve as the initial value. Challenge each cat after 20-22 days virus and canine parvovirus. Vaccinate not fewer than 2 cats
by the intraperitoneal route with a sufficient quantity of a with one dose of the vaccine to be recommended. Maintain
suspension of virulent feline panleucopenia virus. Observe the not fewer than 2 cats as controls. After 21 days, draw a
cats at least daily for 14 days after challenge. Carry out blood sample from each cat and separate the serum from
leucocyte counts on the 4th, 6th, 8th and 10th days after each sample. Inactivate each serum by heating at 56 oC for
challenge. 30 mino To 1 volume of each serum add 9 volumes of a
The test is invalid if during the observation period after 200 giL suspension of light kaolin R in phosphate buffered
saline pH 7.4 R. Shake each mixture for 20 mino Centrifuge,
challenge, fewer than 100 per cent of the control cats show
on not fewer than one occasion a diminution in the number collect the supernatant liquid and mix with 1 volume of a
of leucocytes of at least 75 per cent of the initial value or die concentrated suspension of pig erythrocytes. Allow to stand
from panleucopenia. The vaccine complies with the test if at 4 oC for 60 min and centrifuge. The dilution of the serum
during the observation period after challenge, all the obtained is 1:10. Using each serum, prepare a series of
vaccinated cats survive and show no signs of disease nor twofold dilutions. To 0.025 mL of each of the latter dilutions
leucopenia; that is to say, the diminution in the number of add 0.025 mL of a suspension of canine parvovirus or feline
252 Veterinary Vaccines
panleucopenia virus antigen containing 4 haemagglutinating The following tests for safety (section 2-3-1), increase in
units. Allow to stand at 37 oC for 30 min and add 0.05 mL virulence (section 2-3-2) and irnmunogenicity (2-3-3) may be
of a suspension of pig erythrocytes containing 30 x 106 cells used during the demonstration of safety and efficacy.
per milli1itre. Allow to stand at 4 oC for 90 min and note the 2-3-1 Safety
last dilution of serum that still completely inhibits Carry out the test for each route and method of
haemagglutination. administration to be recommended for vaccination.
The test is invalid if either control cat develops antibodies Use vaccine virus at the least attenuated passage level that
against canine parvovirus or feline panleucopenia virus. will be present between the master seed lot and a batch of
The vaccine complies with the test if both vaccinated cats the vaccine.
have developed titres of at least 1:20. For each test, use not fewer than 5 cats of the minimum age
3-4-2 Test in cats for virus-neutralising antibodies U se for the to be recommended for vaccination and that do not have
test not fewer than 2 cats, 8-12 weeks old, that have antibody specific haemagglutination-inhibiting antibodies against feline
titres less than 4ND so per 0.1 mL of serum measured by the panleucopenia virus and canine parvovirus. Make counts of
method described below. Vaccinate each cat according to the leucocytes in circulating blood on days 8 and 4 before
schedule 10 be recommended. 14 days after vaccination, injection of the vaccine virus and calculate the mean of the 2
examine the serum of each cat as follows. Reat the serum at counts to serve as the initial value. Administer to each cat a
56 oC for 30 min and prepare serial dilutions using a quantity of the vaccine virus equivalent to not less than
medium suitable for feline cells. Add to each dilution an 10 times the maximum virus titre likely to be contained in
equal volume of a virus suspension containing an amount of one dose of the vaccine. Observe the cats at least dai1y for
virus such that when the volume of serum-virus mixture 21 days. Make leucocyte counts on the 4th, 6th, 8th and 10th
appropriate for the assay system is inoculated into cell days after inoculation.
cultures, each culture receives approximately 104 CCID so . The vaccine virus complies with the test if no cat shows
Incubate the mixtures at 37 oC for 1 h and inoculate 4 feline abnormallocal or systemic reactions, signs of disease or dies
cell cultures with a suitable volume of each mixture. Incubate from causes attributable to the vaccine virus and if, for each
the cell cultures at 37 oC for 7 days, passage and incubate for cat and each blood count, the number of leucocytes is not
a further 7 days. Examine the cultures for evidence of less than 50 per cent of the initial value.
specific cytopathic effects and calculate the antibody titre.
2-3-2 Increase in virulence
The vaccine complies with the test if the mean titre is not The test for increase in virulence consists of the
less than 32 ND so per 0.1 mL of serum. If one cat fails to administration of the vaccine virus at the least attenuated
respond, repeat the test using 2 more cats and calculate the passage level that will be present between the master seed lot
result as the mean ofthe titres obtained from all of the 3 cats and a batch of the vaccine to 2 cats of the minimum age to
that have responded. be recommended for vaccination and that do not have
_____________________________________________ ~Ew
each case cats 8-12 weeks old. The quantity of vaccine virus 3-7 POTENCY
to be administered to each cat is not greater than the The vaccine complies with the requirements of the test
minimum virus titre to be stated on the label and the virus is prescribed under Immunogenicity (section 2-3-3) when
at the most attenuated passage level that will be present in a administered by a recommended route and method. It is not
batch of vaccine. necessary to carry out the potency test for each batch of the
U se for the test not fewer than 10 cats that do not have vaccine if it has been carried out on a representative batch
antibodies against feline panleucopenia virus and canine using a vaccinating dose containing not more than the
parvovirus. Vaccinate not fewer than 5 cats, according to the minimum virus titre stated on the label.
schedule to be recommended. Maintain not fewer than 5 cats ____________________________________________ ~E~
no significant difference is observed in vaccinated cats les s than 25 doses of vaccine and 2 passages in the same type
compared with controls. of cellcultures as used for the production of the vaccine orin
2-2-1-2 Repeated administration of vaccine Use for the test cell cultures shown to be at least as sensitive. The inactivated
not fewer than 10 cats of the minimum age to be viral harvest complies with the test if no live virus is detected.
recommended for vaccination. Vaccinate each cat with 2 2-4-2 Batch potency test
dos es of the vaccine to be recommended. At the end of the It is not necessary to carry out the Potency test (section 3-5)
period of time stated in the instructions for use, inject one for each batch of vaccine if it has been carried out using a
dose of vaccine to each cat. Where the instructions for use batch of vaccine with a minimum potency. Where the test is
recommend it, administer a 3rd injection after the period not carried out, an alternative validated method is used, the
indicated. Observe the cats at least daily for 14 days after the criteria for acceptance being set with reference to a batch of
last administration. The vaccine complies with the test if no vaccine that has given satisfactory results in the test described
cat shows abnormal local or systemic reactions or dies from under Potency.
causes attributable to the vaccine. 2-4-3 Bacterial· endotoxins
2-2-1-3 Test in pregnant queens If the vaccine is not contra- For vaccines produced by recombinant DNA technology with
indicated for use in pregnant queens, use not fewer than 10 a bacterial host cell such as Escherichia coli, a test for
queens at different stages of pregnancy. Administer to each bacterial endotoxins (2.6.14) is carried out on each final lot
cat 2 doses of the vaccine. Observe the queens at least daily or, where the nature of the adjuvant prevents performance of
until parturition. a satisfactory test, on the antigen irnmediately before addition
The vaccine complies with the test if no queen shows of the adjuvant. The value found is within the limit approved
abnormal local or systemic reactions or die s from causes for the particular vaccine and which has been shown to be
attributable to the vaccine and if no adverse effects on the safe for cats.
pregnancy or the offspring are noted. 3 BATCH TESTS
2-2-2 Immunogenicity 3-1IDENTIFICATION
A test is carried out for each route and method of When injected into healthy cats that do not have specific
administration to be recommended, using in each case cats of antibodies against the antigen or antigens stated on the label,
the minimum age to be recommended for vaccination. the vaccine stimulates the production of such antibodies.
The vaccine administered to each cat is of minimum
3-2 BACTERIA ANO FUNGI
potency.
The vaccine and, where applicable, the liquid supplied with it
Use for the test not fewer than 25 cats that do not have comply with the test for sterility prescribed in the monograph
antibodies against the antigens of feline leukaemia virus and Vaccines for veterinary use (0062).
against the feline oncogene membrane antigen (anti-FOCMA
3-3 SAFETY
antibodies), and showing no viraemia or antigenaemia at the
U se 2 cats of the minimum age recommended for
time of the test. Vaccinate not fewer than 15 cats according
vaccination and that do not have antibodies against feline
to the schedule to be recommended. Maintain not fewer than
leukaemia virus. Administer to each cat by a recommended
10 cats as controls. Observe the cats at least daily for 14 days
route a double dose of the vaccine. Observe the cats at least
after the last administration. Challenge each cat by the
daily for 14 days.
peritoneal or oronasal route, on one or several occasions,
with a sufficient quantity of suspension of an The vaccine complies with the test if no cat shows notable
epidemiologically relevant virulent strain of feline leukaemia signs of disease or dies from causes attributable to the
virus, consisting predominantly of type A virus. Observe the vaccine.
cats at least daily for 15 weeks and, from the 3rd week 3-4 RESIDUAL UVE VIRUS
onwards, test each week for viraemia or antigenaemia (p27 If the vaccine contains inactivated virus, carry out a test for
protein) by suitable methods such as irnmunofluorescence on residual live feline leukaemia virus by making 2 passages on
circulating leucocytes or enzyme-linked irnmunosorbent susceptible cell cultures. The vaccine complies with the test if
assay. A cat is considered persistently infected if it shows no virus is detected. If the vaccine contains an adjuvant, if
positive viraemia or antigenaemia for 3 consecutive weeks or possible separate the adjuvant from the liquid phase by a
on 5 occasions, consecutively or not, between the 3rd and the method that does not inactivate the virus nor interfere in any
15th week. other way with the detection of virus.
The test is invalid if during the observation period after 3-5 POTENCY
challenge, fewer than 80 per cent of the control cats show The vaccine complies with the requirements of the test
persistant viraemia or antigenaemia. The vaccine complies prescribed under Irnmunogenicity (section-2-2-2) when
with the test if during the observation period after challenge, administered by a recommended route and method.
not fewer than 80 per cent of the vaccinated cats show no _____________________________________________ PhE~
persistent infection.
2-3 IN-PROCESS CONTROL TESTS
During production, suitable immunochemical tests are
carried out for the evaluation of the quality and purity of the
viral antigens included in the vaccine composition.
The values found are within the limits approved for the
particular vaccine.
2-4 MANUFACTURER'S TESTS
2-4-1 Residuallive virus
Where applicable, the test for residual live virus is carried out
using a quantity of inactivated virus harvest equivalent to not
Veterinary Vaccines 255·
Sneezing, paroxysmal
Feline Viral Rhinotracheitis Ocular discharge, slight
2
1
Vaccine, Inactivated Ocular discharge, serious 2
(Feline Viral Rhinotracheitis Vaccine (Inactivated), Conjunctivitis 2
Ph Bur monograph 1207) Weight los s ~ 5.0 per cent 5
~E~ ___________________________________________ Virus excretion (total number of days):
~ 4 days
1 DEFINITION 5-7 days 2
Feline viral rhinotracheitis vaccine (inactivated) is a > 7 days 3
preparation of a suitable strain of feline rhinotracheitis virus
2-4 MANUFACTURER'S TESTS
(feline herpesvirus 1), inactivated while maintaining ap.equate
irnmunogenic properties, or of an inactivated fraction of the
I
2-4-1 Residua1live virus
virus having adequate immunogenic properties. This The test for residual live virus is carried out using 2 passages
monograph applies to vaccines intended for the active in cell cultures of the same type as those used for preparation
immunisation of cats against feline viral rhinotracheitis. of the vaccine or in cell cultures shown to be at least as
sensitive; the quantity of inactivated virus harvest used in the
2 PRODUCTION test is equivalent to not les s than 25 doses of vaccine.
2-1 PREPARATION1)F THE VACCINE The inactivated viral harvest complies with the test if no live
The vaccine virus is grown in cell cultures. The virus harvest virus is detected.
is inactivated; the virus may be disrupted and the fractions
2-4-2 Batch potency test
purifiéd and concentrated. The vaccine may be adjuvanted.
It is not necessary to carry out the Potency test (section 3-5)
2-2 SUBSTRATE FOR VIRUS PROPAGATION for each batch of vaccine if it has been carried out using a
2-2-1 Cell cultures batch of vaccine with a minimum potency. Where the test is
The cell cultures comply with the requirements for cell not carried out, an altemative validated method is used, the
cultures for production of veterinary vaccines (5.2.4). criteria for acceptance being set with reference to a batch of
2-3 CHOICE OF VACCINE COMPOSITION vaccine that has given satisfactory results in the test described
The vaccine is shown to be satisfactory with respect to safety under Potency. The following test may be used.
(5.2.6) and efficacy (5.2.7) for the cats for which it is Use for the test a group of 15 seronegative mice. Administer
intended. to each mouse half adose of the vaccine and, 7 days later,
The following test for irnmunogenicity (2-3-1) may be used repeat the administration. 21 days after the first injection,
during the demonstration of efficacy. take blood samples and determine the level of antibodies
against feline herpesvirus 1 by a suitable irnmunochemical
2-3-1 Immunogenicity method (2.7.1), such as an irnmunofiuorescence technique
A test is carried out for each route and method of using pools of serum from groups of 3 mice. The vaccine
administration to be recommended for vaccination, using in complies with the test if the antibody levels are not
each case cats 8-12 weeks old. The vaccine administered to significantly lower than those obtained with a batch of
each cat is of minimum potency. vaccine that has given satisfactory results in the test described
U se for the test not fewer than 20 cats that do not have under Potency.
antibodies against feline herpesvirus 1 or against a fraction of
the virus. Vaccinate not fewer than 10 cats, according to the 3 BATCH TESTS
schedule to be recommended. Maintain not fewer than 10 3-1IDENTIFICATION
cats as controls. Challenge each cat after 4 weeks by the When administered to animals that do not have specific
intranasal route with a quantity of a suspension of virulent antibodies against feline herpesvirus 1 or against the fraction
feline herpesvirus 1 sufficient to produce typical signs of the of the virus used to produce the vaccine, the vaccine
disease such as fever, nasal discharge and cough in a cat that stimulates the production of such antibodies.
do es not have antibodies against feline herpesvirus 1 or a 3-2 BACTERIA AND FUNGI
fraction of the virus. Observe the cats at least daily for The vaccine and, where applicable, the liquid supplied with it
14 days after challenge. Collect nasal washings daily on days comply with the test for sterility prescribed in the monograph
2 to 14 after challenge to test for virus excretion. Note daily Vaccines for veterinary use (0062).
the body temperature and signs of disease using the scoring 3-3 SAFETY
system shown below. U se 2 cats, 8-12 weeks old and preferably that do not have
The vaccine complies with the test if the score for the antibodies against feline herpesvirus 1 or against a fraction of
vaccinated cats is significantly lower than that for the the virus or, where justified, use cats that have a low level of
controls. such antibodies as long as they have not been vaccinated
Sign Score against feline rhinotracheitis and administration of the
Death 10 vaccine does not cause an anamnestic response. Administer
Depressed state 2 to each cat by a recommended route a double dose of the
Temperature: vaccine. Observe the cats at least daily for 14 days.
39.5 oC - 40.0 oC 1 The vaccine complies with the test if no cat shows notable
~ 40.0 oC 2 signs of disease or dies from causes attributable to the
~ 37.0 oC 3 vaccine.
Glossitis 3
3-4 RESIDUAL UVE VIRUS
Nasal discharge, slight 1
Carry out a test for residual live feline herpesvirus 1 using 10
Nasal discharge, copious 2
doses of vaccine and 2 passages in cell cultures of the same
Cough 2
type as those used for preparation of the vaccine, or in other
Sneezing 1
suitably sensitive cell cultures. The vaccine complies with the
256 Veterinary Vaccines
test if no live virus is detected. If the vaccine contains an properties of the vaccine virus allow sequential passage to
adjuvant that interferes with the test, where possible separate 5 groups via natural spreading, this method may be used,
the adjuvant from the liquid phase by a method that does not otherwise passage as described below is carried out and the
inactivate or otherwise interfere with detection of live virus. maximally passaged virus that has been recovered is tested
3-5 POTENCY for increase in virulence. Care must be taken to avoid
The vaccine complies with the requirements of the test contamination by virus from previous passages.
prescribed under Immunogenicity (section 2-3-1) when Administer to each cat by a route to be recommended a
administered by a recommended route and method. quantity of the vaccine virus that will allow recovery of virus
___________________________________________ ~E~
for the passages described below. Administer the virus by the
route to be recommended for vaccination most likely to lead
to reversion of virulence. After 2-4 days, remove the nasal
mucus, tonsils and local lymphatic ganglia and the trachea of
each cato Mix, homogenise in 10 mL of buffered saline and
Feline Viral Rhinotracheitis decanto Administer 1 mL of the supernatant by the intranasal
Vaccine, Living route to each of 2 other cats. Carry out this passage
operation not fewer than 5 times; verify the presence of the
(Feline Viral Rhinotracheitis Vaccine (Live),
virus at each passage. If the virus is not found at a passage
Ph Eur monograph 1206)
level, carry out a second series of passages.
PhE~ ___________________________________________
Carry out the test for safety (section 2-3-1) using unpassaged
1 DEFINITION vaccine virus and the maximally passaged virus that has been
Feline viral rhinotracheitis vaccine (live) is a preparation of a recovered.
suitable strain of feline rhinotracheitis virus (feline The vaccine virus complies with the test if no indication of
herpesvirus 1). This monograph applies to vaccines intended increased virulence of the maximally passaged virus
for the active immunisation of cats against feline viral compared with the unpassaged virus is observed. If virus is
rhinotracheitis. not recovered at any passage level in the first and second
2 PRODUCTION series of passages, the vaccine virus also complies with the
2-1 PREPARATION OF THE VACCINE test.
The vaccine virus is grown in cell cultures. 2-3-3 Irnrnunogenicity
2-2 SUBSTRATE FOR VIRUS PROPAGATION A test is carried out for each route and method of
administration to be recommended for vaccination, using in
2-2-1 Cell cultures each case cats 8-12 weeks old. The quantity of vaccine virus
The cell cultures comply with the requirements for cell
to be administered to each cat is not greater than the
cultures for production ofveterinary vaccines (5.2.4).
minimum virus titre to be stated on the label and the virus is
2-3 CHOICE OF VACCINE VIRUS at the most attenuated passage level that will be present in a
The vaccine virus is shown to be satisfactory with respect to batch of vaccine.
safety (5.2.6) and efficacy (5.2.7) for the cats for which it is Use for the test not fewer than 20 cats that do not have
intended (including safety for pregnant queens if such use is antibodies against feline herpesvirus 1. Vaccinate not fewer
not contra-indicated). than 10 cats, according to the schedule to be recommended.
The following tests for safety (section 2-3-1), increase in Maintain not fewer than 10 cats as controls. Challenge each
virulence (section 2-3-2) and immunogenicity (2-3-3) may be cat after 4 weeks by the intranasal route with a quantity of a
used during the demonstration of safety and efficacy. suspension of virulent feline herpesvirus 1 sufficient to cause
2-3-1 Safety typical signs of disease such as fever, nasal discharge and
Carry out the test for each route and method of cough. Observe the cats at least daily for 14 days after
administration to be recommended for vaccination, using in challenge. Collect nasal washings daily on days 2 to 14 after
each case cats of the minimum age to be recommended. challenge to test for virus excretion. Note daily the body
Use vaccine virus at the least attenuated passage level that temperature and signs of disease using the scoring system
will be present between the master seed lot and a batch of shown below.
the vaccine. The vaccine virus complies with the test if, during the
For each test, use not fewer than 10 cats that do not have observation period after challenge, the score for the
antibodies against feline herpesvirus 1. Administer to each cat vaccinated cats is significantly lower than that for the
a quantity of the vaccine virus equivalent to not les s than controls.
10 times the maximum virus titre likely to be contained in Sign Score
one dose of the vaccine. Observe the cats at least daily for Death 10
21 days. Depressed state 2
The vaccine virus complies with the test if no cat shows Temperature:
39.5 oC - 40.0 oC
abnormal local or systemic reactions, signs of disease or die s
from causes attributable to the vaccine virus. 2': 40.0\. oC 2
:::; 37.0 oC 3
2-3-2 Increase in virulence Glossitis 3
The test for increase in virulence consists of the Nasal' discharge, slight 1
administration of the vaccine virus at the least attenuated Nasal discharge, copious 2
passage level that will be present between the master seed lot Cough 2
and a batch of the vaccine to 2 cats that do not have Sneezing 1
antibodies against feline herpesvirus 1, sequential passages, Sneezing, paroxysmal 2
5 times where possible, to further similar groups and testing Ocular discharge, slight 1
of the final recovered virus for increase in virulence. If the
Veterinary Vaccines 257
dos e of vaccine, a 1/4 dose of vaccine would be obtained by antibodies neutralising the different serotypes of foot-and-
injecting 0.5 mL, and a 1/10 dose would be obtained by mouth disease virus. Vaccinate not fewer than 5 cattle by a
injecting 0.2 mL. Maintain 2 cattle as controls. Challenge all recommended route. Use a suitable dose of the vaccine for
the cattle after 3 weeks by the intradennal route, into 2 sites each animal. After a defined period, not greater than 28 days
on the upper surface of the tongue (0.1 mL per site), with a following vaccination, draw a blood sample and detennine
dose equivalent to approximately 10 000 IDso of a individually in each serum the level of antibodies against each
suspension of a fully virulent virus, obtained from cattle and serotype used in the preparation of the vaccine by a validated
of the same serotype as that used in the preparation of the technique (e.g. sero-neutralisation test, EUSA). The vaccine
vaccine. Observe the cattle at least daily for 8 days and then complies with the test if titres at least equal to the pass level
euthanise. Unprotected cattle show lesions at sites other than are measured in not fewer than 50 per cent of the cattle.
the tongue. Protected cattle may display linguallesi<~ns. 2-5-4-2 Vaccines far use in ather ruminants The potency of
The test is invalid if both control cattle do not show lesions each batch shall be demonstrated in a suitable, validated test.
on at least 3 feet. From the number of protected cattle in A test in cattle, following the procedures outlined above for
each group, calculate the PDso content of the vaccine. vaccines for use in cattle, may be suitable.
The vaccine complies with the test if the potency is not less EMERGENCY USE: in situations of extreme urgency and
than that to be stated on the label; the minimum potency to subject to agreement by the competent authority, a batch of
be stated on the lihel is not less than 3 PD so per dose for vaccine may be released before completion of the tests and
cattle. the detennination of potency if a test for sterility has been
2-5 MANUFACTURER'S TESTS carried out on the bulk inactivated antigen and all other
2-5-1 Identification components of the vaccine and if the test for safety and the
The bulk inactivated antigen is identified by a suitable detennination of potency have been carried out on a
immunochemical method (2.7.1). representative batch of vaccine prepared from the same bulk
inactivated antigen. In this context, a batch is not considered
2-5-2 Residuallive virus to be representative unless it has been prepared with not
The limit of detection of the cell cultures to be used with more than the amount of antigen or antigens and with the
respect to the virus to be tested is established by detennining same fonnulation as the batch to be released.
the number of CCIDso and the 146S antigen content of a
sample of live virus. The cells are not suitable if an amount 3 BATCH TESTS
of virus corresponding to 1 /lg of 146S antigen has les s than 3-1IDENTIFICATION
106 CCIDso . A proportion of each batch of bulk inactivated The serum of an animal that did not have antibodies against
antigen representing at least 200 doses is tested for freedom foot-and-mouth disease virus prior to being immunised with
from live virus by inoculation into suitable cell cultures. the vaccine neutralises the serotypes of the virus used to
A passage is made during culture of the cells. For this prepare the vaccine, when tested by a suitably sensitive
purpose, the sample of the inactivated antigen may be method.
concentrated to allow testing of such large samples in cell 3-2 BACTERIA ANO FUNGI
cultures. It must be shown that the selected concentration The vaccine and, where applicable, the liquid supplied with it
and assay systems are not detrimental to detection of comply with the test for sterility prescribed in the monograph
infectious virus within the test sample and that the Vaccines far veterinary use (0062).
concentrated inactivated antigen does not interfere with virus 3-3 SAFETY
replication or cause toxic changes. A positive control is U se 2 non-vaccinated animals of one of the species for which
inc1uded in each test. the vaccine is intended, not less than 6 months old, that do
2-5-3 Antigen content not have antibodies against foot-and-mouth disease virus and
The 146S antigen content of each batch of bulk inactivated coming from regions free from foot-and-mouth disease.
antigen is detennined by an in vitro method (for example, by Administer to each animal by a recommended route a double
sucrose density gradient centrifugation and ultraviolet dose of the vaccine. Observe the animals at least daily for
spectrophotometry at 259 nm). 14 days.
2-5-4 Batch potency test The vaccine complies with the test if no animal shows
It is not necessary to carry out the Potency test (section 3-4) notable signs of disease or dies from causes attributable to
for each batch of vaccine if it has been carried out using a the vaccine.
batch of vaccine with a minimum potency. Where the test is 3-4POTENCY
not carried out, an alternative validated method is used, the The vaccine complies with the requirements of the test
criteria for acceptance being set with reference to a batch of mentioned under Immunogenicity (section 2-4-2) when
vaccine that has given satisfactory results in the test described administered by a recommended route and method.
under Potency and has been shown to be satisfactory with ___________________________________________ PhE~
3-4POTENCY with the test if no chicken shows notable clinical signs of fowl
The vaccine complies with the requirements of the test pox or dies from causes attributable to the vaccine virus.
mentioned under Immunogenicity (section 2-2-2) when 2-4-2 Increase in virulence
administered by a recommended route and method. Administer by a suitable route a quantity of the vaccine virus
LABELLING that will allow recovery of virus for the passages described
The label states: below to each of 5 chickens not older than the minimum age
-- the serovar(s) used to prepare the vaccine, to be recommended for vaccination and from an SPF Rock
-- the serovar(s) against which protection is claimed. / (5.2.2). Use the vaccine virus at the least attenuated passage
___________________________________________ ~E~
level that will be present between the master seed lot and a
batch of the vaccine. Prepare 4 to 7 days after administration
a suspension from the induced skin lesions of each chicken
and pool these samples. Administer 0.2 mL of the. pooled
samples by cutaneous scarification of the comb or other
Fowl POX Vaccine, Living unfeathered part of the body, or by another suitable method
to each of 5 other chickens not older than the minimum age
(Fowl-pox Vaccine (Live), Ph Eur monograph 0649) to be recommended for vaccination and from an SPF Rock
PhE~ ___________________________________________ (5.2.2). Carry out this passage operation not fewer than
5 times; verify the presence of the virus at each passage. Care
1 DEFINITION must be taken to avoid contamination by virus from previous
Fowl-pox vaccine (live) is a preparation of a suitable strain of passages. If the virus is not found at a passage level, carry out
avian pox virus. This monograph applies to vaccines intended a second series of passages. Carry out the test for safety
for administration to chickens for active immunisation. (section 2-4-1) using the unpassaged vaccine virus and the
2 PRODUCTION maximally passaged virus that has been recovered.
2-1 PREPARATION OF THE VACCINE Administer the virus by the route recommended for
The vaccine virus is grown in embryonated hens' eggs or in vaccination likely to be the least safe. The vaccine virus
cell cultures. complies with the test if no indication of increase in virulence
of the maximally passaged virus compared with the
2-2 SUBSTRATE FOR vmus PROPAGATION
unpassaged virus is observed. If virus is not recovered at any
2-2-1 Embryonated hens' eggs passage level in the first and second series of passages, the
If the vaccine virus is grown in embryonated hens' eggs, they vaccine virus also complies with the test.
are obtained from Rocks free from specified pathogens (SPF) 2-4-3 Immunogenicity
(5.2.2).
A test is carried out for each route and method of
2-2-2 Cell cultures administration to be recommended using in each case
If the vaccine virus is grown in cell cultures, they comply chickens not older than the youngest age to be recommended
with the requirements for cell cultures for production of for vaccination. The quantity of the vaccine virus
veterinary vaccines (5.2.4). administered to each chicken is not greater than the
2-3 SEED LOTS minimum virus titre to be stated on the label and the virus is
at the most attenuated passage level that will be present in a
2-3-1 Extraneous agents
batch of the vaccine. Use for the test not fewer than 30
The master seed lot complies with the tests for extraneous
chickens ofthe same origin and from an SPF Rock (5.2.2).
agents in seed lots (2.6.24). In these tests on the master seed
Vaccinate by a recommended route not fewer than 20
lot, the organisms used are not more than 5 passages from
chickens. Maintain not fewer than 10 chickens as controls.
the master seed lot at the start of the tests.
Challenge each chicken after 21 days by the feather-follicle
2-4 CHOICE OF VACCINE VIRUS route with a sufficient quantity of virulent fowl-pox virus.
The vaccine virus shall be shown to be satisfactory with Observe the chickens at least daily for 21 days after
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens challenge. Record the deaths and the number of surviving
for which it is intended. chickens that show clinical signs of disease. Examine each
The following tests for safety (section 2-4-1), increase in surviving chicken for macroscopic lesions: cutaneous lesions
virulence (section 2-4-2) and immunogenicity (section 2-4-3) of the comb, wattle and other unfeathered areas of the skin
may be used during demonstration of safety and and diphtherical lesions of the mucous membranes of the
immunogenicity. oro-pharyngeal area.
2-4-1 Safety The test is not valid if:
Carry out the test for each route and method of -- during the observation period after challenge fewer than
administration to be recommended for vaccination using in 90 per cent of the control chickens die or show severe
each case chickens not older than the youngest age to be clinical signs of fowl pox, including notable macroscopical
recommended for vaccination. U se vaccine virus at the least lesions of the skin or mucous membranes of the
attenuated passage level that will be present between the oro-pharyngeal area,
master seed lot and a batch of the vaccine. For each test use -- and/or during the period between vaccination and
not fewer than 20 chickens, from an SPF Rock (5.2.2). challenge, more than 10 per cent of the control or
Administer to each chicken a quantity of the vaccine virus vaccinated chickens show abnormal clinical signs or die
equivalent to not less than 10 times the maximum virus titre from causes not attributable to the vaccine.
likely to be contained in adose of the vaccine. Observe the The vaccine virus complies with the test if during the
chickens at least daily for 21 days. The test is not valid if observation period after challenge not less than 90 per cent
more than 10 per cent of the chickens die from causes not of the vaccinated chickens survive and show no notable
attributable to the vaccine virus. The vaccine virus complies
262 Veterinary Vaccines
adhesions that give the viscera a 'one-unit' appearance ami/or 3 BATCH TESTS
lead to manifest laceration of the peritoneutn following 3-1 Identifcation
evisceration. When injected into fish that do not have specific antibodies
2-2-2 Irnmunogenicity against A. salmonicida, the vaccine stimulates the production
Carry out the test according to a protocol defining limits of of such antibodies.
body mass for the fish, water source, water flow and 3-2 Bacteria and fungi
temperature limits, and preparation of a standardised The vaccine and, where applicable, the liquid supplied with it
challenge. A test is carried out for the route and method of comply with the test for sterility prescribed in the monograph
administration to be recommended. The vaccine Vaccines for veterinary use (0062).
administered to each fish is of minimum potency. 3-3 Safety
Use for the test not fewer than 200 fish. Vaccinate hot fewer U se not fewer than 10 fish of one of the species for which
than 100 fish according to the instructions for use. Perform the vaccine is intended, having, where possible, the minimum
mock vaccination on a control group of not fewer than 100 body mass recommended for vaccination; if fish of the
fish; mark vaccinated and control fish for identification. Keep minimum body mass are not available, use fish not greater
all the fish in the same tank or mix equal numbers of than twice this mass. Use fish from a population that
controls and vaccinates in each tank if more than one tank is preferably does not have specific antibodies against A.
used. Challenge~-ach fish, by injection, at a fixed interval salmonicida subsp. salmonicida or, where justified, use fish
after vaccination, defined according to the statement from a population with a low level of such antibodies as long
regarding deve!opment of immunity, with a sufficient as they have not been vaccinated against or exposed to
quantity of a culture of A. salmonicida subsp. salmonicida furunculosis and administration of the vaccine does not cause
whose virulence has been verified. Observe the fish at least an anamnestic response. Carry out the test in the conditions
daily until at least 60 per cent specific mortality is reached in recommended for the use of the vaccine with a water
the control group. Plot for both vaccinates and controls a temperature not les s than 10 oC. Administer to each fish by
curve of specific mortality against time from challenge and the intraperitoneal route a double dose of the vaccine per
determine by interpolation the time corresponding to mass unit. Observe the fish at least daily for 21 days.
60 per cent specific mortality in controls. The test is invalid if more than 10 per cent of the fish die
The test is invalid if the specific mortality is less than from causes not attributable to the vaccine. The vaccine
60 per cent in the control group 21 days after the first death complies with the test if no fish shows notable signs of
in the fish. Read from the curve for vaccinates the mortality disease or dies from causes attributable to the vaccine.
(M) at the time corresponding to 60 per cent mortality in
3-4 Potency
controls. Calculate the relative percentage survival (RPS)
The vaccine complies with the requirements of the test
using the following expression:
mentioned under Immunogenicity (section 2-2-2) when
administered by the recommended route and method.
4LABELLING
The label states information on the time needed for
development of immunity after vaccination under the range
The vaccine complies with the test if the RPS is not less than of conditions corresponding to the recommended use.
___________________________________________ PhEw
80 per cent.
2-3 MANUF.¡'\CTURER'S TESTS
2-3-1 Batch potency test
The potency test (section 3-4) may be carried out for each
batch of vaccine, using groups of not fewer than 30 fish of Infectious Avian Encephalomyelitis
one of the species for which the vaccine is intended. Where
the test is not carried out, an altemative validated method Vaccine, Living
based on antibody response may be used, the criteria for Epidemic Tremor Vaccine, Living
acceptance being set with reference to a batch of vaccine that (Avian Infectious Bncephalomyelitis Vaccine (Live)J
has given satisfactory results in the test described under Ph Bur monograph 0588)
Potency. The following test may be used. PhEw ___________________________________________
Use not fewer than 35 fish from a population that does not
1 DEFINITION
have specific antibodies against A. salmonicida subsp.
Avian infectious encephalomye!itis vaccine (live) is a
salmonicida and that are within specified limits for body mass.
preparation of a suitable strain of avian encephalomyelitis
Carry out the test at a defined temperature. Inject
virus. This monograph applies to vaccines intended for
intraperitoneally into each of not fewer than 25 fish one dose
administration to non-Iaying breeder chickens to protect
of vaccine, according to the instructions for use. Perform
passively their future progeny and/or to prevent vertical
mock vaccination on a control group of not fewer than 10
transmission of virus via the egg.
fish. Collect blood samples at a defined time after
vaccination. Determine for each sample the leve! of specific 2 PRODUCTION
antibodies against A. salmonicida subsp. salmonicida by a 2-1 PREPARATION OF THE VACClNE
suitable immunochemical method (2.7.1). The test is invalid The vaccine virus is grown in embryonated hens' eggs or in
if the control group shows antibodies against A. salmonicida cell cultures.
subsp. salmonicida. The vaccine complies with the test if the
mean leve! of antibodies in the vaccinates is not significantly
lower than that found for a batch that gave satisfactory
results in the test described under Potency.
264 Veterinary Vaccines
2-2 SUBSTRATE FOR VIRUS PROPAGATION passages. Carry out the test for safety (section 2-4-1) using
2-2-1 Embryonated hens' eggs the unpassaged vaccine virus and the maximally passaged
If the vaccine virus is grown in embryonated hens' eggs, they vaccine virus that has been recovered. The vaccine virus
are obtained from flocks free from specified pathogens (SPF) complies with the test if no indication of increase in virulence
(5.2.2). of the maximally passaged virus compared with the
unpassaged virus is observed. If virus is not recovered at any
2-2-2 Cell cultures passage level in the first and second series of passages, the
If the vaccine virus is grown in cell cultures, they comply vaccine virus also complies with the test.
with the requirements for cell cultures for production of
veterinary vaccines (5.2.4). 2-4-3 Immunogenicity
If the vaccine is recommended for passive protection of
2-3 SEED LOTS future progeny carry out test 2-4-3-1. If the vaccine is
2-3-1 Extraneous agents recommended for prevention of vertical transmission of virus
The master seed lot complies with the tests for extraneous via the egg, carry out test 2-4-3-2. A test is carried out for
agents in seed lots (2.6.24). In these tests on the master seed each route and method of administration to be
lot, the organisms used are not more than 5 passages from recommended, using in each case chickens from an SPF
the master seed lot at the start of the tests. flock (5.2.2) not older than the youngest age to be
2-4 CHOICE OF VACCINE VIRUS recommended for vaccination. The quantity of the vaccine
The vaccine virus shall be shown to be satisfactory with virus administered to each chicken is not greater than the
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens minimum titre to be stated on the label and the virus is at
for which it is intended. the most attenuated passage level that will be present in a
The following tests for safety (section 2-4-1), increase in batch of the vaccine.
virulence (section 2-4-2) and immunogenicity (section 2-4-3) 2-4-3-1 Passive immunity in chickens Vaccinate not fewer
may be used during the demonstration of safety and than 20 breeder chickens from an SPF flock (5.2.2).
immunogenicity. Maintain separately not fewer than 10 breeder chickens of
the same age and origin as controls. At the peak of lay, hatch
2-4-1 Safety
not fewer than 25 chickens from eggs from vaccinated
Carry out the test for each route and method of
breeder chickens and 10 chickens from non-vaccinated
administration to be recommended for vaccination using in
breeder chickens. At 2 weeks of age, challenge each chicken
each case non-laying breeder chickens not older than the
by the intracerebral route with a sufficient quantity of
youngest age to be recommended for vaccination.
virulent avían encephalomyelitis virus. Observe the chickens
U se vaccine virus at the least attenuated passage level that
at least daily for 21 days after challenge. Record the deaths
will be present between the master seed lot and a batch of
and the number of surviving chickens that show clinical signs
the vaccine. For each test, use not fewer than 20 chickens
of disease. The test is not valid if:
from an SPF flock (5.2.2). Administer to each chicken a
- during the observation period after challenge fewer than
quantity of the vaccine virus equivalent to not less than
80 per cent of the control chickens die or show severe
10 times the maximum virus titre likely to be contained in
clinical signs of avían infectious encephalomyelitis,
1 dose of the vaccine. Observe the chickens at least daily for
and/or during the period between the vaccination and
21 days. The test is not valid if more than 10 per cent of the
challenge more than 15 per cent of control or vaccinated
chickens show abnormal clinical signs or die from causes not
chickens show abnormal clinical signs or dIe from causes
attributable to the vaccine virus. The vaccine virus complies
not attributable to the vaccine.
with the test if no chicken shows notable clinical signs of
avian infectious encephalomyelitis or dies from causes The vaccine virus complies with the test if during the
attributable to the vaccine virus. observation period after challenge not fewer than 80 per cent
of the progeny of vaccinated chickens survive and show no
2-4-2 Increase in virulence notable clinical signs of disease.
The test for increase in virulence consists of the
administration of the vaccine virus at the least attenuated 2-4-3-2 Passive immunity in embryos Vaccinate not fewer
passage level that will be present between the master seed lot than 20 breeder chickens from an SPF flock (5.2.2).
and a batch of vaccine to a group of five 1-day-old chickens Maintain separately not fewer than 10 breeder chickens of
from an SPF flock (5.2.2), sequential passages, 5 times where the same age and origin as controls. At the peak of lay,
possible, to further similar groups of 1-day-old chickens, and incubate not fewer than 36 eggs from the 2 groups,
testing of the final recovered virus for increase in virulence. vaccinated and controls, and carry out an embryo sensitivíty
If the properties of the vaccine virus allow sequential passage test. On the sixth day of incubation inocula,te 100 EIDso of
to 5 groups via natural spreading, this method may be used, the Van Roekel strain of avían encephalomyelitis virus into
otherwise passage as described below is carried out and the the yolk sacs of the eggs. 12 days after inoculation examine
maximally passaged virus that has been recovered is tested the embryos for specific lesions of avían encephalomyelitis
for increase in virulence. Care must be takeri to avoid (muscular atrophy). Deaths during the first 24 h are
contaminatiOn by virus from previous passages. Administer considered to be non-specific. The test is not valid if fewer
by a recommended route and method, a quantity of the than 80 Pler cent of the control embryos show lesions of
vaccine virus that will allow recovery of virus for the passages avían encephalomyelitis. The test is not valid if fewer than
described below. 5 to 7 days later, prepare a suspension from 80 per cent of the embryos can be given an assessment.
the brain of each chick and pool these samples. Administer a The vaecine virus complies with the test if not fewer than
suitable volume of the pooled samples by the oral route to 80 per cent of the embryos in the vaccinated group show no
each of 5 other chickens of the same age and origino Carry lesions of avían encephalomyelitis.
out this passage operation not fewer than 5 times; verify the
presence of the virus at each passage. If the virus is not
found at a passage level, carry out a second series of
Veterinary Vaccines 265·
3 BATCH TESTS
3-1 Identification
Infectious Bovine Rhinotracheitis
The vaccine, diluted if necessary and mixed with a Vaccine, Living
monospecific avian encephalomyelitis virus antiserum, no (Infectious Bovine Rhinotracheitis Vaccine (Live)J
longer infects embryonated hens' eggs from an SPF fiock Ph Bur monograph 0696)
(5.2.2) or susceptible cell cultures (5.2.4) into which it is PhE~ ___________________________________________
inoculated.
1 DEFINITION
3-2 Bacteria and fungi
Vaccines intended for administration by injection comply Infectious bovine rhinotracheitis vaccine (live) is a
with the test for sterility prescribed in the monograph preparation of one or more suitable strains of infectious
Vaccines for veterinary use (0062). bovine rhinotracheitis virus (bovine herpesvirus 1). This
monograph applies to vaccines intended for the active
Vaccines not intended for administration by injection either
immunisation of cattle against bovine rhinotracheitis caused
comply with the test for sterility prescribed in the monograph
by bovine herpesvirus l.
Vaccines for veterinary use (0062) or with the following test:
carry out a quantitative test for bacterial and fungal 2 PRODUCTION
contamination; carry out identification tests for 2-1 PREPARATION OF THE VACCINE
microorganisms det~cted in the vaccine; the vaccine does not The vaccine virus is grown in cell cultures.
contain pathogenic microorganisms and contains not more 2-2 SUBSTRATE FOR VIRUS PROPAGATION
than 1 non-pathogenic microorganism per dose.
2-2-1 Cell cultures
Any liquid supplied with the vaccine complies with the test The cell cultures comply with the requirements for cell
for sterility prescribed in the monograph Vaccines for cultures for production ofveterinary vaccines (5.2.4).
veterinary use (0062).
2-3 CHOICE OF VACCINE VIRUS
3-3 Mycoplasmas The vaccine virus is shown to be satisfactory with respect to
The vaccine complies with the test for mycoplasmas (2.6.7). safety (5.2.6) and efficacy (5.2.7) for the cattle for which it is
3-4 Extraneous agents intended.
The vaccine complies with the tests for extraneous agents in The following tests for safety (section 2-3-1), abortigenicity
batches of finished product (2.6.25). and passage through the placenta (section 2-3-2), increase in
3-5 Safety virulence (section 2-3-3) and immunogenicity (2-3-4) may be
Use not fewer dian 10 chickens not older than the minimum used during the demonstration of safety and efficacy.
age recommended for vaccination and from an SPF fiock 2-3-1 Safety
(5.2.2). Administer by a recommended route and method to Carry out the test for each route and method of
each chicken 10 doses of the vaccine. Observe the chickens administration to be recommended for vaccination.
at least daily for 21 days. The test is not valid if more than U se vaccine virus at the least attenuated passage level that
20 per cent of the chickens show abnormal c1inical signs or will be present between the master seed lot and a batch of
die from causes not attributable to the vaccine. The vaccine the vaccine.
complies with the test if no chicken shows notable c1inical For each test, use not fewer than 5 calves, 3 months old, or
signs of disease or dies from causes attributable to the of the minimum age to be recommended for vaccination if
vaccine. this is less than 3 months and that do not have antibodies
3-6 Virus titre against infectious bovine rhinotracheitis virus. Administer to
Titrate the vaccine virus by inoculation into embryonated each calf a quantity of the vaccine virus equivalent to not les s
hens' eggs from an SPF fiock (5.2.2) or into suitable cell than 10 times the maximum virus titre likely to be contained
cultures (5.2.4). The vaccine complies with the test if 1 dose in one dose of the vaccine. Observe the calves at least daily
contains not less than the minimum virus titre stated on the for 21 days.
label. The vaccine virus complies with the test if no calf shows
3-7 Potency abnormal local or systemic reactions or dies from causes
Depending on the indications, the vaccine complies with the attributable to the vaccine virus.
requirements of 1 or both of the tests prescribed under 2-3-2 Abortigenicity and passage through the placenta
Immunogenicity (section 2-4-3-1, 2-4-3-2), when Use not fewer than 24 pregnant cows that do not have
administered by a recommended route and method. It is not antibodies against infectious bovine rhinotracheitis virus: 8 of
necessary to carry out the potency test for each batch of the the cows are in the 4th month of pregnancy, 8 in the 5th and
vaccine if it has been carried out on a representative batch 8 in the 6th or 7th month. Administer to each cow by a route
using a vaccinating dose containing not more than the to be recommended a quantity of the vaccine virus equivalent
minimum virus titre stated on the label. to not less than 10 times the maximum virus titre likely to be
___________________________________________ ~E~
contained in one dose of the vaccine. Observe the cows at
least daily until the end of pregnancy.
The vaccine virus complies with the test if:
-- where abortion occurs, tests show that neither virus nor
viral antigens are present in the fcetus or placenta;
-- on calves born at term before ingestion of colostrums, a
test for antibodies against infectious bovine rhinotracheitis
virus indicates no such antibodies are found.
266 Veterinary Vaccines
intended for use in breeding chickens to protect their infectious bursal disease antigen by a suitable method.
progeny from avian infectious bursal disease. The vaccine complies with the test if 3 or fewer of the
2 PRODUCTION chickens from group B hens show evidence of avian
infectious bursal disease. The test is invalid unless all the
2-1 PREPARATION OF THE VACCINE
chickens from group A hens show evidence of avian
The vaccine virus is grown in embryonated hens' eggs or in
infectious bursal disease.
cell cultures.
Where there is more than one recommended route of
The vaccine may be adjuvanted.
administration, the test described under Potency is carried
2-2 SUBSTRATE FOR VIRUS PROPAGATION out in parallel with the aboye immunogenicity test, using
2-2-1 Embryonated hens' eggs different groups of birds for each recommended route.
If the vaccine virus is grown in embryonated hens' eggs, they The serological response of the birds inoculated by routes
are obtained from healthy fiocks. other than that used in the immunogenicity test is not
2-2-2 Cell cultures significantly less than that of the group vaccinated by that
If the vaccine is grown in cell cultures, they comply with the route.
requirements for cell cultures for production of veterinary 2-5 MANUFACTURER'S TESTS
vaccines (5.2.4)._ 2-5-1 Residuallive virus
2-3 SEED LOTS An amplification test for residuallive avian infectious bursal
2-3-1 Extraneous agents disease virus is carried out on each batch of antigen
Themaster seed lot complies with the tests for extraneous immediately after inactivation to confirm inactivation; the test
agents in seed lots (2.6.24). In these tests on the master seed is carried out in embryonated hens' eggs or in suitable cell
lot, the organisms used are not more than 5 passages from cultures (5.2.4), whichever is the most sensitive for the
the master seed lot at the start of the test. vaccine strain; the quantity of inactivated virus harvest used
in the test is equivalent to not less than 10 doses of the
2-4 CHOICE OF VACCINE COMPOSITION vaccine. The vaccine complies with the test if no live virus is
The vaccine is shown to be satisfactory with respect to safety detected.
(5.2.6) and efficacy (5.2.7) for the birds for which it is
intended. 2-5-2 Batch potency test
It is not necessary to carry out the potency test (section 3-6)
The following test for immunogenicity (2-4-1) may be used
for each batch of the vaccine if it has been carried out using
during the demonstration of efficacy.
a batch of vaccine with a minimum potency. Where the test
2-4-1 Irnmunogenicity is not carried out, an altemative validated method is used,
A test is carried out for each route and method of the criteria for acceptance being set with reference to a batch
administration to be recommended using in each case of vaccine that has given satisfactory results in the test
chickens from a fiock free from specified pathogens (SPF) described under Potency. The following test may be used.
(5.2.2) and not older than the youngest age to be Vaccinate each ofnot fewer than 10 chickens, 14-28 days old
recommended for vaccination (close to the point of lay). and from an SPF fiock (5.2.2), with 1 dose of vaccine by a
The dose of vaccine administered to each chicken contains recommended route. 4-6 weeks later, collect serum samples
not more than the minimum potency to be stated on the from each bird and 10 unvaccinated control birds of the
label. same age and from the same source. Measure the antibody
Where a challenge test is to be carried out, the following test response in a serum-neutralisation test.
may be used. Use 2 groups of not less than 20 hens treated The test is invalid if there are specific antibodies in the sera
as follows: of the unvaccinated birds. The vaccine complies with the test
- Group A: unvaccinated controls; if the mean antibody titre in the sera from the vaccinated
- Group B: vaccinated with inactivated avian infectious birds is equal to or greater than the titres obtained with a
bursal disease vaccine. batch that has given satisfactory results in the test described
Serum samples are collected from each unvaccinated control under Immunogenicity (section 2-4-1).
(group A) hen just before administration of the vaccine,
3 BATCH TESTS
4-6 weeks later, and at the time of egg collection for
hatching. If a serological test is to be carried out for 3-1 Identification
demonstration of immunogenicity by other routes, serum When injected into chickens that do not have antibodies
samples are also collected from each vaccinated (group B) against avian infectious bursal disease virus type 1, the
hen at the time of egg collection for hatching. The antibody vaccine stimulates the production of such antibodies.
response is measured in a serum-neutralisation test. 3-2 Bacteria and fungi
Eggs are collected for hatching not less than 5 weeks after The vaccine, and, where applicable, the liquid supplied with
vaccination and the test described below is carried out with it comply with the test for sterility prescribed in the
chickens at least 3 weeks old from that egg collection. monograph Vaccines for veterinary use (0062).
25 chickens from vaccinated (group B) hens and 10 control 3-3 Safety
chickens of the same breed and age from unvaccinated Use 10 chickens, 14-28 days old, from SPF fiocks (5.2.2).
(group A) hens are challenged with an eye-drop application Administer to each chicken by a recommended route a
of a quantity of a virulent strain of avian infectious bursal double dose of vaccine. Observe the birds at least daily for
disease virus sufficient to produce severe signs of disease, not less than 21 days.
including lesions of the bursa of Fabricius, in all The vaccine complies with the test if no bird shows notable
unvaccinated chickens. 3-4 days after challenge, the bursa of signs of disease or dies from causes attributable to the
Fabricius is removed from each chicken. The bursae are vaccine.
examined for evidence of infection by histological
examination and by testing for the presence of avian
268 Veterinary Vaccines
2-4-2 Damage to the bursa ofFabricius Challenge each chicken of the 3 groups by the intramuscular
Carry out the test for the route to be recommended for route with not less than 105 E1Dso of virulent N ewcastle
vaccination like1y to be the least safe using chickens not older disease virus and note the degree of protection in the
than the youngest age to be recommended for vaccination. 2 groups vaccinated with Hitchner B1 strain Newcastle
U se virus at the least attenuated passage level that will be vaccine compared with the non-vaccinated group. The test is
present between the master seed lot and a batch of the not valid if 1 or more of the non-vaccinated chickens does
vaccine. U se not fewer than 20 chickens from an SPF fiock not die within 7 days of challenge. The degree of
(5.2.2). Administer to each chicken a quantity ofthe vaccine immunosuppression is estimated from the comparative
virus equivalent to 10 times the maximum titre likely to be seroresponses and protection rates of the 2 Hitchner B 1
contained in adose ofthe vaccine. On each of days 7, 14, 21 vaccinated groups. The vaccine complies with the test if there
and 28 after administration of the vaccine virus, euth;anise is no significant difference between the 2 groups.
not fewer than 5 chickens and prepare a section from the site 2-4-4 Increase in virulence
with the greatest diameters of the bursa of Fabricius of each The test for increase in virulence consists of the
chicken. Carry out histological examination of the section administration of the vaccine virus at the least attenuated
and score the degree of bursal damage using the following passage level that will be present between the master seed lot
scale. and a batch of the vaccine to a group of 5 chickens from an
O No lesion, nOm1al bursa. SPF fiock (5.2.2) and not older than the youngest age to be
1 1 per cent to 25 per cent of the follicles show lymphoid recommended for vaccination, sequential passages, 5 times
depletion (i.e. less than 50 per cent depletion in 1 affected where possible, to further similar groups and testing of the
follicle) infiux of heterophils in lesions. final recovered virus for increase in virulence. If the
2 26 per cent to 50 per cent of the follic1es show near1y properties of the vaccine virus allow sequential passage to
complete lymphoid depletion (Le. more than 75 per cent 5 groups via natural spreading, this method may be used,
depletion in 1 affected follicle), affected follicles show otherwise passage as described be10w is carried out and the
necrosis and severe infiux of heterophils may be detected. maximally passaged virus that has been recovered is tested
for increase in virulence. Care must be taken to avoid
3 51 per cent to 75 per cent of the follic1es show lymphoid
contamination by virus from previous passages. Administer
depletion; affected follic1es show necrosis and severe infiux of
by eye-drop a quantity of the vaccine virus that will allow
heterophils is detected.
recovery of virus for the passages described be1ow. Prepare
4 76 per cent to 100 per cent of the follicles show near1y 3 to 4 days after administration a suspension from the bursa
complete lymphoid depletion, hyperplasia and cyst structures of Fabricius of each chicken and pool these samples.
are detected; affected follic1es show necrosis and severe infiux Administer 0.05 mL of the pooled samples by eye-drop to
of heterophils is detected. each of 5 other chickens of the same age and origino Carry
5 100 per cent of the follicles show near1y complete lymphoid out this passage operation not fewer than 5 times; verify the
depletion; complete los s of follicular structure, thickened and presence of the virus at each passage. If the virus is not
folded epithe1ium, fibrosis of bursal tissue. found at a passage level, carry out a second series of
Calculate the average score for each group of chickens. passages. Carry out the test for damage to the bursa of
The vaccine virus complies with the test if: Fabricius (section 2-4-2) using the unpassaged vaccine virus
- no chicken shows notable clinical signs of disease or die s and the maximally passaged virus that has been recovered.
from causes attributable to the vaccine virus, Administer the virus by the route recommended for
- the average score for bursal damage 21 days after vaccination likely to be the least safe. The vaccine virus
administration of the vaccine virus is les s than or equal to complies with the test if no indication of increasing virulence
2.0 and 28 days after administration is less than or equal of the maximally passaged virus compared with the
to 0.6, unpassaged virus is observed. If virus is not recovered at any
- during the 21 days after administration a notable passage leve1 in the first and second series of passages, the
repopulation of the bursae by lymphocytes has taken vaccine virus also complies with the test.
place. 2-4-5 Immunogenicity
2-4-3 Immunosuppression A test is carried out for each route and method of
Carry out the tests for the route recommended for administration to be recommended using in each case
vaccination like1y to be the least safe using chickens not older chickens not older than the youngest age to be recommended
than the youngest age recommended for vaccination. for vaccination. The quantity of vaccine virus administered to
Use vaccine virus at the least attenuated passage leve1 that each chicken is not greater than the minimum virus titre to
will be present between the master seed lot and a batch of be stated on the label and the virus is at the most attenuated
the vaccine. Use not fewer than 30 chickens from an SPF passage level that will be present in a batch of the vaccine.
fiock (5.2.2). Divide them randomly into 3 groups eachiof U se not fewer than 30 chickens of the same origin and from
not fewer than 10 and maintain the groups separate1y. an SPF fiock (5.2.2). Vaccinate by a recommended route not
Administer by eye-drop to each chicken of 1 group a fewer than 20 chickens. Maintain not fewer than 10 chickens
quantity of the vaccine virus equivalent to not less than the as controls. Challenge each chicken after 14 days by eye-drop
maximum titre like1y to be contained in 1 dose of the with a sufficient quantity of virulent avian infectious bursal
vaccine. At the time after administration when maximal disease virus. Observe the chickens at least daily for 10 days
bursal damage is lil,ely to be present, as judged from the after challenge. Record the deaths due to infectious bursal
results obtained in the test for damage to the bursa of disease and the surviving chickens that show clinical signs of
Fabricius (section 2-4-2), administer to each vaccinated disease. At the end of the observation period, euthanise all
chicken and to each chicken of another group 1 dos e of the surviving chickens and carry out histological examination
Hitchner B 1 strain N ewcastle disease vaccine (live). for lesions of the bursa of Fabricius. The test is not valid if
Determine the seroresponse of each chicken of the 2 groups one or more of the following applies:
to the N ewcastle disease virus 14 days after administration.
270 Veterinary Vaccines
-- during the observation period following challenge, fewer using a vaccinating dose containing not more than the
than 50 per cent of the control chickens show minimum virus titre stated on the label.
characteristic signs of avían infectious bursal disease, ___________________________________________ PhE~
such as thymic atrophy and specific bone-marrow lesions. than the minimum age to be recommended for vaccination
Observe the remaining chickens at least daily for 21 days. and from an SPF flock (5.2.2). The test for prevention of
The vaccine virus complies with the test if during the virus excretion is intended to demonstrate reduction of egg
observation period no chicken shows notable signs of chicken transmission through viraemia and virus excretion in the
anaemia or die s from causes attributable to the vaccine virus. faeces. The quantity of the vaccine virus to be administered
2-4-1-2 Safety for young chickens U se not fewer than twenty
to each chicken is not greater than the minimum virus titre
l-day-old chickens from an SPF flock (5.2.2). Administer to to be stated on the label and the virus is at the most
each chicken by the oculonasal route a quantity of the / attenuated passage level that will be present in a batch of
vaccine virus equivalent to not less than the maximum titre vaccine.
likely to be contained in 1 dos e of the vaccine. Observe the 2-4-3-1 Passive immunisation of chickens Vaccinate according
chickens at least daily. Record the incidence of any signs to the recommended schedule not fewer than 10 breeder
attributable to the vaccine virus, such as depression, and any chickens not older than the minimum age recommended for
deaths. 14 days after vaccination, collect blood samples from vaccination and from an SPF flock (5.2.2); keep not fewer
half of the chickens and determine the haematocrit value. than 10 unvaccinated breeder chickens of the same origin
Euthanise these chickens and carry out post-mortem and from an SPF flock (5.2.2) as controls. At a suitable time
examination. Note any pathological changes attributable to after excretion of vaccine virus has ceased, collect fertilised
chicken anaemia virus, such as thymic atrophy and specific eggs from each vaccinated and control breeder chicken and
bone marrow lesions. Observe the remaining chickens at least incubate them. Challenge at least 3 randomly chosen l-day-
daily for 21 days. Assess the extent to which the vaccihe old chickens from each vaccinated and control breeder
strain is pathogenic for l-day-old susceptible chickens from chicken by intramuscular administration of a sufficient
the results of the clinical observations and mortality rates and quantity of virulent chicken anaemia virus. Observe the
the proportion of chickens examined at 14 days that show chickens at least daily for 14 days after challenge. Record the
anaemia (haematocrit value less than 27 per cent) and signs deaths and the surviving chickens that show signs of disease.
of infectious chicken anaemia on post-mortem examination. At the end of the observation period determine the
The results are used to formulate the label statement on haematocrit value of each surviving chicken. Euthanise these
safety for young chickens. chickens and carry out post-mortem examination. Note any
pathological signs attributable to chicken anaemia virus, such
2-4-2 Increase in virulence
as thymic atrophy and specific bone-marrow lesions. The test
The test for increase in virulence consists of the
is not val id if:
administration of the vaccine virus at the least attenuated
- during the observation period after challenge fewer than
passage level that will be present between the master seed lot
90 per cent of the chickens of the control breeder
and a batch of the vaccine to a group of five l-day-old
chickens die or show severe signs of infectious chicken
chickens from an SPF flock (5.2.2), sequential passages,
anaemia, including haematocrit value under 27 per cent,
5 times where possible, to further similar groups of l-day-old
and/or notable macroscopic lesions of the bone marrow
chickens and testing of the final recovered virus for increase
and thymus;
in virulence. If the properties of the vaccine virus allow
- and/or during the period between vaccination and egg
sequential passage to 5 groups via natural spreading, this
collection more than 10 per cent of vaccinated or control
method may be used, otherwise passage as described below is
breeder chickens show notable signs of disease or die
carried out and the maximally passaged virus that has been
from causes not attributable to the vaccine.
recovered is tested for increase in virulence. Care must be
taken to avoid contamination by virus from previous The vaccine complies with the test if during the observation
passages. period after challenge not fewer than 90 per cent of the
chickens of the vaccinated breeder chickens survive and show
Administer to each animal by the intramuscular route a
no notable signs of disease andlor macroscopic lesions of the
quantity of the vaccine virus that will allow recovery of virus
bone marrow and thymus.
for the passages described below. Prepare 7 to 9 days after
administration a suspension from the liver of each chicken 2-4-3-2 Prevention of virus excretion Vaccinate according to
and pool these samples. Depending on the tropism of the the recommended schedule not fewer than 10 chickens not
virus, other tissues such as spleen or bone marrow may be older than the minimum age recommended for vaccination
used. Administer 0.1 mL of the pooled samples by the and from an SPF flock (5.2.2). Maintain separately not fewer
intramuscular route to each of 5 other chickens of the same than 10 chickens of the same age and origin as controls. At a
age and origino Carry out this passage operation not fewer suitable time after excretion of vaccine virus has ceased,
than 5 times; veri:fy the presence of the virus at each passage. challenge all the chickens by intramuscular administration of
If the virus is not found at a passage level, carry out a second a sufficient quantity of virulent chicken anaemia virus.
series of passages. Collect blood samples from the chickens on days 3, 5 and 7
after challenge and faecal samples from the chickens on days
Carry out the tests for safety (section 2-4-1) using the
7, 14 ::¡.nd 21 after challenge and carry out a test for presence
unpassaged vaccine virus and the maximally passaged vaccine
of virus to determine whether or not the chickens are
virus that has been recovered.
viraemic and are excreting the virus. The test is not valid if:
The vaccine virus complies with the test if no indication of - fewer than 70 per cent of the control chickens are
increased virulence of the maximally passaged virus viraemic and excrete the virus at one or more times of
compared with the unpassaged virus is observed. If virus is sampling;
not recovered at any passage level in the first and second - and/or during the period between vaccination and
series of passages, the vaccine virus also complies with the challenge more than 10 per cent of control or vaccinated
test. chickens show abnormal clinical signs or die from causes
2-4-3 Irnmunogenicity not attributable to the vaccine.
A test is carried out for each route and method of
administration to be recommended using chickens not older
272 Veterinary Vaccines
2-4-2 Safety
Carry out the test for each route and method of
administration to be recommended for vaccination, using in
each case chickens not older than the youngest age to be
recommended for vaccination. Use vaccine virus at the least
attenuated passage level that will be present between the
master seed lot and a batch of the vaccine. For each test use
Veterinary Vaccines 273
not fewer than 20 chickens, from an SPF fiock (5.2.2). infiammation of the trachea and orbital sinuses. The test is
Administer to each chicken a quantity of the vaccine virus not valid if:
equivalent to not less than 10 times the maximum virus titre - during the observation period after challenge fewer than
likely to be contained in 1 dos e of the vaccine. Observe the 90 per cent of the control chickens die or show severe
chickens at least daily for 21 days. The test is not valid if c1inical signs of avian infectious laryngotracheitis or
more than 10 per cent of the chickens die from causes not notable macroscopic lesions of the trachea and orbital
attributable to the vaccine virus. The vaccine virus complies sinuses,
with the test if no chicken shows notable c1inical signs /of - or if during the period between the vaccination and
avian infectious laryngotracheitis or dies from causes challenge more than 10 per cent of the vaccinated or
attributable to the vaccine virus. control chickens show notable c1inical signs of disease or
2-4-3 Increase in virulence die from causes not attributable to the vaccine.
The test for increase in virulence consists of the The vaccine virus complies with the test if during ,the
administration of the vaccine virus at the least attenuated observation period after challenge not fewer than 90 per cent
passage level that will be present between the master seed lot of the vaccinated chickens survive and show no notable
and a batch of the vaccine to a group of 5 chickens not more c1inical signs of disease and/or macroscopicallesions of the
than 2 weeks old, from an SPF fiock (5.2.2), sequential trachea and orbital sinuses.
passages, 5 timeswhere possible, to further similar groups 3 BATCH TESTS
and testing of the final recovered virus for increase in 3-1 Identification
virulence. If the properties of the vaccine virus allow The vaccine, diluted if necessary and mixed with a
seqúential passage to 5 groups via natural spreading, this monospecific infectious laryngotracheitis virus antiserum, no
method may be used, otherwise passage as described below is longer infects embryonated hens' eggs from an SPF fiock
carried out and the maximally passaged virus that has been (5.2.2) or susceptible cell cultures (5.2.4) into which it is
recovered is tested for increase in virulence. Care must be inoculated.
taken to avoid contamination by virus from previous
passages. Administer by eye-drop a quantity of the vaccine 3-2 Bacteria and fungi
virus that will allow recovery of virus for the passages Vaccines intended for administration by injection comply
described below. After the period shown to correspond to with the test for sterility prescribed in the monograph
maximum replication of the virus, prepare a suspension from Vaccines far veterinary use (0062).
the mucosae of suitable parts of the respiratory tract of each Vaccines not intended for administration by injection either
chicken and pool these samples. Administer 0.05 mL of the comply with the test for sterility prescribed in the monograph
pooled samples by eye-drop to each of 5 other chickens that Vaccines far veterinary use (0062) or with the following test:
are 2 weeks old and from an SPF fiock (5.2.2). Carry out carry out a quantitative test for bacterial and fungal
this passage operation not fewer than 5 times; verify the contamination; carry out identification tests for micro-
presence of the virus at each passage. If the virus is not organisms detected in the vaccine; the vaccine does not
found at a passage level, carry out a second series of contain pathogenic micro-organisms and contains not more
passages. Determine the index of respiratory virulence than 1 non-pathogenic micro-organism per dose.
(section 2-4-1) using the unpassaged vaccine virus and the Any liquid supplied with the vaccine complies with test for
maximally passaged virus that has been recovered; if the titre sterility in the monograph Vaccines far veterinary use (0062).
of the maximally passaged virus is less than lOs EID sO or 3-3 Mycoplasmas
lOs CCID so, preparethe tenfold, serial dilutions using the The vaccine complies with the test for mycoplasmas (2.6.7).
highest titre available. The vaccine virus complies with the
test if no indication of increase in virulence of the maximally 3-4 Extraneous agents
passaged virus compared with the unpassaged virus is The vaccine complies with the tests for extraneous agents in
observed. If virus is not recovered at any passage level in the batches of finished product (2.6.25).
first and second series of passages, the vaccine virus also 3-5 Safety
complies with the test. Use not fewer than 10 chickens from an SPF fiock (5.2.2)
2-4-4 Immunogenicity and of the youngest age recommended for vaccination.
A test is carried out for each route and method of Administer by eye-drop to each chicken 10 doses of the
administration to be recommended using in each case vaccine. Observe the chickens at least daily for 21 days.
chickens not older than the youngest age to be recommended The test is not valid if more than 20 per cent of the chickens
for vaccination. The quantity of the vaccine virus show abnormal c1inical signs or die from causes not
administered to each chicken is not greater than the attributable to the vaccine. The vaccine complies with the
minimum virus titre to be stated on the label and the virus is test if no chicken shows notable c1inical signs of disease or
at the most attenuated passage level that will be present in a dies from causes attributable to the vaccine.
batch ofthe vaccine. Use for the test not fewer than 30 3-6 Virus titre
chickens of the same origin and from an SPF fiock (5.2.2). Titrate the vaccine virus by inoculation into embryonated
Vaccinate by a recommended route not fewer than 20 hens' eggs from an SPF fiock (5.2.2) or into suitable cell
chickens. Maintain not fewer than 10 chickens as controls. cultures (5.2.4). The vaccine complies with the test if 1 dose
Challenge each chicken after 21 days by the intratracheal contains not les s than the minimum titre stated on the label.
route with a sufficient quantity of virulent infectious 3-7 Potency
laryngotracheitis virus. Observe the chickens at least daily for The vaccine complies with the requirements of the test
7 days after challenge. Record the deaths and the number of prescribed under Immunogenicity (section 2-4-4) when
surviving chickens that show c1inical signs of disease. At the administered according to the recommended schedule by a
end of the observation period euthanise all the surviving recommended route and method. It is not necessary to carry
chickens and carry out examination for macroscopic lesions: out the potency test for each batch of the vaccine if it has
mucoid, haemorrhagic and pseudomembraneous been carried out on a representative batch using a vaccinating
274 Veterinary Vaccines
dose containing not more than the minimum virus titre recommended requires a 2nd dose, administer one dose after
stated on the label. the interval to be recommended. Observe the cows at least
_____________________________________________ PhE~ daily until one day after calving. Record body temperatures
the day before each vaccination, at vaccination, 4 h later and
daily for 4 days.
The vaccine complies with the test if no cow shows notable
Bovine Leptospirosis Vaccine **** signs of disease or dies from causes attributable to the
** * vaccine and if no adverse effects on the pregnancy and
(Inactivated) ***** offspring are noted.
(Ph Eur monograph 1939) 2-2-1-2 PieZd studies The cattle used for the field trials are
PhE~ _____________________________________________ also used to evaluate safety. Use not fewer than 3 groups of
20 cattle with corresponding groups of not fewer than 10
1 DEFINITION controls in 3 different locations. Examine the injection sites
Bovine leptospirosis vaccine (inactivated) is a preparation of for local reactions after vaccination. Record body
inactivated whole organisms andJor antigenic extractes) of temperatures the day before vaccination, at vaccination and
one or more suitable strains of one or more of Leptospira on the 2 days following vaccination.
borgpetersenii serovar hardjo, Leptospira interrogans serovar
The vaccine complies with the test if no animal shows
hardjo or other L. interrogans serovars, inactivated while
notable signs of disease or dies from causes attributable to
maintaining adequate irnmunogenic properties. This
the vaccine. In addition, if the vaccine is for use in pregnant
monograph applies to vaccines intended for the active
cattle, no adverse effects on the pregnancy and offspring are
immunisation of cattle against leptospirosis.
noted.
2 PRODUCTION 2-2-2 Immunogenicity
2-1 PREPARATION OF THE VACCINE
Carry out a separate test for each of the serovars for which a
The seed material is cultured in a suitable mediurn; each claim is made for a beneficial effect on the rates of infection
strain is cultivated separately. During production, various and urinary excretion. If claims are to be made for protection
parameters such as growth rate are monitored by suitable against reproductive or production los ses, further specific
methods; the values are within the limits approved for the studies will be required.
particular producto Purity and identity are verified on the
Each test is carried out for each route and method of
harvest using suitable methods. After cultivation, the bacterial
administration to be recommended, using in each case cattle
harvest is inactivated by a suitable method. The antigen may
be concentrated. The vaccine may be adjuvanted. of the minimurn age to be recommended for vaccination.
The vaccine adrninistered to each animal is of minimum
2-2 CHOICE OF VACCINE COMPOSITION potency.
The vaccine is shown to be satisfactory with respect to safety
2-2-2-1 Immunogenicity against L. borgpetersenii serovar
(5.2.6) and efficacy (5.2.7) for the cattle for which it is
hardjo Use not fewer than 15 cattle that do not have
intended.
antibodies against L. borgpetersenii serovar hardjo and the
The following tests for safety (section 2-2-1) and principal serovars of L. interrogans (icterohaemorrhagiae,
immunogenicity (section 2-2-2) may be used during the canicola, grippotyphosa, sejroe, hardjo, hebdomonadis,
demonstration of safety and efficacy. pomona, australis and auturnnalis). Vaccinatenot fewer than
2-2-1 Safety 10 cattle according to the schedule to be recommended.
2-2-1-1 Laboratory tests Carry out the test for each route Maintain not fewer than 5 cattle as controls. 21 days after
and method of administration to be recommended for the last vaccination, challenge all the cattle by a suitable
vaccination and in cattle of each category for which the mucosal route with a sufficient quantity of a virulent strain of
vaccine is intended (for example, young calves, pregnant the relevant serovar. Observe the cattle at least daily for a
cattle). U se a batch of vaccine containing not less than the fu±ther 35 days. Collect urine samples from each animal on
maximurn potency that may be expected in a batch of days O, 14, 21, 28 and 35 post-challenge. Euthanise surviving
vaccine. cattle at the end of the observation periodo Carry out post-
2-2-1-1-1 General safety. For each test, use not fewer than mortem examination on any animal that dies and on those
10 cattle that do not have antibodies against L. borgpetersenii euthanised at the end of the observation periodo In particular,
serovar hardjo and the principal serovars of L. interrogans examine the kidneys for macroscopic and microscopic signs
(icterohaemorrhagiae, canicola, grippotyphosa, sejroe, hardjo, of leptospira infection. A sample of each kidney is collected
hebdomonadis, pomona, australis and autumnalis). and each urine and kidney sample is tested-for the presence
Administer to each animal a double dose of the vaccine. of the challenge organisms by re-isolation or by another
If the schedule to be recommended requires a 2nd dose, suitable method.
adrninister one dos e after the interval to be recommended. For the test conducted with L. borgpetersenii serovar hardjo,
Observe the cattle at least daily for at least 14 days after the control cattle are regarded as infected if the challenge
last administration. Record body temperatures the day before organism~ are re-isolated from at least 2 samples. The test is
each vaccination, at vaccination, 4 h later and daily for invalid if lnfection has been established in fewer than
4 days. 80 per cent of the control cattle.
The vaccine complies with the test if no animal shows The vaccine complies with the test if the challenge organisms
abnormal local or systemic reactions, signs of disease, or dies are re-isolated from any urine or kidney sample from not
from causes attributable to the vaccine. more than 20 per cent of the vaccinated cattle.
2-2-1-1-2 Safety in pregnant cattle. If the vaccine is intended 2-2-2-2 Immunogenicity against other Zeptospira species For
for use or may be used in pregnant cattle, use not fewerthan leptospiral species other than L. borgpetersenii serovar hardjo,
10 cows at the relevant stages of pregnancy. Administer to the test is conducted as described in 2-2-2-1 but urine
each cow a double dos e of the vaccine. If the schedule to be samples are collected on appropriate days, deterrnined by the
Veterinary Vaccines 275
characteristics of the challenge model. In the case of serovars 3-4 Residuallive bacteria
for which there is published evidence that the serovar has a Carry out a test for live leptospirae by inoculation of a
lower tropism for the urinary tract, a lower rate of infection specific medium. Inoculate 1 mL of the vaccine into 100 mL
may be justified. Depending on their tissue tropism, for sorne of the medium. Incubate at 30 oC for 14 days, sub culture
leptospira serovars, samples from other tissues/body fiuids into a further quantity of the medium and incubate both
can be used to establish whether the cattle are infected or not media at 30 oC for 14 days: the vaccine complies with the
by the challenge organismo test if no growth occurs in either medium. At the same time,
2-3 MANUFACTURER'S TESTS carry out a control test by inoculating a further quantity of
the medium with the vaccine together with a quantity of a
2-3-1 Batch potency test culture containing approximately 100 leptospirae and
It is not necessary to carry out the Potency test (section 3-5) incubating at 30 oC: the test is invalid if growth of
for each batch of vaccine if it has been carried out u~ing a leptospirae does not occur within 14 days.
batch of vaccine with a minimum potency. Where the test is
not carried out, an alternative validated method is used, the 3-5 Potency
criteria for acceptance being set with reference to a batch of The vaccine complies with the requirements of the test
vaccine that has given satisfactory results in the test described mentioned under Immunogenicity (section 2-2-2) when
under Potency. Th~ following test may be used. administered by a recommended route and method.
___________________________________________ PhE~
dos e of the vaccine. If the recommended schedule requires a The vaccine complies with the test if: at least 80 per cent of _
second dose, administer one dose after the recommended the vaccinatesshow no more than mild signs of disease (for
interval. Observe the dogs at least daily until 14 days after example, transient hyperthermia) and, depending on the L.
the last administration. Record body temperatures the day interrogans serovar used for the challenge, one or more of the
before each vaccination, at vaccination, 4 h later and daily for following is also shown:
4 days. - where the vaccine is intended to have a beneficial effect
The vaccine complies with the test if no dog shows abnormal against signs of disease, the clinical scores and
local or systemic reactions, signs of disease or dies from haematological and biochemical scores are statistically
causes attributable to the vaccine. lower for the vaccinates than for the controls,
2-2-1-2 Safety in pregnant bitches If the vaccine is intended - where the vaccine is intended to have a beneficial effect
against infection, the number of days that the organisms
for use or may be used in pregnant bitches, use not fewer
are detected in the blood is statistically lower for the
than 10 bitches at the stage of pregnancy in accordance with
vaccinates than for the controls,
the schedule to be recommended or at different stages of
- where the vaccine is intended to have a beneficial effect
pregnancy. Administer to each bitch a double dos e of the
vaccine. If the recommended schedule requires a second against urinary tract infection and excretion, the number
of days that the organisms are detected in the urine and
dose, administer one dos e after the recommended interval.
Observe the bitches at least daily until one day after the number of kidney samples in which the organisms are
detected is statistically lower for the vaccinates than for
whelping.
the controls.
The vaccine complies with the test if no bitch shows
abnormal local or systemic reactions, signs of disease or dies 2-3 MANUFACTURER'S TESTS
from causes attributable to the vaccine and if no adverse 2-3-1 Batch potency test
effects on the pregnancy or the offspring are noted. It is not necessary to carry out the Potency test (section 3-5)
2-2-2 Irnmunogenicity for each batch of the vaccine if it has been carried out using
F or each type of the serovars against which protective a batch of vaccine with a minimum potency. Where the test
immunity is claimed on the labe!, carry out a separate test is not carried out, an altemative validated method is used,
with a challenge strain representative of that serovar. the criteria for acceptance being set with reference to a batch
of vaccine that has given satisfactory results in the test
Each test is carried out for each route and method of
described under Potency. The following tests may be used.
administration to be recommended for vaccination, using in
each case dogs of the minimum age to be recommended. 2-3-1-1 Por vaccines with or without adJuvants If leptospira
The vaccine administered to each dog is of minimum antigen from more than one serovar (for example L. interrogans
content andlor potency. serovar canicola and serovar icterohaemorrhagiae) has been
used to prepare the vaccine, carry out a batch potency test
Use for the test not fewer than 12 dogs that do not-have
for each serovar against which protective immunity is claimed
antibodies against the principal serovars of L. interrogans
on the label. U se for the test 10 healthy hamsters not more
(icterohaemorrhagiae, canicola, grippotyphosa, sejroe, hardjo,
than 3 months old, that do not have antibodies against the
hebdomo'nadis, pomona, australis and autumnalis). Vaccinate
principal serovars of L. interrogans (icterohaemorrhagiae,
not fewer than 6 dogs, according to the schedule to be
canicola, grippotyphosa, sejroe, hardjo, hebdomonadis,
recommended. Maintain not fewer than 6 dogs as controls.
pomona, australis and autumnalis) and whichhave been
Challenge each dog after 25-28 days by the conjunctival
obtained from a regularly tested and certified leptospira-free
and/or intraperitoneal route with a sufficient quantity of a
source. Administer 1/40 of the dose for dogs by the
suspension of the relevant pathogenic L. interrogans serovar.
subcutaneous route to 5 hamsters. Maintain 5 hamsters as
Observe the dogs at least daily for 28 days after challenge.
controls. Challenge each hamster after 15-20 days by the
Examine the dogs daily and record and score clinical signs intraperitoneal route with a sufficient quantity of a virulent
observed post-challenge and any deaths that occur. If a dog cUlture of leptospirae of the serovar against which protective
shows marked signs of disease, it is euthanised. Monitor immunity is claimed on the label. The vaccine complies with
body temperatures each day for the first week after challenge. the test if not fewer than 4 of the 5 control hamsters die
Collect blood samples from each dog on days O, 2, 3, 4, 5, 8 showing typical signs of leptospira infection within 14 days of
and 11 post challenge. Collect urine samples from each dog receiving the challenge suspension and if not fewer than 4 of
on days O, 3, 5, 8, 11, 14, 21 and 28 post challenge. the 5 vaccinated hamsters remain in good health for 14 days
Euthanise surviving dogs at the end of the observation after the death of 4 control hamsters.
periodo Carry out post-mortem examination on any. dog that
2-3-1-2 Por vaccines with or without adJuvants A suitable
dies during the observation period and on the remamder
validated sero-response test may be carried out. Vaccinate
when euthanised at the end of the observation periodo
each animal in a group of experimental animals with a
In particular, examine the liver and kidneys for macroscopic
suitable dose. Collect blood samples after a suitable, fixed
and microscopic signs of leptospira infection. Take a sample
time after vaccination. For each of the serovars present in the
of each ki~ey and test each blood, urine and kidney sample
vaccine, an in vitro test is carried out on individual blood
for the presence of challenge organisms by re-isolation or by
samples fo determine the antibody response to one or more
another suitable method. Analyse blood samples to detect
antigenic 'components which are indicators of protection and
biochemical and haematological changes indicative of
which are specific for that serovar. The criteria for
infection and score these.
acceptance are set with reference to a batch of vaccine that
The test is invalid if: samples give positive results on day O; has given satisfactory results in the test described under
L. interrogans serovar challenge strain is re-isolated from or Potency.
demonstrated by another suitable method to be present in
2-3-1-3 Por vaccines without adJuvants For each ofthe
fewer than 2 samples on fewer than 2 different days, to show
serovars present in the vaccine, a suitable validated in vitro
infection has been established in fewer than 80 per cent of
test may be carried out to determine the content of one or
the control dogs.
Veterinary Vaccines 277
more antigenic components which are indicators of demonstration of safety, Appendix XV Jevet) 1 and
protection and which are specific for that serovar. immunogenicity, Appendix XV Jevet) 2.
The criteria for acceptance are set with reference to a batch Safety
of vaccine that has given satisfactory results in the test Carry out a test in each category of each species of animal
described under Potency. for which the vaccine is to be recommended and by each
3 BATCH TESTS recommended route of administration. Vaccinate at least five
3-1 Identification animals that do not have antibodies to louping-ill virus.
When injected into healthy animals that do not have specific U se for the test a batch of vaccine with the maximum
antibodies against leptospira serovar(s) present in the vaccine, potency likely to be included in adose of the vaccine.
the vaccine stimulates the production of such antibo~ies. Administer a double dose of vaccine to each animal and
If test 2-3-1-3 is used for batch potency test, it also ~erves to observe them for two weeks. No abnormallocal or systemic
identify the vaccine. reactions occur. If the vaccine is for use or may be used in
pregnant animals,. for the test in this category, administer the
3-2 Bacteria and fungi
vaccine at the relevant stage or stages of pregnancy, prolong
The vaccine and, where applicable, the liquid supplied with it
the observation period up to the time of parturition and note
comply with the test for sterility prescribed in the monograph
any effects on gestation or on the offspring.
Vaccines for veterinary use (0062).
Imrnunogenicity
3-3 Safety
The tests to demonstrate immunogenicity are carried out in
U se 2 dogs of the minimum age recommended for
each category of each species of animal for which the vaccine
vaccination and that do not have antibodies against the
is to be recommended and by each recommended route of
leptospira serovar(s) present in the vaccine. Administer to
administration and using a batch or batches with the
each dog by a recommended route a double dose of the
minimum potency likely to be included in adose of the
vaccine. Observe the dogs at least daily for 14 days.
vaccine. The efficacy claims made on the label
The vaccine complies with the test if no dog shows notable (e.g. protection from clinical signs of the disease) reflect the
signs of disease or dies from causes attributable to the type of data generated.
vaccine.
BATCH TESTING
3-4 Residuallive bacteria
Carry out a test for live leptospirae by inoculation of a Inactivation
specific medium. Inoculate 1 mL of the vaccine into 100 mL Carry out a suitable validated test for residuallouping-ill
ofthe medium.Incubate at 30 oC for 14 days, sub culture virus on the bulk antigen blend immediately before the
into a further quantity of the medium and incubate both addition of the adjuvant. N o live virus is detected.
media at 30 oC for 14 days: the vaccine complies with the The vaccine complies with the requirements stated under
test if no growth occurs in either medium. At the same time, Veterinary Vaccines with the following modijications.
carry out a control test by inoculating a further quantity of IDENTIFICATION
the medium with the vaccine together with a quantity of a When injected into healthy seronegative animals, the vaccine
culture containing approximately 100 leptospirae and stimulates the production of specific haemagglutinating
incubating at 30 oC: the test is invalid if growth of antibodies against louping-ill virus.
leptospirae does not occur within 14 days.
TESTS
3-5 Potency
Extraneous bacteria and fungi
The vaccine complies with the requirements of the test
The vaccine complies with the test for sterility described
mentioned under Immunogenicity (section 2-3-2) when
under Veterinary Vaccines.
administered by a recommended route and method.
___________________________________________ ~Ew
Safety
Use two lambs of the minimum age recommended for
vaccination and that do not have antibodies to louping-ill
virus. Administer a double dose of vaccine to each of the
lambs by a route recommended on the label and observe the
Louping-iII Vaccine animals for two weeks. No abnormallocal or systemic
DEFINITION reactions occur.
Louping-ill Vaccine is a preparation of a suitable strain of POTENCY
louping-ill virus which has been inactivated in such a manner Inject subcutaneously each of no fewer than six healthy
that immunogenic activity is retained. sheep, free from louping-ill haemagglutination-inhibiting (HI)
PRODUCTION antibodies, with the dos e stated on the label. Between 14 and
The virus strain is grown in suitable cell cultures, 28 days after injection the serum of at least five of the sheep
Appendix XV Jevet)l. The viral suspension is harvested and contains HI antibodies at dilutions of 1 in 20 or greater
inactivated. A test for residual infectious louping-ill virus is against 4 to 8 haemagglutinating units.
carried out on each batch of antigen immediately after STORAGE
inactivation. A mouse inoculation test may provide a suitably When stored under the prescribed conditions the vaccine
sensitive test if there is no suitably sensitive in vitro test for may be expected to retain its potency for at least 1 year.
the strain.
The vaccine contains an adjuvant.
CHOICE OF VACCINE COMPOSITION
This vaccine is shown to be satisfactory with respect to safety
and immunogenicity for the animals for which the vaccine is
intended. The following tests may be used during the
278 Veterinary Vaccines
2-2-1-1-2 Safety in pregnant ewes. If the vaccine is intended observations and lung lesions and compare the results
for use or may be used in pregnant ewes, use not fewer than obtained for these parameters and the bacterial re-isolation
10 pregnant ewes at the relevant stages of pregnancy. results for the 2 groups.
Administer to each ewe a double dose of the vaccine, then The test is invalid if signs of M. haemolytica infection occur
one dose after the interval to be recommended. Observe the in less than 70 per cent of the controllambs. The vaccine
ewes at least daily untiI one day after lambing. Record body complies with the test if there is a significant difference
temperatures the day before each vaccination, at vaccination, between the scores obtained for the c1inical and post-mortem
2 h, 4 h and 6 h later and then daily for 4 days; note rp.e observations in the vaccinates compared to the controls.
maximum temperature increase for each ewe. For vaccines with a c1aim for a beneficial effect on the extent
The vaccine complies with the test if: of infection against the serovar, the results for the infection
- no ewe shows abnormal local reactions, notable s'.gns of rates are also significantly better for the vaccinates compared
disease or dies from causes attributable to the vaccine, to the controls.
- the average body temperature increase for all ewes does 2-2-2-2 Passive protection For vaccines with c1aims for
not exceed 1.5 oC and no ewe shows a rise greater than passive protection against mannheimiosis carry out a test for
2 oC, each serovar of M. haemolytica for which protection is to be
- and no adverse effects on the pregnancy and offspring are c1aimed on the label.
noted. A test is carried out for each route and method of
2-2-1-2 Field studies The sheep used for the field trials are administration to be recommended for vaccination.
also used to evaluate safety. Carry out a test in each category The vaccine administered to each ewe is of minimum
of sheep for which the vaccine is intended. Use not fewer potency.
than 3 groups of 20 sheep with corresponding groups of not Use not fewer than 6 ewes that preferably do not have
fewer than 10 controls in 3 different locations. Examine the antibodies against the serovars of M. haemolytica or against
injection sites for local reactions after vaccination. Record the leucotoxin present in the vaccine. Where justified, ewes
body temperatures the day before vaccination, at vaccination with a known history of no previous mannheimia vaccination,
and on the 2 days following vaccination. from a source with a low incidence of respiratory disease and
The vaccine complies with the test if no sheep shows with low antibody titres (measured in a sensitive test system
abnormal local or systemic reactions, notable signs of disease such as an EUSA) may be used. Vaccinate the ewes at the
or dies from causes attributable to the vaccine. The average stages of pregnancy and according to the schedule to be
body temperature increase for all sheep does not exceed recommended. A challenge study is conducted with 20
1.5 oC and no sheep shows a rise greater than 2 oC. newbom, colostrum-deprived lambs. 10 of these lambs are
In addition, if the vaccine is intended for use in pregnant given colostrum from the vaccinated ewes and 10 control
ewes, no adverse effects on the pregnancy and offspring are lambs are given colostrum or colostrum substitute without
demonstrated. detectable antibodies to M. haemolytica. When the lambs are
2-2-2 Irnmunogenicity at the age to be c1aimed for the duration of the passive
2-2-2-1 Active immunisation For vaccines with c1aims for protection, challenge each by the intratracheal route with a
active irnmunisation against mannheimiosis, carry out a test sufficient quantity of a low-passage, virulent strain of a
for each serovar of M. haemolytica for which protection is to serovar of M. haemolytica. Observe the lambs for a further
be c1aimed on the label. 7 days; to avoid unnecessary suffering, severely i1l lambs are
A test is carried out for each route and method of euthanised and are then considered to have died from the
administration to be recommended, using in each case lambs disease. Observe the lambs and assess the effect of the
of the minimum age to be recommended for vaccination. challenge on the offspring of the vaccinates and the controls
The vaccine administered to each lamb is of minimum as described in the test for active irnmunisation.
potency. The test is invalid if signs or lesions of M. haemolytica
U se not fewer than 20 lambs that do not have antibodies infection occur in les s than 70 per cent of the controllambs.
against M. haemolytica and against the leucotoxin of M. The vaccine complies with the test if there is a significant
haemolytica. Vaccinate not fewer than 10 lambs according to difference between the scores obtained for the c1inical and
the schedule to be recommended. Maintain not fewer than post-mortem observations in the lambs from the vaccinates
10 lambs as controls. 21 days after the last vaccination, compared to those from the controls. For vaccines with a
challenge each lamb by the intratracheal route or by another c1aim for a beneficial effect on the extent of infection against
appropriate route, with a sufficient quantity of a low-passage, the serovar, the- results for the infection rates are also
virulent strain of a serovar of M. haemolytica. Where significantly better for the lambs from the vaccinates
necessary for a given serovar, prechallenge with parainfiuenza compared to those from the controls.
type 3 (PI3) virus or another appropriate respiratory 2-3 MANUFACTURER'S TESTS
pathogen may be used. Observe the lambs for a further 2-3-1 Batch potency test
7 days; to avoid unnecessary suffering, severely ill lambs are It is not necessary to carry out the relevant Potency test or
euthanised and are then considered to have died from the tests (section 3-4) for each batch of vaccine if they have been
disease. During the observation period, examine the lambs carried out using a batch of vaccine with a minimum
for signs of disease (for example, increased body temperature, potency. Where the relevant test or tests are not carried out,
dullness, abnormal respiration) and record the mortality. an altemative validated method is used, the criteria for
Euthanise surviving lambs at the end of the observation acceptance being set with reference to a batch of vaccine that
periodo Carry out post-mortem examination on any lamb that has given satisfactory results in the testes) described under
dies and those euthanised at the end of the observation Potency.
periodo Examine the lungs and evaluate the extent of lung
2-3-2 Bacterial endotoxins
lesions due to mannheimiosis. Collect samples of lung tissue
A test for bacteria! endotoxins (2.6.14) is carried out on the
for re-isolation of the challenge organisms. Score the c1inical
finallot or, where the nature of the adjuvant prevents
282 Veterinary Vaccines
performance of a satisfactory test, on the bulk antigen or the grown separately. The vaccine may be freeze-dried or stored
mixture of bulk antigens immediately before addition of the in liquid nitro gen.
adjuvant. The maximum acceptable amount of bacterial 2-2 SUBSTRATE FOR VIRUS PROPAGATION
endotoxins is that found for a batch of vaccine that has been
2-2-1 Cell cultures
shown satisfactory in safety tests 2-2-1-1 given under Choice
The cell cultures comply with the requirements for cell
of vaccine composition or in safety test 3-3 described under
cultures for production ofveterinary vaccines (5.2.4).
Batch tests, carried out using 10 sheep. Where the latter test
is used, note the maximum temperature increase for each 2-3 SEED LOTS
sheep; the vaccine complies with the test if the average body 2-3-1 Extraneous agents
temperature increase for all sheep does not exceed 1.5 oC. The master seed lot complies with the tests for extraneous
The method chosen for determining the amount of bacterial agents in seed lots (2.6.24). In these tests on the master seed
endotoxin present in the vaccine batch used in the safety test lot, the organisms used are not more than 5 passages from
for determining the maximum acceptable level of endotoxin the master seed lot at the start of the tests.
is used subsequently for testing of each batch.
2-4 CHOICE OF VACCINE VIRUS
3 BATCH TESTS The vaccine virus shall be shown to be satisfactory with
3-1 IDENTIFICATION respect to safety (5.2.6) and efficacy (5.2.7) for the chickens
When injected into healthy animals that do not have specific and/or chicken embryos for which it is intended.
antibodies against the serovars M. haemolytica and/or against The tests shown below for residual pathogenicity of the strain
the leucotoxin present in the vaccine, the vaccine stimulates (section 2-4-1), increase in virulence (section 2-4-2) and
the production of such antibodies. immunogenicity (section 2-4-3) may be used during the
3-2 BACTERIA AND FUNGI demonstration of safety and immunogenicity. Additional
The vaccine and, where applicable, the liquid supplied with it testing may be needed to demonstrate safety in breeds of
comply with the test for sterility prescribed in the monograph chickens known to be particularly susceptible to Marek's
Vaccines for veterinary use (0062) disease virus, unless the vaccine is to be contra-indicated.
3-3 SAFETY 2-4-1 Residual pathogenicity of the strain
U se 2 sheep of the minimum age recommended for Carry out the test for the route to be recommended for
vaccination or, if not available, of an age as close as possible vaccination that is likely to be the least safe and in the
to the minimum recommended age, and that have not been category of chickens for which the vaccine is intended that is
vaccinated against mannheimiosis. Administer to each sheep lil,ely to be the most susceptible for Marek's disease. Carry
by a recommended route a double dose of the vaccine. out the test in chickens if the vaccine is intended for
Observe the sheep at least daily for 14 days. Record body chickens; carry out the test in chicken embryos if the vaccine
temperature the day before vaccination, at vaccination, 2 h, is intended for chicken embryos; carry out the test in
4 h and 6 h later and then daily for 2 days. chickens and in chicken embryos if the vaccine is intended
The vaccine complies with the test if no sheep shows notable for both. Use vaccine virus at the least attenuated passage
signs of disease or dies from causes attributable to the level that will be present between the master seed lot and a
vaccine; a transient temperature increase not exceeding 2 oC batch of the vaccine.
may occur. Vaccines in tended for use in chickens Use not fewer than 80
3-4POTENCY
one-day-old chickens from a flock free from specified
The vaccine complies with the requirements of the test or pathogens (SPF) (5.2.2). Divide them randomly into
testes) mentioned under lmmunogenicity (section 2-2-2) 2 groups of not fewer than 40 chickens and maintain the
when administered by a recommended route and method. groups separately. Administer by a suitable route to each
___________________________________________ ~E~
chicken of one group (l) a quantity of the vaccine virus
equivalent to not less than 10 times the maximum virus titre
lil,ely to be contained in 1 dose of the vaccine. Administer by
a suitable route to each chicken of the other group (TI) a
quantity of virulent Marek's disease virus that will cause
mortality and/or severe macroscopic lesions of Marek's
Marek's Disease Vaccine, Living disease in not fewer than 70 per cent of the effective number
Marek's Disease Vaccine (Turkey Herpes Virus) of chickens within 70 days (initial number reduced by the
Marek's Disease Vaccine, Living (HVT) number that die within the first 7 days of the test).
(Marek's Disease Vaccine (Live), Ph Eur monograph 0589) Vaccines intendedfor use in chicken embryos-Use not fewer
PhE~ ___________________________________________ than 150 embryonated eggs from an SPF flock (5.2.2).
Divide them randomly into 3 groups of not fewer than 5 O
1 DEFINITION embryonated eggs and maintain the groups separately but
Marek's disease vaccine (live) is a preparation of a suitable under identical incubation conditions. N ot later than the
strain or strains of Marek's disease virus (gallid herpesvirus 2 recommcrnded day of vaccination, administer by the
or 3) and/or turkey herpesvirus (meleagrid herpesvirus 1). recommended method to each embryonated egg of one
This monograph applies to vaccines intended for group (1) a quantity of the vaccine virus equivalent to not
administration to chickens and/or chicken embryos for active less than 10 times the maximum virus titre likely to be
immunisation. contan;ed in 1 dose of the vaccine. Administer by a suitable
2 PRODUCTION route to each embryonated egg of another group (TI) a
quantity of virulent Marek's disease virus that will cause
2-1 PREPARATION OF THE VACCINE
mortality and/or severe macroscopic lesions of Marek's
The vaccine virus is grown in cell cultures. If the vaccine
disease in not fewer than 70 per cent of the effective number
contains more than one type of virus, the different types are
of hatched chickens within 70 days (initial number reduced
Veterinary Vaccines 283
by the number that die within the first 7 days after hatching). chickens of the youngest age to be recommended for
Keep the last group (HI) non-inoculated. The test is invalid vaccination or chicken embryos. The quantity of the vaccine
if there is a significant difference in hatchability between virus administered to each chicken or chicken embryo is not
groups I and III and the hatchability in any of the 3 groups is greater than the minimum virus titre to be stated on the label
less than 80 per cent. and the virus is at the most attenuated passage level that will
Provided that the chickens and chicken embryos are derived be present in a batch of the vaccine.
from the same flock, a common control group for in ovo and Vaccines intended for use in chickens U se not fewer than 60
parenteral administration can be used. chickens of the same origin and from an SPF flock (5.2.2).
Irrespective of whether the vaccine was administered to Vaccinate by a recommended route not fewer than 30
chickens or chicken embryos, observe the chickens of group chickens. Maintain not fewer than 30 chickens as controls.
Il at least daily for 70 days and those of group I at le\ast daily Vaccines intendedfor use in chicken embryos Use embryonated
for 120 days. The test is invalid if one or more of the chickens of the same origin and from an SPF flock (5.2.2).
following apply: Vaccinate by the in ovo route using the method to be
- more than 10 per cent of the chickens in any of the recommended, 50 per cent of the embryonated eggs.
3 groups die within the first 7 days; Maintain 50 per cent of the embryonated eggs as controls.
- fewer than 70 p_er cent of the effective number of The test is invalid if any group consists of fewer than 30
chickens in group H show macroscopic lesions of Marek's hatched chicks.
disease; Irrespective of whether the vaccine was administered to
The vaccine virus complies with the test if: chickens or chicken embryos, challenge each chicken not
- no chicken of group I shows notable clinical signs or later than 9 days after vaccination by a suitable route with a
macroscopic lesions of Marek's disease or dies from sufficient quantity of virulent Marek's disease virus. Observe
causes attributable to the vaccine virus; the chickens at least daily for 70 days after challenge. Record
- at 120 days the number of surviving chickens of group I the deaths and the number of surviving chickens that show
is not fewer than 80 per cent of the effective number. clinical signs of disease. At the end of the observation period,
2-4-2 Increase in virulence euthanise all the surviving chickens and carry out an
The test for increase in virulence is required for Marek's examination for macroscopic lesions of Marek's disease.
disease virus vaccine strains but not for turkey herpesvirus The test is not valid if:
vaccine strains, which are naturally apathogenic. U se vaccine - during the observation period after challenge, fewer than
virus at the least attenuated passage level that will be present 70 per cent of the control chickens die or show severe
between the master seed lot and a batch of the vaccine. clinical signs or macroscopic lesions of Marek' s disease;
- and/or, during the period between the vaccination and
Vaccines in tended for use in chickens Administer by the
challenge, more than 10 per cent of the control or
intramuscular route a quantity of the vaccine virus that will
vaccinated chickens show abnormal clinical signs or die
allow recovery of virus for the passages described below to
from causes not attributable to the vaccine.
each of 5 one-day-old chickens from an SPF flock (5.2.2).
The vaccine virus complies with the test if the relative
Vaccines intended for use only in chicken embryos or intended for
protection percentage, ca1culated using the following
use in chickens and in chicken embryos Administer by the in
expression, is not les s than 80 per cent:
ovo route, using the recommended method, a quantity of the
vaccine virus that will allow recovery of virus for the passages
described below to each of 5 embryonated eggs not later than v- e x 100
the recommended day for vaccination. 100 - e
5-7 days after administering the vaccine to chickens or
5-7 days after hatching when the vaccine has been v = percentage of challenged vaccinated chickens that
administered in ovo, prepare a suspension of white blood cells survive to the end of the observation period without
from each chicken and pool these samples. Administer a notable clinical signs or macroscopic lesions of Marek's
suitable volume of the pooled samples by the intraperitoneal disease;
route to each of 5 other chickens that are 1 dayold and from e percentage of challenged control chickens that survive
an SPF flock (5.2.2). Carry out this passage operation not to the end of the observation period without notable
fewer than 5 times; verify the presence of the virus at each clinical signs or macroscopic lesions of Marek's disease.
passage. Care must be taken to avoid contamination by virus
from previous passages. If the virus is not found at a passage 3 BATCH TESTS
level, carry out a second series of passages. Carry out the test 3-1 IDENTIFICATION
for residual pathogenicity (section 2-4-1) using the Carry out an immunostaining test in cell cultures using
unpassaged vaccine virus and the maximally passaged virus monoclonal antibodies to demonstrate the presence of each
that has been recovered. Administer the virus by the route to type of virus stated on the label.
be recommended for vaccination that is likely to be the least 3-2 BACTERIA ANO FUNGI
safe for use in these chickens or chicken embryos. The vaccine and, where applicable, the liquid supplied with it
The vaccine virus complies with the test if no indication of comply with the test for sterility prescribed in the monograph
increase in virulence of the maximally passaged virus Vaccines for veterinary use (0062).
compared with the unpassaged virus is observed. If virus is
3-3 MYCOPLASMAS
not recovered at any passage level in the first and second
The vaccine complies with the test for mycoplasmas (2.6.7).
series of passages, the vaccine virus also complies with the
test. 3-4 EXTRANEOUS AGENTS
2-4-3 Immunogenicity The vaccine complies with the tests for extraneous agents in
batches of finished product (2.6.25).
A test is carried out for each route and method of
administration to be recommended, using in each case
284 Veterinary Vaccines
2-3-2 Increase in virulence A vaccine containing Shope fibroma virus complies with the
(This test is performed only for vaccines based on attenuated test if, during the observation period after challenge, not
strains of myxoma virus). The test for increase in virulence fewer than 75 per cent of vaccinated rabbits show no signs of
consists of the administration of the vaccine virus at the least myxomatosis.
attenuated passage level that will be present between the 3 BATCH TESTS
master seed lot and a batch of the vaccine to 2 rabbits,
3-1IDENTIFICATION
5-7 weeks old, that do not have antibodies against myxoma
Carry out an immunofluorescence test in suitable cell
virus, sequential passages, 5 times where possible, to further
cultures, using a monospecific antiserum.
similar groups and testing of the final recovered virus for
increase in virulence. If the properties of the vaccine virus 3-2 BACTERIA ANO FUNGI
allow sequential passage to 5 groups via natural spreading, The vaccine and, where applicable, the liquid supplied with it
this method may be used, otherwise passage as described comply with the test for sterility prescribed in the monograph
be!ow is carried out and the maximally passaged virus that Vaccines for veterinary use (0062).
has been recovered is tested for increase in virulence. Care 3-3 MYCOPLASMAS (2.6.7)
must be taken to avoid contamination by virus from previous The vaccine complies with the test for mycoplasmas.
passages. 3-4 SAFETY
Administer to each rabbit by a route to be recommended a Use not fewer than 2 rabbits, not older than the minimum
quantity of the vaccine virus that will allow recovery of virus age recommended for vaccination and that do not have
for the passages described below. Administer the virus by the antibodies against myxoma virus and rabbit haemorrhagic
route to be recommended for vaccination most likely to lead disease virus and that have been reared in suitable isolation
to reversion of virulence. Euthanise the rabbits 5 to 10 days conditions to avoid contact with myxoma virus. Administer
after inoculation and remove from each rabbit organs, or to each rabbit by a recommended route 10 doses of the
tissues with sufficient virus to allow passage; homogenise the vaccine. Observe the rabbits at least daily for 14 days.
organs and tissues in a suitable buffer solution, centrifuge the The vaccine complies with the test if no rabbit shows notable
suspension and use the supernatant for further passages. signs of disease or dies from causes attributable to the
Inoculate the supernatant into suitable cell culture to verify vaccine.
the presence of virus. Administer by an appropriate route, at
3-5 EXTRANEOUS AGENTS
a suitable rate, a suitable volume of the supematant to each
of 2 other rabbits of the same age. Carry out this passage At the end of the 14 day observation period of the safety test,
operation not fewer than 5 times; verify the presence of the administer by a recommended route to each rabbit, a further
virus at each passage. If the virus is not found at a passage 10 dos es ofvaccine. After 14 days, take a blood sample from
level, carry out a second series of passages. each rabbit and carry out a test for antibodies against rabbit
haemorrhagic disease virus. The vaccine complies with the
Carry out the test for Safety (section 2-3-1) using unpassaged
test if no antibodies are found.
vaccine virus and the maximally passaged virus that has been
recovered. 3-6 vmus TITRE
Titrate the vaccine virus in suitable cell cultures. The vaccine
The vaccine virus complies with the test if no indication of
complies with the test if one dose contains not less than the
increased virulence of the maximally passaged virus
minimum virus titre stated on the label.
compared with the unpassaged virus is observed. If virus is
not recovered at any passage leve! in the first and second 3-7 POTENCY
series of passages, the vaccine virus also complies with the The vaccine complies with the requirements of the test
test. prescribed under Immunogenicity (section 2-3-3) when
administered by a recommended route and method. It is not
2-3-3 Immunogenicity
necessary to carry out the potency test for each batch of the
A test is carried out for each route and method of
vaccine if it has been carried out on a representative batch
administration to be recommended for vaccination using in
using a vaccinating dose containing not more than the
each case rabbits of the mínimum age to be recommended.
minimum virus titre stated on the label.
The quantity of vaccine virus to be administered to each
_____________________________________________ ~E~
DEFINITION
strain of myxoma virus sufficient to cause typical signs of
myxomatosis in a rabbit. Observe the rabbits at least daily for Newcastle Disease and Avian Infectious Bronchitis Vaccine,
Livin~is a mixed preparation derived from separate groups
further 21 days after challenge and monitor each of them.
of eggs infected with suitable strains of Newcastle disease
The test is invalid if fewer than 90 per cent of the control virus and of avian infectious bronchitis_ virus. The Newcastle
rabbits display typical signs of myxomatosis. A vaccine disease virus seed is either a modified strain, such as
containing myxoma virus complies with the test if, during the Hitchner B 1 or La Sota, or a naturally occurring strain of
observation period after challenge, not fewer than 90 per cent low pathogenicity. The avian infectious bronchitis virus seed
of vaccinated rabbits show no signs of myxomatosis. is a strain of the Massachusetts type 1 and it may be used at
Veterinary Vaccines 287
various levels of attenuation. The vaccine is prepared The vaccine contains not less than 103 .5 EIDso of virus per
immediately before use by reconstitution from the dried bird dose.
vaccine with a suitable liquido
POTENCY
PRODUCTION For the Newcasde disease component
For vaccine production the virus is propagated in Vaccinate each of twenty-five 5- to 10-day-old chicks from
embryonated eggs derived from chicken fiocks free from fiocks free from specified pathogens, by the nasal instillation
specified pathogens. The final product is freeze dried. of one dose of vaccine. Twenty-one days later challenge the
Provided that the test for potency described below has been vaccinated chicks as well as 10 control birds by the
performed with satisfactory results on a representative batch intramuscular inoculation of at least 10 6 .0 EIDso of Herts
of vaccine it may be omitted by the manufacturer as ~ (Weybridge 33/56) strain of Newcastle disease virus and
routine control on other batches of vaccine prepared from observe for 10 days. All the control birds die within 6 days
the same seed lot, subject to the agreement of the competent and no fewer than 23 of the vaccinated birds survive the
authority. observation period without showing signs of Newcastle
CAUTION The vaccine is not dangerous to man~ but it is disease.
advisable to avoid undue exposure to the Newcastle disease virus For the avian infectious bronchitis component
and to infected birds~---as the Newcastle disease virus may cause a Vaccinate each of 10 healthy 3- to 4-week-old chickens from
type of conjuctivitis or~ more rarely~ may cause symptoms similar fiocks free from specified pathogens, by nasal or ocular
to those characteristic of influenza. instillation such that each chicken receives one dos e of
The vaccine~ reconstituted with a suitable líquid to provide a vaccine. Twenty-one to twenty-eight days later challenge the
concentration appropriate to the particular test~ complies with the vaccinated chickens, as well as 10 control birds that are kept
requirements stated under Veterinary Vaccines with the following separate from the vaccinated birds, by nasal or ocular
modifications. instillation of 103 .0 to 103 .5. EIDso of the Massachusetts
41 strain of virulent infectious bronchitis virus.
IDENTIFICATION
Between the 4th and 7th day after challenge, take a tracheal
When mixed with a mixture of monospecific N ewcastle swab from each bird. Place each swab in a test tube
disease virus antiserum and avian infectious bronchitis virus containing 3 mL of broth to which suitable antibiotics have
antiserum, the vaccine no longer infects susceptible 10- to been added to inhibit the growth of bacterial contaminants
11-day-old embryonated eggs. and test for the presence of infectious bronchitis virus by the
TESTS inoculation of 0.2 mL of inoculum into the allantoic cavity of
Mycoplasmas 9- to 11-day-old embryonated eggs using at least five eggs for
Complies with the test for absence of mycoplasmas, each swab. A tracheal swab is positive if 20% or more of the
Appendix XVI B(Vet) 3. embryos inoculated from it show lesions typical of infectious
Sterility bronchitis virus. If more than one embryo but fewer than
Carry out the test described under Veterinary Vaccines using 20% of those inoculated from any one swab show lesions
solid media in place of liquid media. The vaccine contains no similar to those of infectious bronchitis, inoculate at least five
pathogenic organisms and not more than one organism of a additional embryonated eggs with allantoic fiuid from each of
non-pathogenic species per bird dose. the suspect embryos. The swab is positive if 20% or more of
these additional embryos show lesions typical of infectious
Absence of extraneous pathogens bronchitis virus.
The neutralised vaccine complies with the test for Avian Live
Not less than 80% of the control birds give positive tracheal
Virus Vaccines: Tests for Extraneous Agents in Batches of
swabs, and no more than 20% of the vaccinated chickens
Finished Product~ Appendix XVI B(Vet) 5.
give positive tracheal swabs.
Safety
Inoculate each of 10 chicks of the minimum age for STORAGE
vaccination ftom a fiod: free from specified pathogens with When stored under the prescribed conditions the dried
10 doses of the vaccine by intranasal instillation and observe vaccine may be expected to retain its potency for not less
for 14 days. No abnormal reaction develops. than 12 months. The reconstituted vaccine should be used
immediate1y.
Virus titre
For the Newcastle disease component Neutralise the vaccine LABELLING
with monospecific avian infectious bronchitis virus antiserum. The label states the names of the strains of virus used in the
Inoculate serial dilutions of neutralised vaccine into the vaccine.
allantoic cavity of 10- to 11-day-old embryonated eggs
derived from chicken fiocks free from specified pathogens. 1 Vaccine prepared using avian infectious bronchitis virus seed of the
Incubate the eggs for 3 days at 37° and then examine the Connecticut type rnay be used in sorne countries but is not permitted
in the United Kingdom.
embryos for evidence of virus infection, which is shown by
the presence of chick red cell haemagglutinins. The vaccine
contains not less than 106 .0 EIDso of virus per bird dose.
For the avían infectious bronchitis component Neutralise the
vaccine with monospecific Newcastle disease virus antiserum.
Inoculate serial dilutions of neutralised vaccine into the
allantoic cavity of 10- to 11-day-old embryonated eggs
derived from chicken fiocks free from specified pathogens.
Incubate the eggs at 37° for 7 days and then examine the
embryos for lesions typical of avian infectious bronchitis.
288 Veterinary Vaccines
2-5-2-1) together with the test for adjuvant (section 2-5-2-2) 3-3 EXTRANEOUS AGENTS
may be conducted if shown to provide a valid potency test. Use 10 chickens, 14-28 days old, from an SPF flock (5.2.2).
2-5-2-1 Antigen content The relative antigen content is Vaccinate each chicken by a recommended route with a
determined by comparing the content of haemagglutinin- double dose of the vaccine. After 3 weeks, administer 1 dos e
neuraminidase antigen per dos e of vaccine with a by the same route. Collect serum samples from each chicken
haemagglutinin-neuraminidase antigen reference preparation, 2 weeks later and carry out tests for antibodies to the
by enzyme-linked immunosorbent assay (2.7.1). For this following agents by the methods prescribed in general
comparison, Newcastle disease virus reference antigen BRP~ chapter 5.2.2. Chicken fiocks free from specified pathogens for the
Newcastle disease virus control antigen BRP~ Newcastle disease production and quality control of vaccines: avian
virus coating antibody BRP and Newcastle disease virus encephalomyelitis virus, avian infectious bronchitis virus,
conjugated detection antibody BRP are suitable. Before\ avian leucosis viruses, egg-drop syndrome virus, avian bursal
estimation, the antigen may be extracted from the emulsion disease virus, avian infectious laryngotracheitis virus,
using isopropyl myristate R or another suitable method. influenza A virus, Marek's disease virus. The vaccine does
The vaccine complies with the test if the estimated antigen not stimulate the formation of antibodies against these
content is not significandy lower than that of a batch that has agents.
been found to be satisfactory with respect to Immunogenicity 3-4 SAFETY
(section 2-4-1). If the vaccine is intended for use in chickens, use 10
2-5-2-2 Adjuvant If the immunochemical assay (section chickens, 14-28 days old, from an SPF flock (5.2.2). If the
2-5-2-1) is performed and if the vaccine is adjuvanted, the vaccine is not for use in chickens, use 10 birds of one of the
adjuvant is tested by suitable physical and chemical methods. species for which the vaccine is intended that do not have
For oil-adjuvanted vaccines, the adjuvant is tested in antibodies against Newcastle disease virus. Administer to
accordance with the monograph Vaccines for veterinary use each bird by a recommended route a double dose of the
(0062). If the adjuvant cannot be adequately characterised, vaccine. Observe the birds at least daily for 21 days.
the antigen content determination cannot be used as the The vaccine complies with the test if no bird shows notable
batch potency test. signs of disease or dies from causes attributable to the
2-5-2-3 Serological assay Use not fewer than 15 chickens, vaccine.
21-28 days old, of the same origin and from an SPF flock 3-5 RESIDUAL UVE VIRUS
(5.2.2). Vaccinate by the intramuscular route not fewer than A test for residual live virus is carried out to confirm
10 chickens with a volume of the vaccine equivalent to 1/50 inactivation of Newcasde disease virus.
of adose. Maintain not fewer than 5 chickens as controls. Inject 2/5 th of adose into the allantoic cavity of each of 10
Collect serurn samples from each chicken after 17-21 days. embryonated hen eggs that are 9-11 days old and from SPF
Measure the antibody levels in the sera by the flocks (5.2.2) (SPF eggs), and incubate. Observe for 6 days
haemagglutination-inhibition (HI) test using the technique and pool separately the allantoic fluid from eggs containing
described below or an equivalent technique with the same live embryos and that from eggs containing dead embryos,
numbers of haemagglutinating units and red blood cells. exc1uding those dying within 24 h of the injection. Examine
The test system used must inc1ude negative and positive embryos that die within 24 h of injection for the presence of
control sera, the latter having an HI titre of 5.0 log2 to 6.0 Newcastle disease virus: the vaccine does not comply with
log2' The vaccine complies with the test if the mean HI titre the test if N ewcastle disease virus is found.
of the vaccinated group is equal to or greater than 4.0 log2 Inject into the allantoic cavity of each of 10 SPF eggs,
and that of the unvaccinated group is 2.0 log2 or less. If the 9-11 days old, 0.2 mL of the pooled allantoic fluid from the
HI titres are not satisfactory, carry out the test for vaccines live embryos and, into each of 10 similar eggs, 0.2 mL of the
for use in chickens (section 2-4-1-1). pooled fluid from the dead embryos and incubate for
Haemagglutination inhibition 5-6 days. Test the allantoic fluid from each egg for the
Inactivate the test sera by heating at 56 oC for 30 min. presence of haemagglutinins using chicken erythrocytes.
Add 25 J..lL .of inactivated serum to the first row of wells in a The vaccine complies with the test if there is no evidence of
microtitre plateo Add 25 J..lL of a buffered 9 gIL solution of haemagglutinating activity and if not more than 20 per cent
sodium chloride R at pH 7.2-7.4 to the rest of the wells. of the embryos die at either stage. If more than 20 per cent
Prepare twofold dilutions of the sera across the plateo of the embryos die at one of the stages, repeat that stage;
To each well add 25 J..lL of a suspension containing 4 the vaccine complies with the test if there is no evidence of
haemagglutinating units of inactivated Newcasde disease haemagglutinating activity and not more than 20 per cent of
virus. Incubate the plate at 4 oC for 1 h. Add 25 J..lL of a the embryos die at that stage.
1 per cent V/V suspension of red blood cells collected from
Antibiotics may be used in the test to control extraneous
chickens that are 3-4 weeks old and free from antibodies
bacterial infection.
against Newcastle disease virus. Incubate the plate at 4 oC
for 1 h. The HI titre is equal to the highest dilution that 3-6 POTENCY
produces complete inhibition. The vaccine complies with the requirements of the test
mentioned under Immunogenicity (section 2-4-1) when
3 BATCH TESTS administered by a recommended route and method.
3-1 IDENTIFICATION ___________________________________________ ~E~
pancreas); pool the samples. Administer 0.05 mL of the - during the observation period after challenge fewer than
pooled samples by eye-drop to each of 5 other birds of the 90 per cent of the control birds die or show severe clinical
same species, age and origino Carry out this passage signs of N ewcastle disease,
operation not fewer than 5 times; verify the presence of the - or if during the period between the vaccination and
virus at each passage. If the virus is not found at a passage challenge more than 10 per cent of the vaccinated or
level, carry out a second series of passages. control birds show abnormal clinical signs or die from
A. Carry out the test for intracerebral pathogenicity index causes not attributable to the vaccine.
(section 2-4-1) using unpassaged vaccine virus and the/ The vaccine virus complies with the test if during the
maximally passaged virus that has been recovered. observation period after challenge not fewer than 90 per cent
B. Carry out the test for amino-acid sequence (section 2-4-2) of the vaccinated birds survive and show no notable clinical
using unpassaged vaccine virus and the maximally passaged signs of Newcastle disease. For species where there is
virus that has been recovered. published evidence that it is not possible to achieve this level
of protection, the vaccine complies with the test if there is a
C. Carry out the test for safety (section 2-4-3) using
significant reduction in morbidity and mortality of the
unpassaged vaccine virus and the maximally passaged virus
vaccinated birds compared with the control birds.
that has been recovered. Administer the virus by the route to
be recommended fQ1: vaccination likely to be the least safe 3 BATCH TESTS
and to the avian species for which the vaccine is intended 3-1IDENTIFICATION
that is likely to be the most susceptible to Newcastle disease. 3-1-1 Identification ofthe vaccine virus
Thevaccine virus complies with the test if, in the tests The vaccine, diluted if necessary and mixed with a
2-4-4A, 2-4-4B and 2-4-4C, no indication of increase in monospecific Newcastle disease virus antiserum, no longer
virulence of the maximally passaged virus compared with the provokes haemagglutination of chicken red blood cells or
unpassaged virus is observed. If virus is not recovered at any infects embryonated hens' eggs from an SPF flock (5.2.2) or
passage level in the first and second series of passages, the susceptible cell cultures (5.2.4) into which it is inoculated.
vaccine virus also complies with the test. 3-1-2 Identification ofthe virus strain
2-4-5 lnununogenicity The strain of vaccine virus is identified by a suitable method,
For each avian species for which the vaccine is intended, a for example using monoclonal antibodies.
test is carried out for each route and method of 3-2 BACTERIA AND FUNGI
administration to be recommended using in each case birds Vaccines intended for administration by injection comply
not older than the youngest age to be recommended for with the test for sterility prescribed in the monograph
vaccination. The quantity of the vaccine virus administered Vaccines for veterinary use (0062).
to each bird is not greater than the minimum titre to be
stated on the label and the virus is at the most attenuated Vaccines not intended for administration by injection either
passage level that will be present in a batch of the vaccine. comply with the test for sterility prescribed in the monograph
Vaccines for veterinary use (0062) or with the following test:
2-4-5-1 Vaccines for use in chickens Use not fewer than 30
carry out a quantitative test for bacterial and fungal
chickens of the same origin and from an SPF flock (5.2.2). contamination; carry out identification tests for
Vaccinate by a recommended route not fewer than 20 microorganisms detected in the vaccine; the vaccine does not
chickens. Maintain not fewer than 10 chickens as controls. contain pathogenic microorganisms and contains not more
Challenge each chicken after 21 days by the intramuscular than 1 non-pathogenic microorganism per dose.
route with not less than 10 5 .0 embryo LD 50 of the Herts
(Weybridge 33/56) strain of Newcastle disease virus. Observe Any liquid supplied with the vaccine complies with test for
the chickens at least daily for 14 days after challenge. Record sterility prescribed in the monograph Vaccines for veterinary
the deaths and the number of surviving chickens that show use (0062).
clinical signs of disease. The test is not valid if 6 days after 3-3 MYCOPLASMAS
challenge fewer than 100 per cent of the control chickens The vaccine complies with the test for mycoplasmas (2.6.7).
have died or if during the period between vaccination and 3-4 EXTRANEOUS AGENTS
challenge more than 10 per cent of the vaccinated or control The vaccine complies with the tests for extraneous agents in
chickens show abnormal clinical signs or die from causes not batches of finished product (2.6.25).
attributable to the vaccine. The vaccine virus complies with
3-5 SAFETY
the test if during the observation period after challenge not
For vaccines recommended for use in chickens, use not fewer
fewer than 90 per cent of the vaccinated chickens survive and
than 10 chickens from an SPF flock (5.2.2) and of the
show no notable clinical signs of Newcastle disease.
youngest age recommended for vaccination. For vaccines
2-4-5-2 Vaccines for use in avian species other than the recommended for use only in avian species other than the
chicken Use not fewer than 30 birds of the species for which chicken, use not fewer than 10 birds of the species likely to
the vaccine is intended for Newcastle disease, of the same be most sensitive to Newcastle disease, that do not have
origin and that do not have antibodies against avian antibodies against N ewcastle disease virus and of the
paramyxovirus 1. Vaccinate by a recommended route not minimum age recommended for vaccination. Administer to
fewer than 20 birds. Maintain not fewer than 10 birds as each bird by eye-drop, or parenterally if only parenteral
controls. Challenge each bird after 21 days by the administration is recommended, 10 dos es of the vaccine in a
intramuscular route with a sufficient quantity of virulent volume suitable for the test. Observe the birds at least daily
avian paramyxovirus 1. Observe the birds at least daily for for 21 days. The test is not valid if more than 20 per cent of
21 days after challenge. Record the deaths and the surviving the birds show abnormal clinical signs or die from causes not
birds that show clinical signs of disease. The test is not valid attributable to the vaccine. The vaccine complies with the
if: test if no bird shows notable clinical signs of disease or dies
from causes attributable to the vaccine.
292 Veterinary Vaccines
2 PRODUCTION 2-2-1-2 Field studies The sheep used for the field trials are
2-1 PREPARATION OF THE VACCINE also used to evaluate safety. Carry out a test in each category
Production of the vaccine is based on a seed-Iot system. of sheep for which the vaccine is intended. U se not fewer
The seed material is cultured in a suitable medium; each than 3 groups of 20 sheep with corresponding groups of not
strain is cultivated separately and identity is verified using a fewer than 10 controls in 3 different locations. Examine the
suitable method. During production, various parameters such injection sites for local reactions after vaccination. Record
as growth rate are monitored by suitable methods; the values body temperatures the day before vaccination, at vaccination
are within the limits approved for the particular produ~t. and on the 2 days following vaccination.
Purity and identity of the harvest are verified using suitable The vaccine complies with the test if no sheep shows
methods. After cultivation, the bacterial suspensions are abnormal local or systemic reactions, notable signs of disease
collected separately and inactivated by a suitable method. or die s from causes attributable to the vaccine. The average
The vaccine may be adjuvanted. \ body temperature increase for all sheep does not exceed
2-2 CHOICE OF VACCINE COMPOSITION 1.5 oC and no sheep shows a rise greater than 2 oC.
The choice of composition and the strains to be included in In addition, if the vaccine is intended for use in pregnant
the vaccine are based on epidemiological data on the ewes, no adverse effects on the pregnancy and offspring are
prevalence of the different serovars of P. trehalosi. demonstrated.
The vaccine is sh~~n to be satisfactory with respect to safety 2-2-2 Immunogenicity
(5.2.6) and efficacy (5.2.7) for the sheep for which it is Carry out a test for each serovar of P. trehalosi for which
intended. protection is to be claimed on the label.
The following tests for safety (section 2-2-1) and A test is carried out for each route and method of
immunogenicity (section 2-2-2) may be used during the administration to be recommended, using in each case lambs
demonstration of safety and efficacy. of the minimum age to be recommended for vaccination.
The vaccine administered to each lamb is of minimum
2-2-1 Safety
2-2-1-1 Laboratory tests Carry out the tests for each route potency.
and method of administration to be recommended for U se not fewer than 20 lambs that do not have antibodies
vaccination and in sheep of each category for which the against P. trehalosi and against the leucotoxin of P. trehalosi.
vaccine is intended (for example, young sheep, pregnant Vaccinate not fewer than 10 lambs according to the schedule
ewes). Use a batch ofvaccine containing not less than the to be recommended. Maintain not fewer than 10 lambs as
maximum potency that may be expected in a batch of controls. 21 days after the last vaccination, challenge each
vaccine. lamb by the subcutaneous or another suitable route, with a
sufficient quantity of a low-passage, virulent strain of a
2-2-1-1-1 General safety. For each test, use not fewer than
10 sheep that preferably do not have antibodies against the serovar of P. trehalosi. Observe the lambs for a further 7 days;
to avoid unnecessary suffering, severely ill lambs are
serovars of P. trehalosi or against leucotoxin present in the
euthanised and are then considered to have died from the
vaccine. Where justified, sheep with a known history of no
disease. During the observation period, examine the lambs
previous pasteurella vaccination and with low antibody titres
for any signs of disease (for example, severe dullness, excess
(measured in a sensitive test system such as an EllSA) may
salivation) and record the mortality. Euthanise surviving
be used. Administer to each sheep a double dose of the
lambs at the end of the observation periodo Carry out post-
vaccine, then one dose after the interval to be recommended.
mortem examination on any lamb that dies and those
Observe the sheep at least daily for at least 14 days after the
euthanised at the end of the observation periodo Examine the
last administration. Record body temperature the day before
lungs, pleura, liver and spleen for haemorrhages and evaluate
vaccination, at vaccination, 2 h, 4 h and 6 h later and then
the extent of lung consolidation due to pasteurellosis. Callect
daily for 4 days; note the maximum temperature increase for
samples of lung, liver and spleen tissue for re-isolation of the
each sheep.
challenge organisms. Score the mortality, clinical observations
The vaccine complies with the test if no sheep shows and the post-mortem lesions and compare the results
abnormal local reactions, notable signs of disease or die s obtained for these parameters and the bacterial re-isolatian
from causes attributable to the vaccine, if the average body results for the 2 groups.
temperature increase for all sheep does not exceed 1.5 oC
The test is invalid if signs or lesions of P. trehalosi infection
and no sheep shows a rise greater than 2 oC.
occur in less than 70 per cent of the controllambs.
2-2-1-1-2 Safety in pregnant ewes. If the vaccine is intended The vaccine complies with the test if there is a significant
for use or may be used in pregnant ewes, use not fewer than difference between the scores obtained for the clinical and
10 pregnant ewes at the relevant stages of pregnancy. post-mortem observations in the vaccinates compared to the
Administer to each ewe a double dose of the vaccine, then controls. For vaccines with a claim for a beneficial effect on
one dose after the interval to be recommended. Observe the the extent of infection against the serovar, the results for the
ewes at least daily until one day after lambing. Record body infection rates are also significantly better for the vaccinates
temperatures the day before each vaccination, at vaccination, compared to the controls.
2 h, 4 h and 6 h later and then daily for 4 days; note the
maximum temperature increase for each ewe. 2-3 MANUFACTURER'S TESTS
The vaccine complies with the test if: 2-3-1 Batch potency test
- no ewe shows abnormal local reactions, notable signs of It is not necessary to carry out the Potency test (section 3-4)
disease or dies from causes attributable to the vaccine, for each batch of vaccine if it has been carried out using a
- the average body temperature increase for all ewes does batch of vaccine with a minimum potency. Where the test is
not exceed 1.5 oC and no ewe shows a rise greater than not carried out, an alternative validated method is used, the
2 oC, criteria for acceptance being set with reference to a batch of
- and no adverse effects on the pregnancy and offspring are vaccine that has given satisfactory results in the test described
noted. under Potency.
294 Veterinary Vaccines
2-3-2 Bacteria! endotoxins properties. This monograph applies to vaccines intended for
A test for bacterial endotoxins (2.6.14) is carried out on the the active irnmunisation of pigs against actinobacillosis.
final lot or, where the nature of the adjuvant prevents 2 PRODUCTION
perfonnance of a satisfactory test, on the bulk antigen or the
2-1 PREPARATION OF THE VACCINE
mixture of bulk antigens immediately before addition of the
The seed material is cultured in a suitable medium; each
adjuvant. The maximum acceptable amount of bacterial
strain is cultivated separately. During production, various
endotoxins is that found for a batch of vaccine that has been
parameters such as growth rate, protein content and quantity
shown satisfactory in safety tests 2-2-1-1 given under Choice
of relevant antigens are monitored by suitable methods;
of vaccine composition or in safety test 3-3 described under
the values are within the limits approved for the particular
Batch tests, carried out using 10 sheep. Where the latter test
producto Purity and identity are verified on the harvest using
is used, note the maximum temperature increase for each
suitable methods. After cultivation, the bacterial suspensions
sheep; the vaccine complies with the test if the average body
are collected separately and inactivated by a suitable method.
temperature increase for all sheep does not exceed 1.5 oC.
They may be detoxified, purified and concentrated.
The method chosen for detennining the amount of bacterial
The vaccine may be adjuvanted.
endotoxin present in the vaccine batch used in the safety test
for determining the maximum acceptable level of endotoxin 2-2 CHOICE OF VACCINE COMPOSITION
is used subsequently for testing of each batch. The choice of strains is based on epidemiological data.
The vaccine is shown to be satisfactory with respect to safety
3 BATCH TESTS (5.2.6) and efficacy (5.2.7) for the pigs for which it is
3-1IDENfIFICATION intended.
When injected into healthy animals that do not have specific
The following tests for safety (section 2-2-1) and
antibodies against the serovars of P. trehalosi and/or against
irnmunogenicity (section 2-2-2) may be used during the
the leucotoxin present in the vaccine, the vaccine stimulates
demonstration of safety and efficacy.
the production of such antibodies.
2-2-1 Safety
3-2 BACTERIA AND FUNGI
2-2-1-1 Laboratory tests Carry out the test for each route
The vaccine and, where applicable, the liquid supplied with it
and method of administration to be recommended for
comply with the test for sterility prescribed in the monograph
vaccination and in each category of pigs for which the
Vaccines for veterinary use (0062).
vaccine is intended. U se a batch containing not less than the
3-3 SAFETY maximum potency that may be expected in a batch of
U se 2 sheep of the minimum age recommended for vaccine.
vaccination or, if not available, of an age as close as possible 2-2-1-1-1 General safety. For each test, use not fewer than
to the minimum recommended age, and that have not been 10 pigs that do not have antibodies against the serotypes of
vaccinated against pasteurella. Administer to each sheep by a A. pleuropneumoniae or its toxins present in the vaccine.
recommended route a double dose of the vaccine. Observe Administer to each pig a double dose of the vaccine, then
the sheep at least daily for 14 days. Record body temperature one dose after the interval to be recommended. Observe the
the day before vaccination, at vaccination, 2 h, 4 h and 6 h pigs at lea~t daily until 14 days after the last administration.
later and then daily for 2 days. Record body temperature the day before vaccination, at
The vaccine complies with the test if no sheep shows notable vaccination, 2 h, 4 h and 6 h later and then daily for 4 days;
signs of disease or dies from causes attributable to the note the maximum temperature increase for each pig.
vaccine; a transient temperature increase not exceeding 2 oC The vaccine complies with the test if no pig shows abnonnal,
may occur. local or systemic reactions or dies from causes attributable to
3-4 POTENCY the vaccine and if the average temperature increase for all
The vaccine complies with the requirements of the test pigs does not exceed 1.5 oC and no pig shows a rise greater
mentioned under Irnmunogenicity (section 2-2-2) when than 2 oC.
administered by a recommended route and method. 2-2-1-1-2 Safety in pregnant sows. If the vaccine is intended
___________________________________________ PhE~
for use in pregnant sows, use not fewer than 10 sows at the
relevant stages of pregnancy. Administer to each sow a
double dose of the vaccine, then one dos e after the interval
to be recommended. Observe the sows at least daily until
farrowing. Record body temperature the day before
Porcine Actinobacillosis Vaccine, vaccination, at vaccination, 2 h, 4 h and 6 _h)ater and then
Inactivated daily for 4 days; note the maximum temperature increase for
(Porcine Actinobacillosis Vaccine (Inactivated) J each sow.
Ph Bur monograph 1360) The vaccine complies with the test if:
PhE~ ____~ _____________________________________ -- no sow shows abnonnal, local or systemic reactions or
dies from causes attributable to the vaccine,
1 DEFINITION -- the av~rage temperature increase for all sows does not
Porcine actinobacillosis vaccine (inactivated) is a preparation exceed 1.5 oC and no sow shows a rise greater than 2 oC,
which has one or more of the following components: -- and no adverse effects on the pregnancy and offspring are
inactivated Actinobacillus pleuropneumoniae of a suitable strain noted.
or strains; toxins, proteins or polysaccharides derived from 2-2-1-2 Field studies The pigs used for field trials are also
suitable strains of A. pleuropneumoniae, and treated to render used to evaluate safety. Carry out a test in each category of
them harrnless while maintaining adequate immunogenic pigs for which the vaccine is intended. Use not fewer than
properties; fractions of toxins derived from suitable strains of 3 groups each of not fewer than 20 pigs with corresponding
A. pleuropneumoniae and treated if necessary to render them groups of not fewer than 10 controls. Examine the injection
harmless while maintaining adequate irnmunogenic
Veterinary Vaccines 295
site for local reactions after vaccination. Record body under Potency. The following test may be used.
temperature the day before vaccination, at vaccination, at the Use 5 mice weighing 18-20 g and that do not have
time interval after which a rise in temperature, if any, was antibodies against the serotypes of A. pleuropneumoniae or its
seen in test 2-2-1-1 and daily during the 2 days following toxins present in the vaccine. Vaccinate each mouse by the
vaccination; note the maximum temperature increase for each subcutaneous route with a suitable dose. Where the
pig. recommended schedule requires a booster injection to be
The vaccine complies with the test if no pig shows abnormal, given, a booster vaccination may also be given in this test
local or systemic reactions or dies from causes attributable to provided it has been demonstrated that this will still provide
the vaccine and if the average temperature increase for aH a suitably sensitive test system. Before the vaccination and at
pigs does not exceed 1.5 oC and no pig shows a rise greater a given interval within the range of 14-21 days after the last
than 2 oC. injection, collect blood from each mouse and prepare serum
2-2-2 Irnmunogenicity samples. Determine individually for each serum the titre of
The challenge strain for the foHowing test is chosen to ensure specific antibodies against each antigenic component stated
challenge with each Ap toxin1 produced by the serotypes to on the label, using a suitable validated test such as enzyme-
be stated on the label; it may be necessary to carry out more linked immunosorbent assay (2.7.1). The vaccine complies
than one test usin~ a different chaHenge strain for each test. with the test if the antibody levels are not significantly lower
than those obtained for a batch that has given satisfactory
Each test is carried out for each route and method of
results in the test described under Potency.
administration to be recommended. The vaccine
administered to each pig is of minimum potency. 2-3-2 Bacterial endotoxins
F or each test, use not fewer than 14 pigs that do not have A test for bacterial endotoxins (2.6.14) is carried out on the
antibodies against A. pleuropneumoniae and Ap toxins. final bulk or, where the nature of the adjuvant prevents
Vaccinate not fewer than 7 pigs according to the schedule to performance of a satisfactory test, on the bulk antigen or
be recommended. Maintain not fewer than 7 pigs as controls. mixture of bulk antigens immediately before addition of the
3 weeks after the last vaccination, challenge all the pigs by adjuvant. The maximum acceptable amount of bacterial
the intranasal or intratracheal route or by aerosol with a endotoxins is that found for a batch of vaccine that has been
sufficient quantity of a virulent serotype of A. shown satisfactory in safety test 2-2-1-1 described under
pleuropneumoniae. Observe the pigs at least daily for 7 days; Choice of vaccine composition or the safety test described
under Tests, carried out using 10 pigs. Where the latter test
to avoid unnecessary suffering, severely ill control pigs are
euthanised ami are then considered to have died from the is used, note themaximum temperature increase for each
disease. Euthanise aH surviving pigs at the end of the animal; the vaccine complies with the test if the average
observation periodo Carry out a post-mortem examination on temperature increase for aH animals does not exceed 1.5 oC.
The method chosen for determining the amount of bacterial
aH pigs. Examine the lungs, the tracheobronchiallymph
endotoxin present in the vaccine batch used in the safety test
nodes and the tonsils for the presence of A. pleuropneumoniae.
Evaluate the extent of lung lesions at post-mortem for determining the maximum acceptable level of endotoxin
examination. Each of the 7 lobes of the lungs is allotted a is used subsequently for batch testing.
maximum possible lesion score2 of 5. The area showing 3 BATCH TESTS
pneumonia ancI/or pleuritis of each lobe is assessed and 3-1IDENTIFICATION
expressed on a scale of O to 5 to give the pneumonic score When injected into healthy animals that do not have specific
per lobe (the maximum total score possible for each antibodies against the antigenic components of A.
complete lung is 35). Calculate separately for the vaccinated pleuropneumoniae stated on the label, the vaccine stimulates
and the control pigs the total score (the maximum score per the production of such antibodies.
group is 245, if 7 pigs are used per group). 3-2 BACTERIA ANO FUNGI
The vaccine complies with the test if the vaccinated pigs, The vaccine and, where applicable, the liquid supplied with it
when compared with controls, show lower incidence of: comply with the test for sterility prescribed in the monograph
mortality; typical signs (dyspnoea, coughing and vomiting); Vaccines for veterinary use (0062).
typical lung lesions; re-isolation of A. pleuropneumoniae from
3-3 SAFETY
the lungs, the tracheobronchiallymph nodes and the tonsils.
U se 2 pigs of the minimum age recommended for
Where possible, the incidence is analysed statisticaHy and
vaccination and that do not have antibodies against the
shown to be significantly lower for vaccinates.
serotypes of A. pleuropneumoniae or its toxins present in the
2-3 MANUFACTURER'S TESTS vaccine. Administer to each pig by a recommended route a
2-3-1 Batch potency test double dose of the vaccine. Observe the pigs at least daily for
It is not necessary to carry out the Potency test (section 3-4) 14 days. Record body temperature the day before
for each batch of vaccine if it has been carried out using a vaccination, at vaccination, 2 h, 4 h and 6 h later and then
batch of vaccine with a minimum potency. Where the test is daily for 2 days.
not carried out, an alternative validated method is used, the The vaccine complies with the test if no pig shows notable
criteria for acceptance being set with reference to a batch of signs of disease or dies from causes attributable to the
vaccine that has given satisfactory results in the test described vaccine; a transient temperature increase not exceeding 2 oC
may occur.
3-4POTENCY
1The nomenclature of the toxins of A. pleuropneumoniae is described The vaccine complies with the requirements of the test
by J. Frey et al., Journal of General Microbiology, 1993, 139, mentioned under Immunogenicity (section 2-2-2) when
1723-1728. administered by a recommended route and method.
2The system of lung scores is described in detail by P.C.T. _____________________________________________ PhE~
which a rise in temperature, if any, was seen in test 2-2-2-1, U se 7 pigs not less than 3 weeks old and that do not have
and daily during the 2 days following vaccination; note the antibodies against the antigens stated on the label. Vaccinate
maximum temperature increase for each pig. each of 5 pigs by the recommended route and according to
The vaccine complies with the test if no pig shows abnormal the recommended schedule. Maintain 2 pigs as controls.
local or systemic reactions or die s from causes attributable to Altematively, if the nature of the antigens allows reproducible
the vaccine, if the average temperature increase for all pigs results to be obtained, a test in laboratory animals (for
does not exceed 1.5 oC and no pig shows a rise greater than example, guinea-pigs, mice, rabbits or rats) may be carried
2 oc. out. To obtain a valid assay, it may be necessary to carry out
a test using several groups of animals, each receiving a
2-2-3 Immunogenicity
different dose. For each dose, carry out the test as follows.
Carry out the test with a challenge strain representing each
Vaccinate not fewer than 5 animal s with a single injection of
type of antigen against which the vaccine is intended to
a suitable dose. Maintain not fewer than 2 animals as
protect: if a single strain with all the necessary antigens is not
controls. Where the recommended schedule requires a
available, repeat the test using different challenge strains.
booster injection to be given, a booster vaccination may also
Each test is carried out for each route and method of be given in this test provided it has been demonstrated that
administration to be recommended for vaccination. this will still provide a suitably sensitive test system. At a
The vaccine administered to each gilt is of minimum given interval within the range of 14-21 days after the last
potency. injection, collect blood from each animal and prepare serum
U se not fewer than 8 gilts susceptible to E. coli infections, samples. Use a suitable validated test such as an enzyme-
that do not have antibodies against the antigens to be stated linked immunosorbent assay (2.7.1) to measure the antibody
on the label. Take not fewer than 4 at random and vaccinate response to each of the antigens stated on the label.
these at the stage of pregnancy and according to the schedule The vaccine complies with the test if the antibody levels in
to be recommended. Maintain not fewer than 4 gilts as the vaccinates are not significantly less than those obtained
controls. Within 12 h of their giving birth, take not fewer with a batch that has given satisfactory results in the test
than 15 healthy piglets from the vaccinated gilts and 15 described under Potency and there is no significant increase
healthy piglets from the controls, taking at least 3 from each in antibody titre in the controls.
litter. Challenge each piglet by the oral route with a sufficient Where animals that do not have antibodies against the
quantity of a virulent strain of E. coli before or after antigens stated on the label are not available, seropositive
colostrum feeding and using the same conditions for animals may be used in the above test. During the
vaccinated piglets and controls. The strain used must not be development of a test with seropositive animals, particular
one used in the manufacture of the vaccine. Return the care will be required during the validation of the test system
piglets to their dam and observe at least daily for 8 days. to establish that the test is suitably sensitive and to specify
On each day, note signs in each piglet and score using the acceptable pass, fail and retest criteria. It will be necessary to
following scale: take into account the range of possible prevaccination titres
O no signs and establish the acceptable minimum titre rise after
slight diarrhoea vaccination in relation to these.
2 marked diarrhoea (watery faeces) 2-3-2 Bacteria! endotoxins
A test for bacterial endotoxins (2.6.14) is carried out on the
3 dead
finallot or, where the nature of the adjuvant prevents
Calculate total scores for each piglet over 8 days. performance of a satisfactory test, on the bulk antigen or the
The test is invalid if fewer than 40 per cent of the piglets mixture of bulk antigens immediately before addition of the
from the control gilts die and more than 15 per cent of the adjuvant. The maximum acceptable amount of bacterial
piglets from the control gilts show no signs of illness. endotoxins is that found for a batch of vaccine that has been
The vaccine complies with the test if there is a significant shown satisfactory in safety test 2-2-2-1 given under Choice
reduction in score in the group of piglets from the vaccinated of vaccine composition or in the safety test described under
gilts compared with the group from the unvaccinated Tests, carried out using 10 piglets. Where the latter test is
controls. used, note the maximum temperature increase for each
For sorne adhesins (for example, F5 and F41), there is piglet; the vaccine complies with the test if the average
published evidence that high mortality cannot be achieved temperature increase for all piglets does not exceed 1.5 oc.
under experimental conditions. If challenge has to be carried The method chosen for determining the amount of bacterial
out with a strain having such adhesins: the test is invalid if endotoxin present in the vaccine batch used in the safety test
fewer than 70 per cent of the control piglets show signs for determining the maximum acceptabletevel of endotoxin
expected with the challenge strain; the vaccine complies with is used subsequently for testing of each batch.
the test if there is a significant reduction in score in the 3 BATCH TESTS
group of piglets from the vaccinated gilts compared with the
3-1IDENTIFICATION
group from the unvaccinated controls.
In animals that do not have antibodies against the antigens
2-3 MANUFACTURER'S TESTS stated oQ the label, the vaccine stimulates the production of
2-3-1 Batch potency test such antihodies.
It is not necessary to carry out the Potency test (section 3-4) 3-2 BACTERIA AND FUNGI
for each batch of vaccine if it has been carried out using a The váccine and, where applicable, the liquid supplied with it
batch of vaccine with a minimum potency. Where the test is comply with the test for sterility prescribed in the monograph
not carried out, an alternative validated method is used, the Vaccines for veterinary use (0062).
criteria for acceptance being set with reference to a batch of
3-3 SAFETY
vaccine that has given satisfactory results in the test described
U se 2 pigs, preferably, that do not have antibodies against
under Potency. The following test may be used.
the antigens stated on the label or, where justified, use pigs
Veterinary Vaccines 299
with a low leve1 of such antibodies as long as they have not each pig a double dos e of the vaccine, then one dose after
been vaccinated against colibacillosis and adrninistration of 14 days. Observe the pigs at least daily until 14 days after the
the vaccine does not cause an anamnestic response. last adrninistration.
Administer to each pig by a recommended route a double The vaccine complies with the test if no pig shows notable
dose of the vaccine. Observe the pigs at least daily for signs of disease or dies from causes attributable to the
14 days. Record bodYctemperature before vaccination, at vaccine during the 28 days of the test.
vaccination, 2 h, 4 h and 6 h later and then daily for 2 days.
2-3-1-1-2 Safety in pregnant sows. If the vaccine is intended
The vaccine complies with the test if no pig shows notable for use in pregnant sows, use for the test not fewer than 10
signs of disease or dies from causes attributable to the pregnant sows at the stage or at different stages of pregnancy
vaccine; a transient temperature increase not exceeding 2 oC according to the schedule to be recornmended. Administer to
may occur. each sow a double dose of the vaccine, then one dose after
3-4 POTENCY 14 days. Observe the sows at least daily until farrowing.
The vaccine complies with the requirements of the test The vaccine complies with the test if no sow shows abnormal
mentioned under Immunogenicity (section 2-2-3) when local or systemic reactions or dies from causes attributable to
adrninistered by a recommended route and method. the vaccine and if no adverse effects on the pregnancy or the
____________ ~ _____________________________ PhE~ offspring are noted.
2-3-1-1-3 Safety in the pigs used in test 2-3-2 for
immunogenicity. The pigs used in the test for
immunogenicity are also used to evaluate safety. Measure the
rectal temperature of each vaccinated pig at the time of
Porcine Parvovirus Vaccine, vaccination 24 h and 48 h latero Examine the injection site
Inactivated after vaccination and at slaughter for local reactions.
(Porcine Parvovirosis Vaccine (Inactivated) , The vaccine complies with the test if no pig shows:
Ph Bur monograph 0965) -- abnormal body temperature;
PhE~ ___________________________________________ -- other systemic reactions (for example, anorexia);
-- abnormallocal reactions attributable to the vaccine.
1 DEFINITION 2-3-1-2 PieZd studies The pigs used for field trials are also
Porcine parvovirosis vaccine (inactivated) is a preparation of used to evaluate safety. Carry out a test in each category of
a suitable strain of porcine parvovirus, inactivated while pigs for which the vaccine is intended (sows, gilts).Use not
maintaining adequate immunogenic properties or of a fewer than 3 groups each of not fewer than 20 pigs with
noninfectious fraction of the virus. This monograph applies corresponding groups of not fewer than 10 controls. Measure
to vaccines intended for the active immunisation of sows and the rectal temperature of each vaccinated pig at the time of
gilts for protection of their progeny against transplacental vaccination, 24 h and 48 h latero Examine the injection site
infection. after vaccination and at slaughter for local reactions.
2 PRODUCTION The vaccine complies with the test if no pig shows:
2-1 PREPARATION OF THE VACCINE -- abnormal body temperature;
The vaccine virus is grown in cell cultures. The viral -- abnormallocal reactions attributable to the vaccine.
suspension is harvested; the virus is inactivated by a suitable 2-3-2 Immunogenicity
method and may be fragmented (inactivation may be by A test is carried out for each route and method of
fragmentation); the virus or viral fragments may be purified administration to be recommended, using in each case gilts
and concentrated at a suitable stage of the process. of 5-6 months old. The vaccine adrninistered to each gilt is
The vaccine may be adjuvanted. of minimum potency.
2-2 SUBSTRATE FOR VIRUS PROPAGATION Use for the test not fewer than 12 gilts that do not have
2-2-1 Cell cultures antibodies against porcine parvovirus or against a fraction of
The cell cultures comply with the requirements for cell the virus. Vaccinate not fewer than 7 gilts according to the
cultures for production of veterinary vaccines (5.2.4). schedule to be recommended. Maintain not less than 5
2-3 CHOICE OF VACCINE COMPOSITION unvaccinated gilts of the same age as controls. The interval
The vaccine is shown to be satisfactory with respect to safety between vaccination and service is that to be recommended.
(5.2.6) (including absence of adverse effects on fertility, Mate aH the gilts on 2 consecutive days immediately
gestation, farrowing or offspring) and efficacy (5.2.7) for the following signs of oestrus. At about the 40th day of gestation,
pigs for which it is intended. challenge each gilt with a suitable quantity of a virulent strain
of porcine parvovirus. Euthanise the gilts at about the 90th
The following tests for safety (section 2-3-1) and
day of gestation and examine their foetuses for infection with
irnmunogenicity (section 2-3-2) may be used during the
porcine parvovirus as demonstrated by the presence of either
demonstration of safety and efficacy.
virus or antibodies.
2-3-1 Safety The test is invalid if:
2-3-1-1 Laboratory tests Carry out the tests for each route -- fewer than 7 vaccinated gilts and 5 control gilts are
and method of administration to be recommended for challenged;
vaccination and where applicable, in pigs of each category for -- fewer than 90 per cent of piglets from the control gilts are
which the vaccine is intended. Use a batch of vaccine infected;
containing not less than the maximum potency that may be -- and the average number of piglets per litter for the
expected in a batch of vaccine. vaccinated gilts is fewer than 6.
2-3-1-1-1 General safety. For each test, use not fewer than
10 pigs that do not have antibodies against porcine
parvovirus or against a fraction of the virus. Administer to
300 Veterinary Vaccines
The vaccine complies with the test if not fewer than non-confiuent cells; after incubation for 7 days, make a
80 per cent of the total number of piglets from vaccinated sub culture using trypsinised cells. After incubation for a
gilts are protected from infection. further 7 days, examine the cultures for residuallive
2-4 MANUFACTURER'S TESTS parvovirus by an immunfiuorescence test. The vaccine
complies with the test if no live virus is detected.
2-4-1 Residuallive virus
A test for residual live virus is carried out on each batch of 3-5 EXTRANEOUS AGENfS
antigen immediately after inactivation. The quantity of On the pigs used for the safety test carry out tests for
inactivated viral harvest used in the test is equivalent to not antibodies. The vaccine complies with the test if it does not
les s than 100 doses of the vaccine. The bulk harvest is stimulate the formation of antibodies - other than those
inoculated into suitable non-confiuent cells; after incubation against porcine parvovirus - against viruses pathogenic for
for 7 days, a sub culture is made using trypsinised cells. After pigs or against virus es which could interfere with the
incubation for a further 7 days, the cultures are examined for diagnosis of infectious diseases in pigs (including the virus es
residual live parvovirus by an irnmunfiuorescence test. of the pestivirus group).
The inactivated viral harvest complies with the test if no live 3-6 POTENCY
virus is detected. The vaccine complies with the requirements of the test
2-4-2 Batch potency test mentioned under Immunogenicity (section 2-3-2) when
It is not necessary to carry out the Potency test (section 3-6) administered by a recommended route and method.
_____________________________________________
for each batch of the vaccine if it has been carried out using PhE~
fewer than 6 controls, challenge each piglet at 7 days of age 2-5-2 Bacterial endotoxins
by the intranasal route with a sufficient quantity of B. A test for bacterial endotoxins (2.6.14) is carried out on the .
bronchiseptica. In addition, challenge each piglet at 10 days of batch or, where the nature of the adjuvant prevents
age by the intranasal route with a sufficient quantity of a performance of a satisfactory test, on the bulk antigen or the
toxigenic strain of P. multocida. At the age of 42 days, mixture of bulk antigens irnmediately before addition of the
euthanise the piglets of the 4 groups and dissect the nose of adjuvant. The maximum acceptable amount of bacterial
each of them transversally at premolar-l. Examine the ventral endotoxins is that found for a batch of vaccine shown
and dorsal turbinates and the nasal septum for evidence of satisfactory in safety test 2-4-2-1 given under Choice of
atrophy or distortion and grade the observations on the scale vaccine composition or in the safety test described under
described aboye. Tests, carried out using 10 pigs. Where the latter test is used,
The test is invalid if fewer than 80 per cent of the progeny of note the maximum temperature increase for each pig;
each litter of the unvaccinated breeder pigs have a total score the vaccine complies with the test if the average temperature
of at least 10. The vaccine complies with the test if a increase for all pigs does not exceed 1.5 oC. The method
significant reduction in the total score has been demonstrated chosen for determining the amount of bacterial endotoxin
in the groups from the vaccinated breeder pigs compared to present in the vaccine batch used in the safety test for
the corresponding group from the unvaccinated breeder pigs. determining the maximum acceptable level of endotoxin is
used subsequently for testing of each batch.
2-5 MANUFACTURER'S TESTS
2-5-1 Batch potency test 3 BATCH TEST
It is not necessary to carry out the Potency test (section 3-4) 3-1 IDENTIFICATION
for each batch of vaccine if it has been carried out using a In animals that do not have specific antibodies against the
batch of vaccine with a minimum potency. Where the test is antigens stated on the label, the vaccine stimulates the
not carried out, an alternative validated method is used, the production of such antibodies.
criteria for acceptance being set with reference to a batch of 3-2 BACTERIA AND FUNGI
vaccine that has given satisfactory results in the test described The vaccine and, where applicable, the liquid supplied with it
under Potency. The following test may be used. comply with the test for sterility prescribed in the monograph
Use not fewer than 7 pigs not less than 3 weeks old and that Vaccines for veterinary use (0062).
do not have antibodies against the components of the 3-3 SAFETY
vaccine. Vaccinate not fewer than 5 pigs by a recommended Use not fewer than 2 pigs that do not have antibodies against
route and according to the recommended schedule. Maintain P. multocida and that preferably do not have antibodies
not fewer than 2 pigs of the same origin as controls under against B. bronchiseptica. Administer to each pig by a
the same conditions. Alternatively, if the nature of the recommended route a double dose of the vaccine. Observe
antigens allows reproducible results to be obtained, a test in the pigs at least daily for 14 days. Record body temperature
laboratory animals that do not have antibodies against the the day before vaccination, at vaccination, 2 h, 4 h and 6 h
components of the vaccine may be carried out. To obtain a later and then daily for 2 days.
valid assay, it may be necessary to carry out a test using The vaccine complies with the test if no pig shows notable
several groups of animals, each receiving a different quantity signs of disease or dies from causes attributable to the
of vaccine. For each quantity of vaccine, carry out the test as vaccine; a transient temperature increase not exceeding 2 oC
follows: vaccinate not fewer than 5 animal s with a suitable may occur.
quantity of vaccine. Maintain not fewer than 2 animals of the
3-4POTENCY
same species and origin as controls. Where the recommended
The vaccine complies with the requirements of the tests
schedule requires a booster injection to be given, a booster
mentioned under Irnmunogenicity (section 2-4-3) when
vaccination may also be given in this test provided it has
administered by a recommended route and method.
been demonstrated that this will still provide a suitably
_____________________________________________ ~E~
lot may be pooled and considered as a single harvest. It may virus complies with the test if not more than 2 of 25
be mixed with a suitable stabiliser. vaccinated foxes (or a statistically equivalent number if more
2-2 SUBSTRATE FOR VIRUS PROPAGATION than 25 vaccinated foxes are challenged) show signs of rabies.
2-2-1 Cell cultures 3 BATCH TESTS
The cell cultures comply with the requirements for cell 3-1IDENTIFICATION
cultures for production- of veterinary vaccines (5.2.4); if the 3-1-1 When mixed with a monospecific rabies antiserum, the
cell cultures are of mammalian origin, they are shown to be vaccine is no longer able to infect susceptible cell cultures
free from rabies virus. / into which it is inoculated.
2-3 CHOICE OF VACCINE VIRUS 3-1-2 A test is carried out to demonstrate the presence of the
The vaccine virus is shown to be satisfactory with respect to genetic marker.
safety (5.2.6) and efficacy (5.2.7) for the foxes for whlch it is 3-2 BACTERIA AND FUNGI
intended. The vaccine and, where applicable, the liquid supplied with it
The following characteristics (section 2-3-1) may be used comply with the test for sterility prescribed in the monograph
during the demonstration of safety and the following tests for Vaccines for veterinary use (0062).
Immunogenicity (2-3-2) may be used during the 3-3 MYCOPLASMAS (2.6.7)
demonstration of efficacy. The vaccine complies with the test for mycoplasmas.
2-3-1 Virus strain characteristics 3-4 EXTRANEOUS AGENTS
Only a virus strain shown to be satisfactory with respect to 3-4-1 N eutralise the vaccine virus with a suitable
the following characteristics may be used in the preparation monospecific neutralising rabies virus antiserum and
of the vaccine: inoculate into susceptible cell cultures. The vaccine complies
-- when administered orally at the dos e and by the method with the test if it no longer provokes cytopathic effects in
to be recommended for use to 40 foxes, it causes no sign susceptible cell cultures, and it shows no evidence of
of rabies within 180 days of administration, haemagglutinating or haemadsorbing agents.
-- when administered orally at 10 times the dos e to be 3-4-2 Inoculate 1 in 10 and 1 in 1000 dilutions of the
recommended to each of 10 foxes, it causes no sign of vaccine into susceptible cell cultures. Incubate at 37 oC.
rabies within 180 days of administration, After 2, 4 and 6 days, stain the cells with a panel of
-- when administered orally at 10 times the dose to be monoclonal antibodies that do not react with the vaccine
recommended to each of 10 dogs, it causes no sign of strain but that react with other strains of rabies vaccine (for
rabies within -180 days of administration, example, street virus, Pasteur strain). The vaccine complies
-- when administered orally at 10 times the dos e to be with the test if it shows no evidence of contaminating rabies
recommended to each of 10 cats, it causes no sign of virus.
rabies within 180 days of administration,
-- in natural and experimental conditions, the virus strain 3-5 VIRUS TITRE
does not spread from one animal to another in wild Titrate the vaccine virus in suitable cell cultures. The vaccíne
rodents, complies with the test if one dose contains not less than the
-- the virus strain has one or more stable genetic markers mínimum virus titre stated on the labe!'
that may be used to discriminate the vaccine strain from 3-6 POTENCY
other rabies virus strains. The vaccine complies with the requirements of the test
2-3-2 lnununogenicity prescribed under Immunogenicity (section 2-3-2) when
A test is carried out for the oral route of administration and admínistered by a recommended route and method. It is not
with the bait to be stated on the label using foxes. necessary to carry out the potency test for each batch of the
The quantity of vaccine to be administered to each fox is not vaccine if it has been carried out on a representative batch
greater than the minimum virus titre to be stated on the label using a vaccinating dose containing not more than the
and the virus is at the most attenuated passage level that will mínimum virus titre stated on the labe!'
be present in a batch of vaccine. 4 LABELLING
Use for the test not fewer than 35 foxes at least 3 months The label states the nature of the genetic marker of the virus
old, that do not have antibodies against rabies. Vaccinate not strain.
fewer than 25 foxes, according 10 the schedule to be ___________________________________________ ~E~
repeat the test. The vaccine complies with the test if, from Determination oj potency oj the vaccine to be examined Prepare
the 3rd to the 21 st days following the injection, the animals at least 3 serial dilutions of the vaccine to be examined and 3
show no signs of rabies and immunofluorescence tests carried similar dilutions of the reference preparation. Prepare the
out on the brains of the animals show no indication of the dilutions such that those containing the largest quantity of
presence of rabies virus. vaccine may be expected to protect more than 50 per cent of
3-4 Safety the animals into which they are injected and those containing
If the vaccine is intended for more than one species including the smallest quantities of vaccine may be expected to protect
one belonging to the order of Carnivora, carry out the test in less than 50 per cent of the animals into which they are
dogs. Otherwise use one of the species for which the vaccine injected. Allocate each dilution to a different group of mice
is intended. Use 2 animals, that preferably do not have and inject by the intraperitoneal route into each mouse
antibodies against rabies virus. Administer to each apimal by 0.5 mL of the dilution allocated to its group. 14 days after
a recommended route a double dos e of the vaccine. Observe the injection prepare a suspension of the challenge virus such
the animals at least daily for 14 days. that, on the basis of the pre1iminary titration, it contains
about 50 IDso in each 0.03 mL. Inject intracerebrally into
The vaccine complies with the test if no animal shows
each vaccinated mouse 0.03 mL of this suspension. Prepare
notable signs of disease or dies from causes attributable to
3 suitable serial dilutions of the challenge suspension.
the vaccine.
Allocate the challenge suspension and the 3 dilutions one to
3-5 Potency each of 4 groups of 10 unvaccinated mice and inject
The potency of rabies vaccine is determined by comparing intracerebrally into each mouse 0.03 mL of the suspension or
the dose necessary to protect mice against the clinical effects one of the dilutions allocated to its group. Observe the
of the dose of rabies virus defined below, administered animals in each group at least daily for 14 days. The test is
intracerebrally, with the quantity of a reference preparation, invalid if more than 2 mice of any group die within the first
calibrated in International Units, necessary to provide the 4 days after challenge. Record the numbers in each group
same protection. that show signs of rabies in the period 5 days to 14 days after
The International Unit is the activity of a stated quantity of challenge.
the International Standard. The equivalence in International The test is invalid unless:
Units of the International Standard is stated by the World - for both the vaccine to be examined and the reference
Health Organisation. preparation the 50 per cent protective dose lies between
Rabies vaccine (inactivated) jor veterinary use BRP is calibrated the smallest and the largest dose given to the mice;
in International Units against the International Standard. - the titration of the challenge suspension shows that
The test described be10w uses a parallel-line mode1 with at 0.03 mL ofthe suspension contained at least 10 IDso;
least 3 points for the vaccine to be examined and the - the confidence limits (P = 0.95) are not less than
reference preparation. Once the analyst has experience with 25 per cent and not more than 400 per cent of the
the method for a given vaccine, it is possible to carry out a estimated potency; when this validity criterion is not met,
simplified test using one dilution of the vaccine to be the lower limit of the estimated potency must be at least
examined. Such a test enables the analyst to determine that 1 IU in the smallest prescribed dose;
the vaccine has a potency significant1y higher than the - the statistical analysis shows a significant slope (P = 0.95)
required minimum but will not give full information on the and no significant deviations from linearity or parallelism
validity of each individual potency determination. It allows a of the dose-response curves (P = 0.99).
considerable reduction in the number of animals required for The vaccine complies with the test if the estimated potency is
the test and should be considered by each laboratory in not less than 1 IU in the smallest prescribed dose.
accordance with the provisions of the European Convention Application oj alternative end-points Once a laboratory has
for the Protection ofVertebrate Animals used for established the above assay for routine use, the lethal
Experimental and other Scientific Purposes. end-point is replaced by an observation of clinical signs and
Selection and distribution oj the test animals U se in the test application of an end-point earlier than death to reduce
healthy female mice about 4 weeks old and from the same animal suffering. The following is given as an example.
stock. Distribute the mice into at least 10 groups of not fewer The progress of rabies infection in mice following
than 10 mice. intracerebral injection can be represented by 5 stages defined
Preparation oj the challenge suspension Inoculate a group of by typical clinical signs:
mice intracerebrally with the CVS strain of rabies virus and Stage 1: ruffled fur, hunched back;
when the mice show signs of rabies, buí: before they die, Stage 2: slow movements, los s of alertness (circular
euthanise the mice and remove the brains and prepare a movements may also occur);
homogenate of the brain tissue in a suitable diluent. Separate
Stage 3: shaky movements, trembling, convulsions;
gross particulate matter by centrifugation and use the
supernatant liquid as challenge suspension. Distribute the Stage 4: signs of paresis or paralysis;
suspension in small volumes in ampoules, seal and store at a Stage 5: moribundstate.
temperature be10w - 60 oc. Thaw one ampoule of the Mice are observed at least twice daily from day 4 after
suspension and make serial dilutions in a suitable diluent. challenge. Clínical signs are recorded using a chart such as
A1locate each dilution to a group of mice and inject that shown in Table 0451.-1. Experience has shown that
intracerebrally into each mouse 0.03 mL of the dilution using stage 3 as an end-point yields assay results equivalent
allocated to its group. Observe the animals at least daily for to those found when a lethal end-point is used. This must be
14 days and recordthe number in each group that, between verified by each laboratory by scoring a suitable number of
the 5th and the 14th days, deve10p signs of rabies. Calculate assays using both clinical signs and the lethal end-point.
the IDso of the undiluted suspension.
306 Vaccines
Table 0451.-1. - Example of a chart used to record clinical 2-3 CHOICE OF VACCINE COMPOSITION
signs in the rabies vaccine potency test The vaccine is shown to be satisfactory with respect to safety
Days after challenge (5.2.6) and efficacy (5.2.7) for the rabbits for which it is
intended.
Clinical signs 4 5 6 7 8 9 10 11
The following tests for safety (section 2-3-1) and
Ruffled fur
immunogenicity (section 2-3-2) may be used during the
Hunched back
demonstration of safety and efficacy.
2-3-1 Safety
Slow movements
2-3-1-1 General safety test Carry out the test for each route
Loss of alertness
Circular movements
and method of administration to be recommended. Use not
fewer than 10 healthy rabbits from the same stock, not older
than the minimum age to be recommended for vaccination
Shaky movements and free from antibodies against RHDV. Administer to each
Trembling rabbit by a route and method to be recommended 2 doses of
Convulsions
the vaccine. Observe the animals for 21 days. Record the
body temperature the day before vaccination, at vaccination,
Paresis 4 h after vaccination and then daily for 4 days; note the
Paralysis maximum temperature increase for each animal. The vaccine
complies with the test if no rabbit shows notable signs of
Moribund state disease or die s from causes attributable to the vaccine, the
average body temperature increase for aH animals does not
exceed 1.5 oC, and no animal shows a temperature rise
greater than 2 oc.
4 LABELLING
The label states: 2-3-1-2 Safety in pregnant animals If the vaccine is intended
- the type of cell culture used to prepare the vaccine and for use in pregnant rabbits, administer the vaccine to not
the species of origin; fewer than 10 pregnant rabbits according to the schedule to
- the minimum number of International Units per dose; be recommended on the label. Prolong the observation
- the minimum period for which the vaccine provides period until 1 day after parturition. The vaccine complies
protection. with the test if no rabbit shows notable signs of disease or
___________________________________________ ~E~
dies from causes attributable to the vaccine, and no adverse
effects on the pregnancy or on the offspring are noted.
2-3-2 Immunogenicity
A test is carried out for each route and method of
administration to be stated on the label. Use not fewer than
Rabbit Haemorrhagic Disease 15 healthy, susceptible rabbits not les s than 10 weeks old,
free from antibodies against RHDV, from the same healthy
Vaccine (Inactivated) stock, and reared in suitable isolation conditions to ensure
(Ph Eur monograph 2325) absence of contact with RHDV. Administer 1 dose of vaccine
PhE~ __________________________________________ to each of not fewer than 10 of the rabbits according to the
instructions for use to be stated on the label. Maintain not
1 DEFINITION fewer than 5 other rabbits as controls. Not les s than 7 days
Rabbit haemorrhagic disease vaccine (inactivated) is a after vaccination, challenge each rabbit by a suitable route
preparation of a suitable strain of rabbit haemorrhagic disease with a quantity of a virulent strain of RHDV sufficient to
virus (RHDV), inactivated while maintaining adequate cause signs of rabbit haemorrhagic disease (RHD) in a
immunogenic properties. This monograph applies to vaccines susceptible rabbit. Observe the rabbits for a further 14 days.
intended for active immunisation of rabbits.
The test is invalid if fewer than 80 per cent of control rabbits
2 PRODUCTION die with typical signs of RHD within 120 h of challenge.
2-1 PREPARATION OF THE VACCINE The vaccine complies with the test if not fewer than
The vaccine virus is grown in rabbits. The rabbits must be 90 per cent ofvaccinated rabbits show no signs of RHD.
healthy, not vaccinated against RHDV, free from antibodies
2-4 MANUFACTURER'S TESTS
against RHDV, not treated with antibiotics within at least
15 days of their use and from a healthy and monitored 2-4-1 Residuallive virus
breeding unit. A suspension is prepared from a homogenate A test for residuallive virus is carried out on the bulk harvest
of suitable internal organs of those rabbits that are euthanised of each batch to confirm inactivation of the RHDV. The test
or that succumb to the infection within 120 h of inoculation. for inactivation is carried out in healthy, susceptible rabbits,
The virus in the suspension may be purified and not less than 10 weeks old, free from antibodies against
concentrated, and is inactivated by a suitable method. RHDV aI\ld from the same healthy stock. 5 rabbits are
2-2 SEED LOTS
inoculated by a suitable parenteral route (subcutaneous or
intramuscular) with at least a 5 mL dos e of the suspension.
2-2-1 Extraneous agents The rabbits are observed for not less than 7 days. At the end
Each master seed lot complies with the tests for extraneous of the observation period, the animals are euthanised and
agents in seed lots prescribed in the monograph Vaccines for liver extracts are tested by a suitable method for freedom
veterinary use (0062). from RHDV.
The vaccine complies with the test if no rabbit die s and no
RHDV antigen is detected in the livers.
Veterinary Vaccines 307
administration to be recommended for vaccination, using in and there is no significant increase in antibody titre in the
each case animals of each species for which the vaccine is controls.
intended. The vaccine administered to each animal is of Where animals that do not have antibodies against the
minimum potency. antigens stated on the label are not available, seropositive
For each test, use not fewer than 15 animals that do not animals may be used in the aboye test. During the
have antibodies against the antigens to be stated on the labe!' development of a test with seropositive animals, particular
Take not fewer than 10 at random and vaccinate these at the care will be required during the validation of the test system
stage of pregnancy and according to the schedule to be to establish that the test is suitably sensitive and to specify
recommended. Maintain not fewer than 5 animals as acceptable pass, fail and retest criteria. It will be necessary to
controls. Collect colostrum from all animals after parturition take into account the range of possible prevaccination titres
and store the samples individually in conditions that maintain and establish the acceptable minimum titre rise after
antibody levels. Take not fewer than 15 newbom unsuckled vaccination in relation to these.
animals and house them in an environment ensuring absence 2-3-2 Bacteria! endotoxins
of enteric pathogens. Allocate a colostrum sample from not A test for bacterial endotoxins (2.6.14) is carried out on the
fewer than 10 vaccinated dams and not fewer than 5 controls finallot or, where the nature of the adjuvant prevents
to the offspring. After birth, feed the animals with the performance of a satisfactory test, on the bulk antigen or the
colostrum sample allocated to it. After feeding the colostrum mixture of bulk antigens immediately before addition of the
and within 12 h of birth, challenge all the animals by the oral adjuvant. The maximum acceptable amount of bacteria!
route with a sufficient quantity of a virulent strain of E. cali endotoxins is that found for a batch of vaccine that has been
and observe at least daily for 10 days. The strain must not be shown satisfactory in safety test 2-2-2-1 given under Choice
one used in the manufacture of the vaccine. of vaccine composition or in the safety test described under
On each day, note daily signs in each animal and score using Tests, carried out using 10 animals. Where the latter test is
the following scale: used, note the maximum temperature increase for each
O no signs animal; the vaccine complies with the test if the average
slight diarrhoea temperature increase for all animals does not exceed 1.5 oC.
The method chosen for determining the amount of bacterial
2 marked diarrhoea (watery faeces)
endotoxin present in the vaccine batch used in the safety test
3 dead for determining the maximum acceptable level of endotoxins
Calculate total scores for each animal over 10 days. is used subsequently for testing of each batch.
The test is invalid if fewer than 80 per cent of the animals 3 BATCH TESTS
given colostrum from the controls die or show severe signs of 3-1IDENTIFICATION
disease. The vaccine complies with the test if there is a In animals that do not have antibodies against the antigens
significant reduction in score in the group of animals given stated on the label, the vaccine stimulates the production of
colostrum from vaccinated dams compared with the group such antibodies.
given colostrum from the unvaccinated controls.
3-2 BACTERIA ANO FUNGI
2-3 MANUFACTURER'S TESTS The vaccine and, where applicable, the liquid supplied with it
2-3-1 Batch potency test comply with the test for sterility prescribed in the monograph
It is not necessary to carry out the Potency test (section 3-4) Vaccines far veterinary use (0062).
for each batch of vaccine if it has been carried out using a 3-3 SAFETY
batch of vaccine with a minimum potency. Where the test is U se 2 animal s of one of the species for which the vaccine is
not carried out, an altemative validated method is used, the intended and preferably, that do not have antibodies against
criteria for acceptance beingset with reference to a batch of the antigens stated on the label or, where justified, use
vaccine that has given satisfactory results in the test described animal s with a low level of such antibodies as long as they
under Potency. The following test may be used. have not been vaccinated against colibacillosis and
To obtain a valid assay, it may be necessary to carry out a administration of the vaccine does not cause an anamnestic
test using several groups of animals, each receiving a different response. Administer to each animal by a recommended
dose. For each dose required, carry out the test as follows. route a double dose of the vaccine. Observe the animals at
Use not fewer than 7 animals (for example rabbits, guinea- least daily for 14 days. Record body temperature before
pigs, rats or mice) that do not have antibodies against the vaccination, at vaccination, 2 h, 4 h and 6 h later and then
antigens stated on the labe!' Vaccinate not fewer than daily for 2 days.
5 animals, using one injection of a suitable dose. Maintain The vaccine complies with the test if no animal shows
2 animals as controls. Where the recommended schedule notable signs of disease or dies from causes attributable to
requires a booster injection to be given, a booster vaccination the vaccine; a transient temperature increase not exceeding
may also be given in this test provided it has been 2 oC may occur.
demonstrated that this will still provide a suitably sensitive
test system. At a given interval within the range of 3-4POTENCY
14-21 days after the last injection, collect blood from each The vacdne complies with the requirements of the test
animal and prepare serum samples. Use a suitable validated mentioned under Immunogenicity (section 2-2-3) when
test such as an enzyme-linked immunosorbent assay (2.7.1) administered by a recommended route and method.
_____________________________________________
to measure the antibody response to each of the protective PhE~
vaccine that has given satisfactory results in the test described 2-2 SUBSTRATE FOR VIRUS PROPAGATION
under Potency. The following test may be used. Cell cultures
U se 10 mice of a suitable strain (for example, NMRI) Cell cultures comply with the requirements for cell cultures
weighing 17-20 g, from a uniform stock and that do not have for production ofveterinary vaccines (5.2.4).
antibodies against swine erysipelas. Vaccinate each mouse by 2-3 CHOICE OF VACCINE VIRUS
the subcutaneous route with a suitable dos e (usualIy 1/10 of The vaccine virus is shown to be satisfactory with respect to
the pig dose). At a given interval (for example, 21-28 days), safety (5.2.6) and efficacy (5.2.7) for the swine for which it is
depending on the vaccine to be examined, bleed the mice intended.
under anaesthesia. Pool the sera, using an equal volume from
The following tests described under Safety test in piglets
each mouse. Determine the level of antibodies by a suitable
(section 2-3-1), Safety test in pregnant sows and test for
irnmunochemical method (2.7.1), for example, enzyme-linked
transplacental transmission (section 2-3-2), Non-
irnmunosorbent assay with erysipelas ELISA coating
transmissibility (section 2-3-3), Increase in virulence (2-3-4)
antigen BRP. The vaccine complies with the test if the
and Irnmunogenicity (2-3-5) may be used during the
antibody level is not significantly less than that obtained with
demonstration of safety and immunogenicity.
a batch that has given satisfactory results in the test described
under Potency. 2-3-1 Safety test in piglets
Carry out the test for each recommended route using in each
3. BATCH TESTS case piglets not older than the minimum age recommended
3-1IDENTIFICATION for vaccination. U se vaccine virus at the least attenuated
Injected into animals that do not have antibodies against passage level that will be present in a batch of the vaccine.
E. rhusiopath;iae, the vaccine stimulates the production of
Use not fewer than 10 healthy piglets that do not have
such antibodies.
antibodies against pestiviruses. Administer to not fewer than
3-2 BACTERIA AND FUNGI 10 piglets a quantity of the vaccine virus equivalent to not
The vaccine and, where applicable, the liquid supplied with it less than 10 times the maximum virus titre likely to be
comply with the test for sterility prescribed in the monograph contained in 1 dose of the vaccine. Observe the piglets at
Vaccines for veterinary use (0062). least daily for 21 days. The body temperature of each
3-3 SAFETY vaccinated piglet is measured on at least the 3 days preceding
U se 2 pigs of the minimum age recommended for administration of the vaccine, at the time of administration,
vaccination and preferably that have no antibodies against 4 h after and then daily for at least 14 days. The vaccine
swine erysipelas or, where justified, use pigs with a low level complies with the test if the average body temperature
of such antibodies as long as they have not been vaccinated increase for all piglets does not exceed 1.5 oC, no piglet
against swine erysipelas and administration of the vaccine shows a temperature rise greater than 1.5 oC for a period
does not cause an anamnestic response. Administer to each exceeding 3 days, and no piglet shows notable signs of
pig by a recommended route a double dose of the vaccine. disease or dies from causes attributable to the vaccine.
Observe the pigs at least daily for 14 days. 2-3-2 Safety test in pregnant sows and test for
The vaccine complies with the test if no pig shows notable transplacental transmission
signs of disease or dies from causes attributable to the Carry out the test by a recommended route using not fewer
vaccine. than 10 healthy sows or gilts of the same age and origin,
3-4 POTENCY between the 55 th and 80th days of gestation, and that do not
The vaccine complies with the requirements of the tests have antibodies against pestiviruses. Use vaccine virus at the
rnentioned under Immunogenicity (section 2-1-1) when least attenuated passage level that will be present in a batch
administered by a recommended route and method. of the vaccine.
___________________________________________ ~E~ Administer to not fewer than 10 sows or gilts a quantity of
the vaccine virus equivalent to not less than the maximum
virus titre likely to be contained in 1 dose of the vaccine.
Record the body temperature on at least the 3 days
preceding administration of the vaccine, at the time of
Swine-Fever Vaccine (Live, administration, 4 h after and then daily for at least 15 days.
Observe until farrowing.
Prepared in Cell Cultures), Carry out tests for serum antibodies against c1assical swine-
Classical fever virus. No antibodies against c1assical~wine-fever virus
(Ph Eur monograph 0065) are found in sera taken from the newbom piglets before
PhE~ ____________________________________ ~ _____ ingestion of colostrum. The test is invalid if the vaccinated
sows do not seroconvert. The vaccine virus complies with the
1 DEFINITION test if no abnormalities in the gestation or in the piglets are
Classical swine-fever vaccine (live, prepared in cell cultures) noted, no sow or gilt shows a temperature rise greater than
is a preparation obtained from a strain of c1assical swine-fever 1.5 oC for a period exceeding 5 days, and no sow or gilt
virus that has lost its pathogenicity for the pig by in vivo shows notable signs of disease or dies from causes
and/or in vitro passage and has been adapted to growth in attributable to the vaccine.
cell cultures.
2-3-3 Non-transmissibility
2 PRODUCTION Keep together for the test not fewer than 12 healthy piglets,
2-1 PREPARATION OF THE VACCINE 6-10 weeks old and of the same origin,and that do not have
The vaccine virus is grown in cell cultures. antibodies against pestiviruses. U se vaccine virus at the least
attenuated passage level that will be present between the
master seed lot and a batch of the vaccine. Administer by a
Veterinary Vaccines 313·
recommended route to not fewer than 6 piglets a quantity of disease within the 21 days following challenge. The vaccine
the vaccine virus equivalent to not less than the maximum complies with the test if the minimum dose stated on the
virus titre likely to be contained in 1 dose of the vaccine. label corresponds to not less than 100 PD so .
Maintain not fewer than 6 piglets as contact controls. 2-3-5-2 Protection against transplacental infection Use 8 sows
The mixing of vaccinated piglets and contact piglets is done that do not have antibodies against pestiviruses, randomly
24 h after vaccination. allocated to either the vaccine group (n = 6) or the control
After 45 days, euthanise all piglets. Carry out appropriate group (n = 2).
tests on the piglets to detect antibodies to classical swilJ.e- Between the 40th and 50th day of gestation, all sows allocated
fever. Carry out appropriate tests on the control piglets to to the vaccine group are vaccinated once with 1 dos e of
detect classical swine-fever virus in the tonsils. The vaccine vaccine containing not more than the minimum titre stated
complies with the test if antibodies are found in all, on the label. On day 60 of gestation, all sows are challenged
vaccinated piglets and if no antibodies and no virus ~re found by a recommended route with a suitable strain of virulent
in the control piglets. virus. Just before farrowing and about 5-6 weeks after
2-3-4 Increase in virulence challenge, the sows are euthanised and their foetuses are
The test for increase in virulence consists of the examined for classical swine-fever virus. Serum samples from
administration of the vaccine virus at the least attenuated sows and foetuses are tested for the presence of antibodies
passage level that will be present between the master seed lot against classical swine-fever virus. Isolation of classical swine-
and a batch of the vaccine to piglets that do not have fever virus is carried out from blood of the sows (collected
antibodies against pestiviruses. 7 and 9 days after challenge and at euthanasia), and from
Administer to each of 2 healthy piglets, 6-10 weeks old, by a homogenised organ material (spleen, kidneys, lymph nodes)
recommended route, a quantity of the vaccine virus of the foetuses.
equivalent to not less than the maximum virus titre likely to The test is invalid if one or more of the vaccinated sows do
be contained in 1 dose of the vaccine. Collect an appropriate not seronconvert after the vaccination and the control sows
quantity of blood from each piglet daily between day 2 and do not seroconvert after the challenge, or if no virus is found
day 7 after administration of the vaccine virus, and pool the in more than 50 per cent of the foetuses from the control
samples taken on the same day. Administer 2 mL of the sows (excluding mummified foetuses).
pooled blood with the highest virus titre by a recommended The vaccine complies with the test if no virus is found in the
route to each of 2 other piglets of the same age and origino blood of vaccinated sows and in foetuses from the vaccinated
If no virus is found, repeat the test once again with another 2 sows, and no antibodies against classical swine-fever virus are
piglets. If virusis found, carry out a 2nd series of passages by found in the serum of the foetuses from the vaccinated sowS.
administering 2 mL of positive blood by a recommended
3 BATCH TESTS
route to each of 2 other piglets of the same age and origino
3-1 Identification
Carry out this passage operation not fewer than 5 times,
Specific classical swine-fever monoclonal antibodies are used
verifying the presence of the virus at each passage. Care must
to identify the vaccinal strain.
be taken to avoid contamination by virus from previous
passages. 3-2 Bacteria and fungi
The vaccine virus complies with the test if no indication of The vaccine complies with the test for sterility prescribed in
increasing virulence of the maximally passaged virus the monograph Vaccines for veterinary use (0062).
compared with the unpassaged virus is observed. If virus is 3-3 Mycoplasmas (2.6. 7)
not recovered at any passage level in the 1st and 2nd series of The vaccine complies with the test for mycoplasmas.
passages, the vaccine virus also complies with the test. 3-4 Extraneous agents
2-3-5 Immunogenicity N eutralise the vaccine virus using monoclonal antibodies to
2-3-5-1 Protective dose The efficacy of the vaccine is the vaccine virus. Inoculate into cell cultures known to be
expressed by the number of 50 per cent protective doses sensitive to viruses pathogenic for pigs and to pestiviruses.
(PD so) for pigs contained in the vaccinal dos e as indicated Maintain these cultures for not less than 14 days and carry
on the label. The vaccine contains at least 100 PD so per out at least 3 passages during this periodo The vaccine
dose. complies with the test if no cytopathic effect is produced;
U se 1 or more groups of piglets aged 6-10 weeks and that do the cells show no evidence of the presence of haemadsorbing
not have antibodies against pestiviruses, and use an agents.
additional group of piglets of the same age and origin as U se monoclonal antibodies that can identify possible
controls. Each group of piglets is vaccinated with 1 dilution contamination with pestiviruses. No virus is detected by an
of the vaccine dose. 14 days after the single injection of appropriate method.
vaccine, challenge the piglets by a suitable route with a 3-5 Safety
suitable strain of virulent virus and adose that kills not fewer Use 2 healthy piglets, 6-10 weeks old, that do not have
than 50 per cent of the non-vaccinated piglets in less than antibodies against pestiviruses. Administer to each piglet by a
21 days. Observe the piglets for 21 days and record the body recommended route 10 doses of the vaccine. Observe the
temperature 3 days before challenge and daily after challenge piglets at least daily for 14 days. The vaccine complies with
for 21 days. The PD so is calculated by the usual statistical the test if no piglet shows notable signs of disease or dies
methods (for example, 5.3), taking into account the surviving from causes attributable to the vaccine.
piglets that have no clinical signs of swine fever, including
3-6 Virus titre
cutaneous lesions or an increase in body temperature.
Titrate the vaccine virus in suitable cell cultures (5.2.4).
The test is invalid if fewer than 50 per cent of the control The vaccine complies with the test if 1 dose contains not les s
piglets display typical signs of serious infection with swine- than the minimum virus titre stated on the label.
fever virus, including cutaneous lesions, or die, and if fewer
than 100 per cent of the control piglets show clinical signs of
314 Veterinary Vaccines
3-7 Potency 2-3-1-1-2 Safety in the pigs used in test 2-3-2 for
The vaccine complies with the requirements of the test immunogenicity. The pigs used in the test for
prescribed under Irnmunogenicity (section 2-3-5) when immunogenicity are also used to evaluate safety. Measure the
administered by a recommended route and method. It is not rectal temperature of each vaccinated pig at the time of
necessary to carry out the potency test for each batch of the vaccination and 24 h and 48 h latero Examine the injection
vaccine if it has been carried out on a representative batch site at slaughter for local reactions.
using a vaccinating dose containing not more than the The vaccine complies with the test if no pig shows:
minimum virus titre stated on the labe!' -- abnormal body temperature;
___________________________________________ ~E~
-- other systemic reactions (for example, anorexia);
-- abnormallocal reactions attributable to the vaccine.
2-3-1-2 Field studies The pigs used for field trials are also
used to evaluate safety. Carry out a test in each category of
pigs for which the vaccine is intended (sows, fattening pigs).
Swine Influenza Vaccine, Use not fewer than 3 groups each of not fewer than 20 pigs
Inactivated in at least 2 locations with corresponding groups of not fewer
(Porcine Influenza Vaccine (Inactivated) J than 10 controls. Measure the rectal temperature of each
Ph Eur monograph 0963) vaccinated pig at the time of vaccination and 24 h and 48 h
PhE~ ___________________________________________ latero Examine the injection site at slaughter for local
reactions.
1 DEFINITION The vaccine complies with the test if no pig shows:
Porcine influenza vaccine (inactivated) is a preparation of one -- abnormal body temperature;
or more suitable strains of swine or human influenza virus -- abnormallocal reactions attributable to the vaccine.
inactivated while maintaining adequate immunogenic
2-3-2 Immunogenicity
properties. Suitable strains contain both haemagglutinin and
The following test carried out using an epidemiologically
neuraminidase. This monograph applies to vaccines intended
relevant challenge strain or strains is suitable to demonstrate
for the active immunisation of pigs against porcine influenza.
the irnmunogenicity of the vaccine. It is carried out for each
2 PRODUCTION subtype used in the preparation of the vaccine.
2-1 PREPARATION OF THE VACCINE A test is carried out for each route and method of
The vaccine virus is grown in embryonated hens' eggs or in administration to be recommended, using in each case pigs
cell cultures. Each virus strain is cultivated separately. After of the minimum age to be recommended for vaccination.
cultivation, the viral suspensions are collected separately and The vaccine administered to each pig is of minimum
inactivated by a suitable method. If necessary, they may be potency.
purified. The vaccine may be adjuvanted. U se for the test not fewer than 20 pigs that do not have
2-2 SUBSTRATE FOR VIRUS PROPAGATION antibodies against swine influenza virus. Vaccinate not fewer
2-2-1 Embryonated hens' eggs than 10 pigs according to the schedule to be recommended.
If the vaccine virus is grown in embryonated hens' eggs, they Maintain not fewer than 10 pigs as controls. Take a blood
are obtained from a healthy flock. sample from all control pigs irnmediately before challenge.
3 weeks after the last administration of vaccine, challenge all
2-2-2 Cell cultures
the pigs by the intratracheal route with a sufficient quantity
If the vaccine virus is grown in cell cultures, they comply
of a virulent influenza field virus. Euthanise half of the
with the requirements for cell cultures for production of
vaccinated and control pigs 24 h after challenge and the
veterinary vaccines (5.2.4).
other half 72 h after challenge. For each pig, measure the
2-3 CHOICE OF VACCINE COMPOSITION q"llantity of influenza virus in 2 lung tissue homogenates, one
The choice of strains is based on the antigenic types and sub- from the left apical, cardiac and diaphragmatic lobes, and the
types observed in Europe. The vaccine is shown to be other from the corresponding right lung lobes. Take
satisfactory with respect to safety (5.2.6) and efficacy (5.2.7) equivalent samples from each pig.
for the pigs for which it is intended.
The test is invalid if antibodies against influenza virus are
The following tests for safety (section 2-3-1) and found in any control pig irnmediately before challenge.
irnmunogenicity (section 2-3-2) may be used during the The vaccine complies with the test if, at both times of
demonstration of safety and efficacy. measurement, the mean virus titre in the pooled lung tissue
2-3-1 Safety samples of vaccinated pigs is· significantlylower than that for
2-3-1-1 Laboratory tests Carry out the tests for each route control pigs, when analysed by a suitable statistical method
and method of administration to be recommended for such as the Wilcoxon Mann-Whitney test.
vaccination and where applicable, in pigs of each category for 2-4 MANUFACTURER'S TESTS
which the vaccine is intended (sows, fattening pigs). Use a
2-4-1 Residuallive virus
batch of vaccine containing not less than the maximum
An ampljfication test for residual live virus is carried out on
potency that may be expected in a batch of vaccine.
each batch of antigen immediately after inactivation by
2-3-1-1-1 General safety. For each test, use not fewer than passage in the same type of substrate as that used for
10 pigs that do not have antibodies against swine influenza produetion (eggs or cell cultures) or a substrate shown to be
virus. Administer to each pig a double dose of the vaccine, at least as sensitive. The quantity of inactivated virus harvest
then one dose after 14 days. Observe the pigs at least daily used in the test is equivalent to not les s than 10 doses of the
until 14 days after the last administration. vaccine. The inactivated viral harvest complies with the test if
The vaccine complies with the test if no pig shows abnormal no live virus is detected.
local or systemic reactions or dies from causes attributable to
the vaccine during the 28 days of the test.
Veterinary Vaccines 315
2-4-2 Batch potency test do not diminish the capacity to detect residual infectious
It is not necessary to carry out the Potency test (section 3-6) influenza virus.
for each batch of vaccine if it has been carried out using a 3-5 EXTRANEOUS AGENTS
batch of vaccine with a minimum potency. Where the test is On the pigs used for the safety test, carry out tests for
not carried out, an alternative validated method is used, the antibodies. The vaccine complies with the test if it does not
criteria for acceptance being set with reference to a batch of stimulate the formation of antibodies other than those against
vaccine that has given satisfactory results in the test described influenza virus. In particular, no antibodies against viruses
under Potency. The following test may be used. pathogenic for pigs or against viruses which could interfere
U se 5 guinea-pigs, 5-7 weeks old and that do not have with the diagnosis of infectious diseases of pigs (including
antibodies against swine influenza virus. Vaccinate each virus es of the pestivirus group) are detected.
guinea-pig by the subcutaneous route with a quarter: of the 3-6 POTENCY
recommended dose. Collect blood samples before th'e The vaccine complies with the requirements of the test
vaccination and 21 days after vaccination. Determine for mentioned underlmmunogenicity (section 2-3-2) when
each sample the level of specific antibodies against each virus administered by a recommended route and method.
subtype in the vaccine by haemagglutination-inhibition or ___________________________________________ PhE~
The test is invalid if more than 10 per cent of the fish die intraperitoneal route a double dose of the vaccine. Observe
from causes not attributable to the vaccine. The vaccine the fish at least daily for 21 days.
complies with the test if no fish shows notable signs of The test is invalid if more than 6 per cent of the fish die
disease or die s from causes attributable to the vaccine. from causes not attributable to the vaccine. The vaccine
3-4 Potency complies with the test if no fish shows abnormal local or
The vaccine complies -with the requirements of the test systemic reactions or dies from causes attributable to the
mentioned under Irnmunogenicity (section 2-2-2) when vaccine.
administered by a recommended route and method. / 2-2-1-1-2 Vaccines intended for administration by
4 LABELLING immersion. Use not fewer than 50 fish from a population
The label states information on the time needed for l that does not have specific antibodies against L. anguillarum
development of immunity after vaccination under the range or where applicable V. ordalii and has not been vaccinated
of conditions corresponding to the recommended use. against or exposed to vibriosis. Prepare an irnmersion bath at
twice the concentration to be recommended. Bathe the fish
___________________________________________ ~E~
Sterility
DIAGNOSTIC PREPARATIONS It complies with the test for sterility prescribed in the
monograph Vaccines for veterinary use (0062).
POTENCY
Avian Tuberculin Purified Protein The potency of avian tuberculin purified protein derivative is
determined by comparing the reactions produced in
Derivative sensitised guinea-pigs by the intradermal injection of a series
Avian Tuberculin P.P.D. of dilutions of the preparation to be examined with those
(Ph Eur monograph 0535) produced by known concentrations of a reference preparation
~E~ ____________________________________+-_____ calibrated in International Units.
The International Unit is the activity contained in a stated
DEFINITION amount of the International Standard. The equivalence in
Avian tuberculin purified protein derivative (avian tuberculin International Units of the Intemational Standard is stated by
PPD) is a preparation obtained from the heat-treated the W orld Health Organisation.
products of growth and lysis of Mycobacterium avium capable
Sensitise not fewer than 8 albino guinea-pigs, each weighing
of revealing a delayed hypersensitivity in an animal sensitised
400-600 g, by the deep intramuscular injection of a suitable
to micro-organisms·üf the same species.
dos e of inactivated or live M. avium. Not les s than 4 weeks
PRODUCTION after the sensitisation of the guinea-pigs, shave their flanks to
It is obtained from the water-soluble fractions prepared by provide space for not more than 4 injection sites on each
heating in free-flowing steam and subsequendy filtering side. Prepare dilutions of the preparation to be examined and
cultures of M. avium grown in a liquid synthetic medium. of the reference preparation using isotonic phosphate-
The active fraction of the filtrate, consisting mainly of buffered saline (pH 6.5-7.5) containing 0.005 gIL of
protein, is isolated by precipitation, washed and re-dissolved. polysorbate 80 R. U se not fewer than 3 doses of the reference
An antimicrobial preservative that does not give rise to false preparation and not fewer than 3 doses of the preparation to
positive reactions, such as phenol, may be added. The final be examined. Choose the doses such that the lesions
sterile preparation, free from mycobacteria, is distributed produced have a diameter of not less than 8 mm and not
aseptically into sterile tamper-proof glass containers, which more than 25 mm. Allocate the dilutions randomly to the
are then c10sed so as to prevent contamination. sites, for example using a Latin square designo Inject each
The preparation may be freeze-dried. dose intradermally in a constant volume of 0.1 mL or
The identificationJ the tests and the determination of potency apply 0.2 mL. Measure the diameters of the lesions after 24-28 h
to the liquid form and to the freeze-dried form after reconstitution and calculate the results of the test using the usual statistical
as stated on the label. methods (for example, 5.3) and assuming that the diameters
of the lesions are direcdy proportional to the logarithm of the
IDENTIFICATION concentration of the tuberculins.
Inject a range of graded doses intradermally at different sites
The test is not valid unless the confidence limits (P = 0.95)
into suitably sensitised albino guinea-pigs, each weighing not
are not less than 50 per cent and not more than 200 per cent
less than 250 g. After 24-28 h, reactions appear in the form
of the estimated potency. The estimated potency is not les s
of oedematous swellings with erythema, with or without
than 75 per cent and not more than 133 per cent of the
necrosis, at the points of injection. The size and severity of
stated potency. The stated potency is not less than
the reactions vary according to the dose. Unsensitised guinea-
20 000 IU/rnL.
pigs show no reactions to similar injections.
STORAGE
TESTS
pH (2.2.3)
Protected from light, at a temperature of 5 ± 3 oC.
6.5 to 7.5. LABELLING
Phenol (2.5.15) The label states:
Maximum 5 gIL, if the preparation to be examined contains - the potency in International Units per millilitre;
phenol. - the na me and quantity of any excipient;
- for freeze-dried preparations:
Sensitising effect - the name and volume of the reconstituting liquid to be
U se a group of 3 guinea-pigs that have not been treated with added;
any material that will interfere with the test. On 3 occasions - that the product is to be used irnmediately after
at intervals of 5 days, inject intradermally into each guinea- reconstitution.
pig adose of the preparation to be examined equivalent to ___________________________________________ PhEw
500 IU in 0.1 mL. 15-21 days after the 3rd injection, inject
the same dos e (500 ID) intradermally into these animals and
into a control group of 3 guinea-pigs of the same mass,
which have not previously received injections of tuberculin.
24-28 h after the last injections, the reactions of the 2 groups
are not significantly different.
Toxicity
Use 2 guinea-pigs, each weighing not less than 250 g, that
have not previously been treated with any material that will
interfere with the test. Inject subcutaneously into each
guinea-pig 0.5 mL of the preparation to be examined.
Observe the animal s for 7 days. N o abnormal effects occur
during the observation periodo
320 Diagnostic Preparations
POTENCY
Bovine Tuberculin Purified Protein The potency of bovine tuberculin purified protein derivativé
Derivative is determined by comparing the reactions produced in
Bovine Tuberculin P.P.D. sensitised guinea-pigs by the intradermal injection of a series
of dilutions of the preparation to be examined with those
(Ph Bur monograph 0536)
PhE~ ___________________________________________
produced by known concentrations of a reference preparation
calibrated in International Units.
DEFINITION The International Unit is the activity contained in a stated
Bovine tuberculin purified protein derivative (bovine amount of the International Standard. The equivalence in
tuberculin PPD) is a preparation obtained from the heat- International Units of the International Standard is stated by
treated products of growth and lysis of Myeobaeterium bovis the World Health Organisation.
capable of revealing a delayed hypersensitivity in an animal Sensitise not fewer than 8 albino guinea-pigs, each weighing
sensitised to micro-organisms of the same species. 400-600 g, by the deep intramuscular injection of 0.0001 mg
PRODUCTION of wet mass of living M. bovis of strain AN5 suspended in
It is obtained fram the water-soluble fractions prepared by 0.5 mL of a 9 gIL solution of sodium ehloride R. Not les s than
heating in free-flowing steam and subsequently filtering 4 weeks after the sensitisation of the guinea-pigs, shave their
cultures of M. bovis grown in a liquid synthetic medium. flanks to provide space for not more than 4 injection sites on
The active fraction of the filtrate, consisting mainly of each side. Prepare dilutions of the preparation to be
protein, is isolated by precipitation, washed and re-dissolved. examined and of the reference preparation using isotonic
An antimicrobial preservative that does not give rise to false phosphate-buffered saline (pH 6.5-7.5) containing 0.005 giL
positive reactions, such as phenol, may be added. The final of polysorbate 80 R. Use not fewer than 3 doses of the
sterile preparation, free from mycobacteria, is distributed reference preparation and not fewer than 3 doses of the
aseptically into sterile, tamper-proof glass containers, which preparation to be examined. Choose the doses such that the
are then closed so as to prevent contamination. lesions produced have a diameter of not less than 8 mm and
The preparation may be freeze-dried. not more than 25 mm. Allocate the dilutions randomly to the
sites, for example using a Latin square designo Inject each
The identifieationJ the tests and the determination of poteney apply
to the liquid form and to the freeze-dried form after reeonstitution
dose intradermally in a constant volume of 0.1 mL or
0.2 mL. Measure the diameters of the lesions after 24-28 h
as stated on the labe!'
and calculate the results of the test using the usual statistical
IDENTIFICATION methods (for example, 5.3) and assuming that the diameters
Inject a range of graded dos es intradermally at different sites of the lesions are directly proportional to the logarithm of the
into suitably sensitised albino guinea-pigs, each weighing not concentration of the tuberculins.
less than 250 g. After 24-28 h, reactions appear in the form The test is not valid unless the confidence limits (P = 0.95)
of oedematous swellings with erythema, with or without are not less than 50 per cent and not more than 200 per cent
necrosis, at the points of injection. The size and severity of of the estimated potency. The estimated potency is not les s
the reactions vary according to the dose. Unsensitised guinea- than 66 per cent and not more than 150 per cent of the
pigs show no reactions to similar injections. stated potency. The stated potency is not less than
TESTS 20 000 IU/mL.
pH (2.2.3) STORAGE
6.5 10 7.5. Protected from light, at a temperature of 5 ± 3 oc.
Phenol (2.5.15) LABELLING
Maximum 5 gIL, if the preparation to be examined contains
The label states:
phenol.
--:'- the potency in International Units per millilitre;
Sensitising effect -- the name and quantity of any excipient;
U se a group of 3 guinea-pigs that have not been treated with -- for freeze-dried preparations:
any material that will interfere with the test. On 3 occasions -- the name and volume of the reconstituting liquid to be
at intervals of 5 days, inject intradermally into each guinea- added;
pig adose of the preparation to be examined equivalent to -- that the product is to be used immediately after
500 IU in 0.1 mL. 15-21 days after the 3rd injection, inject reconstitution.
the same dose (500 IV) intradermally into these animals and
______________________---------=~---------~E~
into a control group of 3 guinea-pigs of the same mass,
which have not previously received injections of tuberculin.
24-28 h after the last injections, the reactions of the 2 groups
are not significantly different.
Toxicity . Mallein Purified Protein Derivative
Use 2 guinea-pigs, each weighing not less than 250 g, that MalleiniP.P.D.
have not previously been treated with any material that will
interfere with the test. Inject subcutaneously into each DEFINITION
guinea-pig 0.5 mL of the preparation to be examined. Mallein Purified Protein Derivative is a preparation of the
Observe the animals for 7 days. No abnormal effects occur heat treated products of growth and lysis of Pseudomonas
during the observation periodo mallei. It contains not less than 0.95 mg per mL and not
more than 1.05 mg per mL of purifiecl protein derivative.
Sterility
It complies with the test for sterility prescribed in the PRODUCTION
monograph Vaeeines for veterinary use (0062). It is prepared from the water-soluble fractions obtained by
heating in free-flowing steam and subsequently filtering
Diagnostic Preparations 321
cultures of the glanders bacillus grown in a liquid synthetic Repeat the operation using 2.5 mL of water in place of the
medium. The active fraction of the filtrate, which is preparation being examined. The difference between the
predominantly protein, is isolated by precipitation, washed titrations represents the arnmonia liberated by the substance
and redissolved in phosphate buffered saline at neutral pH. being tested. Each mL of 0.00447M sulfuric acid VS is
It is then distributed in sterile containers that are inert equivalent to 0.875 mg of purified protein derivative.
towards the contents" and sealed so as to exc1ude micro- STORAGE
organisms. A suitable preservative may be added.
Mallein Purified Protein Derivative should be protected from
CAUTION Mallein P.P.D. is not dangerous to man, but the light and stored at a temperature between 2° and 8°. Under
organism from which it is prepared is pathogenic to man and may these conditions it may be expected to retain its potency for
be fatal ij an infection is untreated. 1f an infection is suspected not les s than 6 months.
treatment should begin without delay. .
LABELLING
IDENTIFICATION The labe1 states (1) the volume of the contents; (2) the date
Inject small doses intradermally into suitable guinea-pigs that after which the preparation is not intended to be used;
have been sensitised with killed P. mallei in oily adjuvant. (3) that the preparation is to be used for animals only;
Oedematous swellings occur at the point of injection after (4) the conditions under which it should be stored; (5) the
48 hours. name and percentage of any added preservative; (6) the dose.
TESTS ANNEX
Acidity or alkalinity Guidance to manufacturers performing the test for sterility.
pH, 6.5 to 7.5, Appendix V L. In determining the number of containers to be tested, the
Phenol manufacturer should have regard to the environmental
For preparations containing phenol as a preservative, not conditions of manufacture, the volume of preparation per
more than 0.5% w/v, determined by the method described container and any other special considerations applying to
under Veterinary Antisera. the preparation concerned. With respect to diagnostic
Sterility preparations for veterinary use, 1% of the containers in a
Complies with the test for sterility, Appendix XVI A, using batch, with a minimum of three and a maximum of 10 is
Method 1: Membrane filtration, whenever possible and considered a suitable number assuming that the preparation
particular1y when the volume in a container is greater than has been manufactured under appropriately validated
100 mL, withthe following modifications. conditions designed to exc1ude contamination.
Incubate the media for not less than 14 days at 30° to 35° in 1 Guidance to manufacturers on the number of containers is provided
the test intended to detect bacteria and at 20° to 25° in the in the Annex to this monograph.
test intended to detect fungi.
U se the quantities stated under Application of the test to
injectable preparations except that when the quantity in each
container! is 20 mL or more of a liquid, the minimum
quantity to be used for each medium is 10% of the contents
or 5 mL, whichever is the less.
Abnonnal toxicity
Inject 0.5 mL subcutaneously into each of two guinea-pigs.
Neither shows a significant local or systemic reaction within
7 days.
ASSAY
To 2.5 mL add 2.5 mL of water and 2.5 mL of a 40% w/v
solution of trichloroacetic acid, mix, allow to stand for
30 minutes and centrifuge for 15 minutes. Discard the
supernatant liquid and dissolve the residue in 0.5 mL of
5M sodium hydroxide. Transfer the solution to a Kjeldahl fiask
with the aid of 6 mL of water and add about 0.1 g of a
mixture of 100 parts of potassium sulfate, 10 parts of cOpper(lI)
sulfate and 5 parts of selenium dioxide and 1 mL of nitrogen-
free sulfuric acid. Evaporate the water and continue heating
until a brown deposit appears. Dissolve the deposit by the
addition of 0.5 mL of hydrogen peroxide solution (100 vol),
continue heating until white fumes of sulfur trioxide appear
and boil rapidly for at least 10 minutes. (If while heating a
brown deposit again appears, add a further 0.5 mL of
hydrogen peroxide solution (100 vol). Transfer to an arnmonia
distillation apparatus with the aid of 5 mL of water and add
5 mL of a 50% w/v solution of sodium hydroxide to form a
lower layer. Distil for 3 minutes, collecting the distillate in a
mixture of 5 mL of a 2% w/v solution of boric acid and
0.05 mL of a solution containing 0.066% w/v of methyl red
and 0.033% w/v of bromocresol green in ethanol (96%) and
titrate with 0.00447M sulfuric acid VS (prepared by diluting
89.3 mL of 0.05M sulfuric acid VS to 1000 mL with water).
Monographs
Surgical Materials
Surgical Materials 325
hand over the other end held in the left hand, passing one
SUTURES end over the strand and through the loop so formed (see
Figure 0660.-1) and pulling the kilot tight.
DEFINITION
Sterile catgut in distributor for veterinary use consists of
strands prepared from collagen taken from the intestinal
membranes of mammals. After cleaning, the membranes are
split longitudinally into strips of varying width, which, when
assembled in small numbers, according to the diameter
required, are twisted under tension, dried, polished, selected
and sterilised. The strands may be treated with chemical
substances such as chromium salts to prolong absorption and Figure 0660.-1. - Simple knof
glycerol to make them supple, provided such substances do
not reduce tissue acceptability. Make not fewer than one measurement per 2 m of length.
The strand is presented in a distributor that allows the If the strand consists of several sections joined by kilots,
withdrawal and use of all or part of it in aseptic conditions. make not fewer than three measurements per section and, in
The design of the distributor is such that with suitable any case, not fewer than one measurement per 2 m of length
handling the sterility of the content is maintained even when at points evenly spaced along the strand or along each
part of the strand has been withdrawn. Ir may be stored dry section. Determine the breaking load using a suitable
or in a preserving liquid to which an antimicrobial tensilometer. The apparatus has two clamps for holding the
preservative but not an antibiotic may be added. strand, one of which is mobile and is driven at a constant
TESTS rate of 30 cm per minute. The clamps are designed so that
1f stored in a preserving liquid, remove the strand from the the strand being tested can be attached without any
distributor and measure promptly and in succession the length, possibility of slipping. At the beginning of the test the length
diameter and breaking load. 1f stored in the dry state, immerse the of strand between the clamps is 12.5 cm to 20 cm and the
strand in alcohol R or a 90 per cent V/V solution of 2-propanol R knot is midway between the clamps. Set the mobile clamp in
for 24 h and proceed with the measurements as indicated above. motion and note the force required to break the strand. If the
strand breaks in a clamp or within 1 cm of it, the result is
Length discarded and the test repeated on another part of the strand.
Measure the length without applying to the strand more The average of all the results, excluding those legitirnately
tension than is necessary to keep it straight. The length is not discarded, is equal to or greater than the value in colurnn C
less than 95 per cent of the length stated on the label. If the and no value is less than that given in column D in
strand consists of several sections joined by knots, the length Table 0660.-1 for the gauge number concerned.
of each section is not les s than 2.5 m.
Diameter Table 0660.-1. - Diamefers and breaking loads
Carry out the test using a suitable instrument capable of
Diameter Breaking load
measuring with an accuracy of at least 0.002 mm and having (millimetres) (newtons)
a circular pressor foot 10 mm to 15 mm in diameter. Gauge
number A B e D
The pressof foot and the moving parts attached to it are
mino max. mino max.
weighted so as to apply a total load of 100 ± 10 g to the
strand being tested. When making the measurements, lower 1 0.100 0.149 0.085 0.175 1.8 0.4
the pressor foot slowly to avoid crushing the strand. Make 1.5 0.150 0.199 0.125 0.225 3.8 0.7
not fewer than one measurement per 2 m of length. If the
2 0.200 0.249 0.175 0.275 7.5 1.8
strand consists of several sections joined by kilots, make not
fewer than three measurements per section. In any case make 2.5 0.250 0.299 0.225 0.325 10 3.8
not fewer than twelve measurements. Make the 3 0.300 0.349 0.275 0.375 12.5 7.5
measurements at points evenly spaced along the strand or
3.5 0.350 0.399 0.325 0.450 20 10
along each section. The strand is not subjected to more
tension than is necessary to keep it straight during 4 0.400 0.499 0.375 0.550 27.5 12.5
measurement. The average of the measurements carried out 0.500 0.599 0.450 0.650 38.4 20.0
5
on the strand being tested and not less than two-thirds of the
individual measurements are within the limits given in the 6 0.600 0.699 0.550 0.750 45.0 27.5
column under A in Table 0660.-1 for the gauge number 7 0.700 0.799 0.650 0.850 60.0 38.0
concemed. N one of the measurements is outside the limits 0.950 70.0 45.0
8 0.800 0.899 0.750
given in the columns under B in Table 0660.-1 for the gauge
number concemed.
Minimum breaking load Soluble chromium compounds
The minimum breaking load is deterrnined over a simple Place 0.25 g in a conical fiask containing 1 mL of water R
kilot formed by placing one end of a strand held in the right per 10 mg of catgut. Stopper the fiask, allow to stand at
326 Surgical Materials
37 ± 0.5 oC for 24 h, cool and decant the liquido Transfer necessary to keep it straight. The length of the strand is not
5 mL to a small test tube and add 2 mL of a 10 giL solution less than 95 per cent of the length stated on the label.
of diphenylcarbazide R in alcohol R and 2 mL of dilute sulfuric Diameter
acid R. The solution is not more intensely coloured than a Unless otherwise prescribed, measure the diameter by the
standard prepared at the same time using 5 mL of a solution following method using the strand in the condition in which
containing 2.83 ¡..tg of potassium dichromate R per millilitre, it is presented. Use a suitable instrument capable of
2 mL of dilute sulfuric acid R and 2 mL of a 10 giL solution measuring with an accuracy of at least 0.002 mm and having
of diphenylcarbazide R in alcohol R (1 ppm of Cr). a circular pressor foot 10 mm to 15 mm in di ame ter.
Sterility (2.6.1) The pressor foot and the moving parts attached to it are
It complies with the test for sterility as applied to catgut and weighted so as to apply a total load of 100 ± 10 g to the
other surgical sutures. Carry out the test on three sections, strand being tested. When making the measurements, lower
each 30 cm long, cut off respectively from the beginning, the the pressor foot slowly to avoid crushing the strand. Make
centre and the end of the strand. not fewer than one measurement per 2 m of length and in
any case not fewer than 12 measurements at points evenly
STORAGE
spaced along the strand. During the measurement submit
Store protected from light and heat.
monofilament strands to a tension not greater than that
LABELLING required to keep them straight. Submit multifilament strands
The label states: to a tension not greater than one-fifth of the minimum
- the gauge number, breaking load shown in column C of Table 0605.-1
- the length in centimetres or in metres. appropriate to the gauge number and type of material
___________________________________________ ~E~ con cerned or 10 N whichever is less. For multifilament
strands of gauge number above 1.5 make two measurements
at each point, the second measurement being made after
rotating the strand through 90°. The diameter of that point is
the average of the two measurements. The average of the
Sterile Non-absorbable Strands in measurements carried out on the strand being tested and not
less than two-thirds of the individual measurements are
Distributor within the limits given in the columns under A in
(Strands, Sterile Non-absorbable, in Distributor for Table 0605.-1 for the gauge number concerned. None of the
Veterinary Use, Ph Bur monograph 0605) measurements is outside the limits given in the columns
PhE~ ___________________________________________ under B in Table 0605.-1 for the gauge number concerned.
DEFINITION
Table 0605.-1. - Diamefers and minimum breaking loads
The statements in this monograph are intended to be read in
conjunction with the individual monographs on sterile non- Diameter Minimum breaking load
(millimetres) (newtons)
absorbable strands in distributor for veterinary use in the
Gauge AH other
Phannacopoeia. The requirements do not necessarily apply to number A B Linen thread non-absorbable
sterile non-absorbable strands which are not the subject of such strands
monographs. mino max. mino max. e D e D
Sterile non-absorbable strands in distributor for veterinary 0.5 0.050 0.069 0.045 0.085 1.0 0.35
use are strands which, when introduced into a living 0.7 0.070 0.099 0.060 0.125 1.0 0.3 1.5 0.60
organism, are not metabolised by that organismo Sterile non-
1 0.100 0.149 0.085 0.175 2.5 0.6 3.0 1.0
absorbable strands vary in origin, which may be animal,
vegetable or synthetic. They occur as cylindrical 1.5 0.150 0.199 0.125 0.225 5.0 1.0 5.0 1.5
monofilaments or as multifilament strands. Multifilament 2 0.200 0.249 0.175 0.275 8.0 2.5 9.0 3.0
strands consist of elementary fibres which are assembled by
2.5 0.250 0.299 0.225 0.325 9.0 5.0 13.0 5.0
twisting, cabling or braiding. Such strands may be sheathed.
Sterile non- absorbable strands may be treated to render 3 0.300 0.349 0.275 0.375 11.0 8.0 15.0 9.0
them non-capillary, and they may be coloured with colouring 3.5 0.350 0.399 0.325 0.450 15.0 9.0 22.0 13.0
matter or pigments authorised by the competent authority.
4 0.400 0.499 0.375 0.550 18.0 11.0 27.0 15.0
The strands are sterilised.
They are presented in a suitable distributor that allows the 5 0.500 0.599 0.450 0.650 26.0 ·15.0 35.0 22.0
withdrawal and use of all or part of the strand in aseptic 6 0.600 0.699 0.550 0.750 37.0 18.0 50.0 27.0
conditions. The design of the distributor is such that with 0.700 0.799
7 0.650 0.850 50.0 26.0 62.0 35.0
suitable handling the sterility of the content is maintained
even whenpart of the strand has been removed. They may 8 0.800 0.899 0.750 0.950 65.0 37.0 73.0 50.0
be stored dry or in a preserving liquid to which an
antimicrobial preservative but not an antibiotic may be
Minimum breaking load
added.
Unless otherwise prescribed, determine the minimum
TESTS breaking load by the following method using the strand in
Remove the strand from the distributor and measure promptly and the condition in which it is presented. The minimum
in succession the length, diameter and minimum breaking load. breaking load is determined over a simple knot formed by
Length placing one end of a strand he1d in the right hand over the
Measure the length in the condition in which the strand is other end held in the left hand, passing one end over the
presented and without applying more tension than is strand and through the loop so formed (se e Figure 0605.-1)
and pulling the knot tight.
Surgical Materials 327
clamps. Set the mobile clamp in motion and note the force
required to break the strand. If the strand breaks in'¡ a clamp
or within 1 cm of it, the result is discarded and the test
repeated on another part of the strand. The average of aH the
results, excluding those legitimately dis-carded, is equal to or
greater than the value in column C and no value is less than Sterile Linen Thread in Distributor
that given in column D in Table 0605.-1 for the gauge (Linen Thread, Sterile, in Distributor for Veterinary
number and type-of material concemed. Use, Ph Eur monograph 0608)
PhE~ ___________________________________________
DEFINITION
Sterile linen thread in distributor for veterinary use consists
of the pericyclic fibres of the stem of Linum usitatissimum L.
The elementary fibres, 2.5 cm to 5 cm long, are assembled in
bundles 30 cm to 80 cm long and spun into continuous
lengths of suitable diameter. The thread may be creamy-
white or may be coloured with colouring matter authorised
by the competent authority. The thread is sterilised.
IDENTIFICATION
A. Dissect the end of a thread, using a needle or fine
tweezers, to isolate a few individual fibres. Examined under a
microscope, the fibres are seen to be 12 ~m to 31 ~m wide
and, along the greater part of their length, have thick walls,
Figure 0605.-1. - Simple knot sometimes marked with fine longitudinal striations, and a
narrow lumen. The fibres graduaHy narrow to a long, fine
point. Sometimes there are unilateral sweHings with
Sterility (2.6.1)
transverse Hnes.
They comply with the test for sterility as appHed to catgut
and other surgical sutures. Carry out the test on three B. Impregnate isolated fibres with iodinated zinc chloride
sections each 30 cm long, cut off respectively from the solution R. The fibres are coloured violet-blue.
beginning, the centre and the end of the strand. TESTS
Extractable colour It complies with the tests prescribed in the monograph on
Strands that are dyed and intended to remain so during use Strands, sterile non-absorbable, in distributor for veterinary use
comply with the test for extractable colour. Place 0.25 g of (0605).
the strand to be examined in a conical flask, add 25.0 mL of 1f stored in a dry state, expose to an atmosphere with a relative
water R and cover the mouth of the flask with a short- humidity of 65 ± 5 per cent at 20 ± 2 oC for 4 h immediately
sternmed funnel. Boil for 15 min, cool and adjust to the before measuring the diameter and for the determination of
original volume with water R. Depending on the colour of the minimum breaking load immerse in water R at room temperature
strand, prepare the appropriate reference solution as for 30 min immediately before carrying out the test.
described in Table 0605.-2 using the primary colour
STORAGE
solutions (2.2.2).
See the monograph on Strands, sterile non-absorbable, in
distributor for veterinary use (0605).
Table 0605.-2. - Colour reference solutions
LABELLING
Colour of strand CompositioD of reference SOlutiOD
(parts by volume) See the monograph on Strands, sterz'le non-absorbable, in
Red Yellow Blue Water distributor for veterinary use (0605).
primary primary primary ___________________________________________ ~E~
LABELLING
See the monograph on Strands, sterile non-absorbable, in
distributor jor veterinary use (0605)
__________________________________________ ~E~
Sterile Polyamide 6/6 Suture in
Distributor
(Polyamide 6/6 Suture, Sterile, in Distributor jor
Veterinary Use, Ph Bur monograph 0610)
Sterile Polyamide 6 Suture in NOTE: The name Nylon 6/6 as a synonym for Polyamide 6/6
IDENTIFICATION LABELLING
A. In contact with a flame it melts and burns, forming a hard See the monograph on Strands, sterile non-absorbable, in
globule of residue and gives off a characteristic odour distributor for veterinary use (0605).
resembling that of celery. ____ ~ _____________________________________ PhE~
DEFINITION
Sterile braided silk suture in distributor for veterinary use is
obtained by braiding a variable number of threads, according
to the diameter required, of degurnmed silk obtained from
the cocoons of the silkworm Bombyx mori L. It may be
coloured with colouring matter authorised by the competent
authority. The suture is sterilised.
IDENTIFICATION
A. Dissect the end of a strand, using a needle or fine
tweezers, to isolate a few individual fibres. The fibres are
sometimes marked with very fine longitudinal striations
parallel to the axis of the strand. Examined under a
microscope, a cross-section is more or less triangular or semi-
circular, with rounded edges and without a lurnen.
B. Impregnate isolated fibres with iodinated potassium iodide
solution R. The fibres are coloured pale yellow.
TESTS
It complies with the tests prescribed in the monograph on
Strands, sterile non-absorbable, in distributor for veterinary use
(0605).
STORAGE
See the monograph on Strands, sterile non-absorbable, in
distributor for veterinary use (0605).
Infrared Reference Spectra
S2 Infrared Reference Spectra
80 f\ AA
\ f\/\ rvV\ (lA
1\
A
60
r ¡f\ AI\ n A A fr \fl
IV
V '{ . \I
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2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
S4 Infrared Reference Spectra
~ 20., .................................................................................................................. ,. . . . . . lA
e
ro
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4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm- 1
)
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Wavenumber (cm-1)
S6 Infrared Reference Spectra
~
ID
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c
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::::::
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
~
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Amprolium Hydrochloride RSV07 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0 ,·-r'·,··...•·.. ···~··..···......·..·••........~....~..·i··.....~···"·,,· ..·,,.....................................y ................... "............................................., ..., ............." .........." .." ....................··"..,,·········c"···,,·····,,·,,·,,···...·...···..··..··..···..··•·..................,....~." ................." ........".~" ••.."., .."··T···..···,,··,,···....·....·~··,,··,,·· ..··....·•..·,,·,,"""··.... ·,···· ....•" ......................................................".~.,
Wavenumber (cm-1)
Infrared Reference Spectra S7
rYV A~
80 /'1,11
~ l\
(V\
"(\ ~I! r/ N
V
11
60 ~j
1/ .~ ···N···········¡ N·········v IV
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1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
60 ..,.............................................+ ..........f
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
80
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
S8 Infrared Reference Spectra
Cloprostenol Sodium RSVll Instrument: Fourier Transform Phase: Liquid Paraffin Mull
1OO. O. ,.~.............",.'~"~~'_"W""";'''''··'··'·''·'·''~'' ···~·"~····""T···""··"···-"··""·""··""·""··"·,"""··"·· .........~.•.~"'· ....- ....·T..·..·............'" ..·..·..• __..·_.·.. y .. · ............ ~.............- ..........,..............~ ............_~ ......., ........~........~~....._ ................."
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Cloxacillin Benzathine RSV12 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
~
ID
ü 20
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
60.+ .....................................................q
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Infrared Reference Spectra S9
8e 20.,..................... .., ...... ··Iy· .. ··•.. ·····)-··· .. ···~ ....··• ... ·... ;·········..······•···· ......... , ............................... 1" ........................ ,............................................;. ..................................\
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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80 ~ D
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
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Wavenumber (cm-1)
S 1O Infrared Reference Spectra
'-
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80
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60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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\\ (\
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Diprenorphine Hydrochloride RSV18 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0" .................................... ;......................... ,................................................................................................................... " .............................................. , ............................... , .......................................,
40 . ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \. w.w"..................w... ······.·.·.·.....l" 1I·.•• ··..........······"w~ j .............. w...w.w..'..........................w.wll ........·..·.. ·..·w...... ···ll· . ·¡·I .... ·..·····I..!lw···II..·..v· f·· ......A/····u· ..·w· ..I....I.JI· u. · · :..·...... ··· . ·. · ...\j ...w. . . .w. ·1 .. ·.1 ............., · ·... · .. ·.... . w.)
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
80 .......... I
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
n~
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
S 12 Infrared Reference Spectra
l~
80
60
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1\
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40 ......... ~ ....W o
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Flunixin Meglumine RSV23 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0."' ................................, ............................................ ,." .................................................................. ,................................................ , ...................................... ......................................... .................................. ,
60; ....................................... ; ............. ·······················1' ....············ ............... } .............................................. j ............ d ••••••.•• , ••••••• 'llI
Griseofulvin RSV24 Instrument: Fourier Transform Phase: 1.5% w/v Solution in Chloroform Thickness: 0.1 mm
r
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
S 14 Infrared Reference Spectra
Meclofenamic Acid RSV26 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
80
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Infrared Reference Spectra S 15
80
'11 rf--'
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60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Infrared Reference Spectra SI 7
80 -- /
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I ~rf{ ~r\ A
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60 \ I ~ !
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2000,0 1800 1600 1400 1200 1000 800 600 400_0
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Sulfathiazole Sodium RSV42 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
....
~
80 \ \n ~ (
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¡~ (\A
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Infrared Reference Spectra SI 9
40
o~
ID
ü 20
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:::::
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(J)
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¡.=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
80
~
,-
60
\
)
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rn .r~ ~~'f\\;V r~
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:::::
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¡.=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Tylosin RSV46 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 , ............................................... , ............................................... ,............................................................................................................................................................... ,.............................................. , ................................................ ; .........................
The following appendices are included in this section of the British Pharmacopoeia
(Veterinary) .
APPENDIX XVI
B (Vet) 1. Vacant
B (Vet) 2. Vacant
B (Vet) 3. Mycoplasmas
B (Vet) 4. Avían Viral Vaccines: Tests for Extraneous Agents in Seed Lots
B (Vet) 5. Avian Live Virus Vaccines: Tests for Extraneous Agents in Batches of
Finished Product
B (Vet) 6. Vacant
B (Vet) 7. Vacant
B (Vet) 8. Vacant
APPENDIX XXI
B (Vet). Approved Synonyms (Veterinary)
European Pharmacopoeia Equivalent Texts A3
t Reproduced in juil as Pan 111 of the General Notices of the British Pharmacopoeia and British Pharmacopoeia (Veterinary).
A4 European Pharmacopoeia Equivalent Texts
Table 5.2.2.-1
Agent Test Vertical Rapid/slow
to be used** transmission spread
Avian adenoviruses, group 1 AGP, EIA yes slow
Avian encephalomyelitis virus AGP, EIA yes rapid
Avian infectious bronchitis virus HI, EIA no rapid
Avian infectious laryngotracheitis virus VN, EIA no slow
Avian leucosis virus es EIA for virus, yes slow
VN, EIA for antibody
Avian nephritis virus IS no slow
Avian orthoreoviruses IS, EIA yes slow
Avian reticuloendotheliosis virus AGP, IS, EIA yes slow
Chicken anaemia virus IS, EIA, VN yes slow
Egg drop syndrome virus HI, EIA yes slow
Infectious bursal disease virus Serotype 1: AGP, EIA, VN no rapid
Serotype 2: VN
Influenza A virus AGP, EIA, HI no rapid
Marek's disease virus AGP no rapid
NewcastIe disease virus HI, EIA no rapid
Turkey rhinotracheitis virus EIA no slow
Mycoplasma gallisepticum Agg and HI to confirm a yes slow
positive test,
EIA, HI
Mycoplasma synoviae Agg and HI to confirm a yes rapid
positive test,
EIA, HI
Salmonella pullorum Agg yes slow
Agg: agglutination HI: haemagglutination inhibition
AGP: agar gel precipitation; the technique is suitable where testing is carried IS: immunostaining
out weekly VN: virus neutralisation
EIA: enzyme irnrnunoassay
**Subject to agreement by the competent authority, other types of test may be used provided they are at least as sensitive as those indicated and of
appropriate specificity.
conditions appropriate to SPF flocks. The SPF flock is serotypes of the viruses. Samples for testing are taken at
housed within an isolator or in a building with filtered air random.
under positive pressure. Appropriate measures are taken to A positive result for chicken anaemia virus (CAV) does not
prevent entry of rodents, wild birds, insects and unauthorised necessarily exclude use of material derived from the flock,
personnel. but live vaccines for use in birds les s than 7 days old shall be
Personnel authorised to enter the facility must have no produced using material from CAV-negative flocks.
contact with other birds or with agents potentially capable of Inactivated vaccines for use in birds less than 7 days old may
infecting the flock. It is advisable for personnel to shower and be produced using material from flocks that have not been
change clothing or to wear protective clothing before entering shown to be free from CAV, provided it has been
the controlled facility. demonstrated that the inactivation process inactivates CAVo
Wherever possible, items taken into the facility are sterilised.
In particular it is recommended that the feed is suitably Establishment of an SPF flock
treated to avoid introduction of undesirable micro-organisms A designated SPF flock is derived from chickens shown to be
and that water is at least of potable quality, for example from free from vertically-transmissible agents listed in
a chlorinated supply. No medication is administered to birds Table 5.2.2-1. This is achieved by testing of 2 generations
within the flock that might interfere with detection of any prior to the designated SPF flock. A general scheme for the
disease. procedure to be followed in establishing and maintaining an
A permanent record is kept of the general health of the flock SPF floc~ is shown diagrammatically in Table 5.2.2.-2.
and any abnormality is investigated. Factors to be monitored In order f.o establish a new SPF flock, a series of tests must
include morbidity, mortality, general physical condition, feed be conducted on 3 generations of birds. All birds in the 1st
consumption, daily egg production and egg quality, fertility generation must be tested at least once before the age of 20
and hatchability. Records are maintained for a period of at weeks for freedom from avian leucosis group-antigen and
least 5 years. Details of any deviation from normal in these tested by an enzyme immunoassay (EIA) or by virus
performance parameters or detection of any infection are neutralisation (VN) for freedom of antioodies to avian
notified to the users of the eggs as soon as practicable. leucosis virus subtypes A, B and J. All birds must also be
tested for freedom from antibodies to the vertically-
The tests or combination of tests described below must have
transmissible agents listed in Table 5.2.2-1. From the age of
suitable specificity and sensitivity with respect to relevant
Appendix XV AII
Table 5.2.2-2. - Schematic description of the establishment and maintenance of SPF flocks
NEWSTOCK Establish freedom from vertically·transmissible agents
Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general cJinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
nd
2 GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general cJinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
"
3rd GENERATION T~st all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general.cJinical observation from 8 weeks of age
3rd GENERATION Carry out routine testing for specified agents from 20 weeks of age
SUBSEQUENT GENERATIONS Test two 5 per cent samples for avian leucosis antigen and for antibodies against specified
a~ents between 12 and 20 weeks of a!!e
Test for Salmonella spp. and perform general cJinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
8 weeks the fiock is tested for freedom from Salmonella. Routine testing of designated SPF flocks
Clínical examination is carried out on the fiock from 8 weeks General examination and necropsy
of age and the birds must not exhibit any signs of infectious
Clinical examination is carried out at least once per week
disease. The test methods to be used for these tests are given
throughout the life of the fiock in order to verify that the
in the table and further guidance is also given in the section
birds are free from fowl-pox virus and signs of any other
below on routine testing of designated SPF fiocks. From 20
infection. In the event of mortality exceeding 0.2 per cent per
weeks of age, the fiock is tested as described under Routine
week, necropsy is performed on aH available carcasses to
testing of designated SPF fiocks. AH stages of this testing
verify that there is no sign of infection. Where appropriate,
regime are also applied to the subsequent 2 generations,
histopathological and/or microbiological/virological studies are
except the testing of every bird before lay for verticaHy-
performed to confirm diagnosis. Specific examination for
transmissible agents. AH test results must indicate freedom
tuberculosis lesions is carried out and histological samples
from pathogens in aH 3 generations for the fiock consisting of
from any suspected lesions are specificaHy stained to verify
the 3rd generation to be designated as SPF.
freedom from Mycobacterium avium. Caecal contents of aH
SPF embryos derived from another designated SPF fiock available carcas ses are examined microbiologicaHy for the
contained within a separate facility on the same site may be presence of Salmonella spp. using the techniques described
introduced. From 8 weeks of age, these replacement birds are below. Where appropriate, caecal samples from up to 5 birds
regarded as a fiock and are tested in accordance with test may be pooled.
procedures described above.
Cultural testing lor Salmonella spp
lnitial testing requirements for subsequent Cultural testing for Salmonella spp. is performed either by
generations derived from a designated SPF flock testing samples of droppings or cloacal swabs or by testing of
Where a replacement fiock is derived exclusively from a fuHy drag swabs. Where droppings or cloacal swabs are tested, a
established SPF fiock the new generation is tested prior to total of 60 samples within each 4-week period is tested
being designated as SPF. In addition to the tests for throughout the entire life of the fiock. Tests may be
Salmonella and monitoring of the general health and performed on pools of up to 10 samples. Where drag swabs
performance of the fiock, further specific testing from the age are tested, a minimum of 2 drag swabs are tested during each
of 8 weeks is required. Tests are performed on two 4-week period throughout the entire life of the fiock.
5 per cent samples of the fiock (minimum 10, maxÍnium 200 Detection of Salmonella spp. in these samples is performed by
birds) taken with an interval of at least 4 weeks between the pre-enrichment of the samples foHowed by culture using
ages of 12-16 weeks and 16-20 weeks. Salmonella-selective media.
AH samples are coHected and tested individuaHy. Blood Tests lor avian leucosis antigen
samples for antibody tests and suitable samples for testing for Prior to the commencement of laying, cloacal swabs or blood
leucosis antigen are coHected. The test methods to be used samples (using buffy coat cultivation) are tested for the
are as described under Routine testing of designated SPF presence of group-specific leucosis antigen. A total of
fiocks. Only when aH tests have confirmed the absence of 5 per cent (minimum 10, maximum 200) of the fiock is
infection may the new generation be designated as SPF. sampled during each 4-week periodo During lay, albumen
samples from 5 per cent (minimum 10, maximum 200) of
the eggs are tested in each 4-week periodo Tests are
performed by EIA for group-specific antigen using methods
A12 Appendix XV
that are capable of detecting antigen from subgroups A, B Any progeny derived from that Rock during or after the
and J. 4-week period prior to the last negative sample being
Test/or antibodies to other agents collected may not be designated as SPF.
Tests for antibodies to all agents listed in Table 5.2.2.-1 are
performed throughout the laying period of the Rock. In each
4-week period, samples are taken from 5 per cent (minimum J (Vet) 1. Cell Cultures for the
10, maximum 200) of the Rock. It is recommended that
1.25 per cent of the Rock is sampled each week since sorne Production of Veterinary Vaccines
test methods for sorne agents must be conducted on a weekly (Ph. Eur. method 5.2.4)
basis. Table 5.2.2.-1 classifies the agents into those that Cell cultures for the production of vaccines for veterinary use
spread rapidly through the Rock and those that spread slowly comply with the requirements of this section. It may also be
or may not infect the entire Rock. For those agents listed as necessary that cell cultures used for testing of vaccines for
slowly spreading, each sample is tested individually. veterinary use also comply with sorne or all of these
For those agents listed as rapidly spreading, at least requirements.
20 per cent of the samples collected in each 4-week period
For most mammalian virus es, propagation in celllines is
are tested individually or, where serum neutralisation or
possible and the use of primary cells is then not acceptable.
EUSA tests are employed, all of the samples may be tested
individually or by preparing pools of 5 samples, collected at Permanently infected cells used for production of veterinary
the same time. vaccines comply with the appropriate requirements described
below. The cells shall be shown to be infected only with the
Suitable methods to be used for detection of the agents are
agent stated.
shown in Table 5.2.2.-1. Subject to agreement by the
competent authority, other test methods may be used Celllines
provided they are shown to be at least as sensitive as those Cell lines are normally handled according to a cell-seed
indicated and of appropriate specificity. system. Each master cell seed is assigned a specific code for
identification purposes. The master cell seed is stored in
Tests to be conducted at the end of the laying period aliquots at - 70 oC or lower. Production of vaccine is not
Following the last egg collection, final testing to confirm the normally undertaken on cells more than twenty passages
absence of vertically-transmissible agents indicated in from the master cell seed. Where suspension cultures are
Table 5.2.2.-1 is performed. After the last egg collection, a used, an increase in cell numbers equivalent to approximately
minimum of 5 per cent of the Rock (minimum 10, maximum three population doublings is considered equivalent to one
200) is retained for at least 4 weeks. Blood samples are passage. If cells beyond twenty passage levels are to be used
collected from every bird in the group during the 4-week for production, it shall be demonstrated, by validation or
period with at least 1.25 per cent of the birds (25 per cent of further testing, that the production cell cultures are
the sample) being bled not earlier than 4 weeks after the final essentially similar to the master cell seed with regard to their
egg collection. Serum samples are tested for vertically- biological characteristics and purity and that the use of such
transmissible agents (as defined by Table 5.2.2.-1) using the cells has no deleterious effect on vaccine production.
methods indicated. Where sampling is performed on a weekly The history of the cell line shall be known and recorded in
basis, at least 1.25 per cent of the birds (25 per cent of the detail (for example, origin, number of passages and media
sample) are tested each week during this periodo used for multiplication, storage conditions).
Altematively, within 4 weeks of the final egg collection blood The method of storing and using the cells, including details
and/or other suitable sample materials are collected from at of how it is ensured that the maximum number of passages
least 5 per cent of the Rock and tested for the presence of permitted is not exceeded during product manufacture, are
vertically-transmissible agents using validated nucleic acid recorded. A sufficient quantity of the master cell seed and
amplification techniques (2.6.21). each working cell seed are kept for analytical purposes.
Action to be taken in the event of detection of a The tests described below are carried out (as prescribed in
specified agent Table 5.2.4.-1) on a culture of the master cell seed and the
working cell seed or on cell cultures from the working cell
If evidence is found of contamination of the Rock by an
seed at the highest passage level used for production and
agent listed as slowly spreading in Table 5.2.2.-1, all
derived from a homogeneous sample demonstrated to be
materials derived from the Rock during the 4-week period
representative.
irnmediately preceding the date on which the positive sample
was collected are considered unsatisfactory. Similarly, if Characteristics of Culture The appearance of cell
evidence is found of contamination of the Rock by an agent monolayers, before and after histological staining, is
listed as rapidly spreading in Table 5.2.2.-1, all materials described. Information, if possible numerical data, is
derived from the Rock during the 2-week period irnmediately provided especially on the speed and rate of growth.
preceding !he date on which the positive sample was Similarly, the presence or absence of contact inhibition,
collected are considered unsatisfactory. Any product polynucl~ated cells and any other cellular abnormalities are
manufactured with such materials, and for which the use of specified:
SPF materials is required, is considered unsatisfactory and Karyotype A chromosomal examination is made of not
must be discarded; any quality control tests conducted using fewer than fifty cells undergoing mitosis in the master cell
the materials are invalido seed and at a passage level at least as high as that to be used
Producers must notify users of all eggs of the evidence of in production. Any chromosomal marker present in the
contamination as soon as possible following the outbreak. master cell seed must also be found in the high passage cells
and the modal number of chromosomes in these cells must
Any Rock in which an outbreak of any specified agent is
not be more than 15 per cent higher than of cells of the
confirmed may not be redesignated as an SPF Rock.
master cell seed. The karyotypes must be identical. If the
Appendix XV A13
modal number exceeds the level stated, if the chromosomal Detection of Haemadsorbent Virus es Monolayers
markers are not found in the working cell seed at the highest totalling at least 70 cm2 are washed several times with an
level used for production or if the karyotype differs, the cell appropriate buffer and a sufficient volume of a suspension of
line shall not be used for manufacture. suitable red blood cells added to cover the surface of the
monolayer evenly. After different incubation times cells are
Table 5.2.4.-1. "" Cell culture stage at which tests examined for the presence of haemadsorption.
are carried out Detection of Specified Viruses Tests are carried out for
Master ceIl Working CeIl from working freedom from contaminants specific for the species of origin
seed ceIl seed ceIl seed at highest of the cell line and for the species for which the product is
passage level
General microscopy + + + intended. Sufficient cells on suitable supports are prepared to
carry out tests for the agents specified. Suitable positive
Bacteria and fungi + +
controls are inc1uded in each test. The cells are subjected to
Mycoplasmas + + suitable tests, for example using fluorescein-conjugated
Viruses + + antibodies or similar reagents.
+ +
Tests in Other Cell Cultures Monolayers totalling at
Identification of species
least 140 cm2 are required. The cells are frozen and thawed
Karyotype + +
at least three times and then centrifuged to remove cellular
Tumorigenicity + debris. Inoculate aliquots onto the following cells at any time
up to 70 per cent confluency:
- primary cells of the source species;
Identification of the Species It shall be shown, by one - cells sensitive to viruses pathogenic for the species for
validated method, that the master cell seed and the cells which the vaccine is intended;
from the working cell seed at the highest passage level used
- cells sensitive to pestiviruses.
for production come from the species of origin specified.
When a fluorescence test is carried out and the The inoculated cells are maintained in culture for at least
corresponding serum to the species of origin of cells is used 7 days, after which freeze-thawed extracts are prepared as
and shows that all the tested cells are fluorescent, it is not aboye and inoculated onto sufficient fresh cultures of the
necessary to carry out other tests with reagents able to detect same cell types to aHow for the testing as described below.
contamination by cells of other species. The cells are incubated for at least a further 7 days.
The cultures are examined regularly for the presence of any
Bacterial and Fungal Contamination The cells comply
cytopathic changes indicative of living organisms.
with the test for sterility (2.6.1). The sample of cells to be
examined consists of not less than the number of cells in a At the end of this period of 14 days, the inoculated cells are
monolayer with an area of 70 cm2 or, for cells grown in subjected to the following checks:
suspension, an approximately equivalent number of cells. - freedom from cytopathic and haemadsorbent organisms,
The cells are maintained in culture for at least 15 days using the methods specified in the relevant paragraphs
without antibiotics before carrying out the test. aboye,
Mycoplasmas (2.6.7) The cells comply with the test for - absence of pestiviruses and other specific contaminants by
mycoplasmas. The cells are maintained in culture for at least immunofluorescence or other validated methods as
15 days without antibiotics before carrying out the test. indicated in the paragraph aboye on Detection of
Absence of Contaminating Virus es The cells must not Specified Viruses.
be contaminated by viruses; suitably sensitive tests, inc1uding Tumorigenicity The risk of a cell line for the target
those prescribed below, are carried out. species must be evaluated and, if necessary, tests are carried
The monolayers tested shall have an area of at least 70 cm2 , out.
and shall be prepared and maintained using medium and
additives, and grown under similar conditions to those used Primary CelIs
for the preparation of the vaccine. The monolayers are For most mammalian vaccines, the use of primary cells is not
maintained in culture for a total of at least 28 days. acceptable for the manufacture of vaccines since cell lines can
Subcultures are made at 7-day intervals, unless the cells do be used. If there is no altemative to the use of primary cells,
not survive for this length of time, when the sub cultures are the cells are obtained from a herd or flock free from specified
made on the latest day possible. Sufficient cells, in suitable pathogens, with complete protection from introduction of
containers, are produced for the final sub culture to carry out diseases (for example, disease barriers, filters on air inlets,
the tests specified below. suitable quarantine before introduction of animals). Chicken
The monolayers are examined regularly throughout the flocks comply with the requirements prescribed in general
incubation period for the possible presence of cytopathic chapter 5.2.2. Chicken Flocks Free from Specified Pathogens for
effects and at the end of the observation period for cytopathic the Production and Quality Control of Vaccines. For aH other
effects, haemadsorbent viruses and specific viruses by species, the herd or flock is shown to be free from relevant
irnmuno-fluorescence and other suitable tests as indicated specified pathogens. All the breeding stock in the herd or
below. flock intended to be used to produce primary cells for
vaccine manufacture is subject to a suitable monitoring
Detection of Cytopathic Viruses Two monolayers of at
procedure inc1uding regular serological checks carried out at
least 6 cm2 each are stained with an appropriate cytological
least twice ayear and two supplementary serological
stain. The entire area of each stained monolayer is examined
examinations performed in 15 per cent of the breeding stock
for any inc1usion bodies, abnormal numbers of giant cells or
in the herd between the two checks mentioned aboye.
any other lesion indicative of a cellular abnormality which
might be attributable to a contaminant. Wherever possible, particularly for mammalian cells, a seed-
lot system is used with, for example, a master cell seed
A14 Appendix XV
formed after les s than five passages, the working cell seed effects, haemadsorbent virus es and specific virus es by
being no more than five passages from the initial preparation immunofiuorescence and other suitable tests as indicated
of the cell suspension from the animal tissues. below.
Each master cell seed, working cell seed and cells of the Detection of Cytopathic Viruses Two monolayers of at
highest passage of primary cells are checked in accordance least 6 cm2 each are stained with an appropriate cytological
with Table 5.2.4.-2 and the procedure described below. stain. Examine the entire area of each stained monolayer for
The sample tested shall cover all the sources of cells used for any inc1usion bodies, abnormal numbers of giant cells or any
the manufacture of the batch. No batches of vaccine other lesion indicative of a cellular abnonnality that might be
manufactured using the cells may be released if any one of attributable to a contaminant.
the checks perfonned produces unsatisfactory results. Detection of Haemadsorbent Viruses Monolayers
totalling at least 70 cm 2 are washed several times with a
Table 5.2.4.-2. - Cel! culture stage at which tests suitable buffer solution and a sufficient volume of a
are carried out suspension of suitable red blood cells added to cover the
Master ceU Working Highest passage surface of the monolayer evenly. After different incubation
seed cell seed level times, examine cells for the presence of haemadsorption.
General microscopy + + +
Detection of Specified Virus es Tests are be carried out
Bacteria and fungi + + for freedom of contaminants specific for the species of origin
Mycoplasmas + + of the cells and for the species for which the product is
intended.
Virus es + +
Sufficient cells on suitable supports are prepared to carry out
Identification of species + tests for the agents specified. Suitable positive controls are
inc1uded in each test. The cells are subjected tosuitable tests
using fiuorescein-conjugated antibodies or similar reagents.
Characteristics of Cultures The appearance of cell
monolayers, before and after histological staining, is Tests in Other Cell Cultures Monolayers totalling at
described. Information, if possible numerical data, is least 140 cm2 are required. The cells are frozen and thawed
recorded, especially on the speed and rate of growth. at least three times and then centrifuged to remove cellular
Similarly, the presence or absence of contact inhibition, debris. Aliquots are inoculated onto the following cells at any
polynuc1eated cells and any other cellular abnormalities are time up to 70 per cent confiuency:
specified. - primary cells of the source species;
Identification of Species It shall be demonstrated by one - cells sensitive to viruses pathogenic for the species for
validated test that the master cell seed comes from the which the vaccine is intended;
specified species of origino - cells sensitive to pestiviruses.
When a fiuorescence test is carried out and the The inoculated cells are maintained in culture for at least
corresponding serum to the species of origin of cells is used 7 days, after which freeze-thawed extracts are prepared as
and shows that all the tested cells are fiuorescent, it is not above, and inoculated onto sufficient fresh cultures of the
necessary to carry out other tests with reagents able to detect same cell types to allow for the testing as described below.
contamination by cells of other species. The cells are incubated for at least a further7 days.
Bacterial and Fungal Sterility The cells comply with the AlI cultures are regularly examined for the presence of any
test for sterility (2.6.1). The sample of cells to be examined cytopathic changes indicative of living organisms.
consists of not less than the number of cells in a monolayer At the end of this period of 14 days, the inoculated cells are
with an area of 70 cm2 or for cells grown in suspension an subjected to the following checks:
approximately equivalent number of cells. The cells are ¡ - freedom from cytopathic and haemadsorbent organisms is
maintained in culture for at least 15 days without antibiotics demonstrated using the methods specified in the relevant
before carrying out the test. paragraphs above;
Mycoplasmas (2.6.7) The cells comply with the test for - relevant substrates are tested for the absence of
mycoplasmas. The cells are maintained in culture for at least pestiviruses and other specific contaminants by
15 days without antibiotics before carrying out the test. immunofiuorescence or other validated methods as
Absence of Contaminating Virus es The cells must not indicated in the paragraph above on Detection of
be contaminated by viruses; suitably sensitive tests, inc1uding Specified Viruses.
those prescribed below are carried out.
The monolayers tested shall be at least 7 O cm2, and shall be
prepared and maintained in culture using the same medium
and additives, and under similar conditions to those used for J (Vet) 2. Substances of Animal Origin
the preparation of the vaccine. for the Production of Immunological
The monolayers are maintained in culture for a total of at
least 28 days or for the longest period possible if culture for
Veterinary Medicinal Products
28 days is impossible. Sub cultures are made at 7-day (Ph. Eur. method 5.2.5)
intervals, unless the cells do not survive for this length of
time when the sub cultures are made on the latest day 1. SCOPE
possible. Sufficient cells, in suitable containers are produced Substances of animal origin (for example serum, trypsin and
for the final subculture to carry out the tests specified below. serum albumin) may be used during the manufacture of
The monolayers are examined regularly throughout the immunological veterinary medicinal products.
incubation period for the possible presence of cytopathic The requirements set out in this chapter apply to substances
effects and at the end of the observation period for cytopathic of animal origin produced on a batch basis, for use at all
Appendix XV A15·
stages of manufacture, for example in culture media or as extraneous agents needs to be assessed. The risk of
added constituents of products during blending. These contamination of the substance and the resultant
requirements are not intended for the control of seed immunological veterinary medicinal product with inactivated
materials or substrates of animal origin that are covered by extraneous agents may also need to be taken into account.
requirements in other pharmacopoeial texts such as the This would be the case if, for example, the contaminant was
monograph Vaccines for- veterinary use (0062) and chapter one from which a European country is officially free and/or is
5.2.4. Gel! cultures for the production of veterinary vaccines. the subject of a specific disease control program in a
European country and where the presence of the inactivated
2. General principIes and requirements agent could lead to the stimulation of a detectable irnmune
Substances of animal origin comply with the requirements of the response in recipient animals.
European Pharmacopoeia (where a relevant monograph eJfists). As part of the risk assessment, the presence in the substance
Restrictions are placed on the use of substances of animal of antibodies that can interfere with the detection andlor
origin because of safety concerns associated with pathogens inactivation of living extraneous agents must also be taken
that may be present in them and epidemiological and/or into account.
regulatory concerns associated with the presence of particular The risk assessment may need to be repeated and the risk
antigens (either live or inactivated). management steps described below re-evaluated and revised
General principIes: in order to take account of changes:
- it is recommended to minimise, wherever practicable, the - in the incidence of diseases occurring in the country or
use of substances of animal origin; countries of origin of animals used as the source for the
- unless otherwise justified, the use of substances of animal substance, inc1uding emerging diseases (new pathogens);
origin as constituents in the formulation of medicinal - in the incidence of diseases and of disease control
products is not acceptable except where such substances measures applied in the European countries in which
are subject to a treatment validated for the inactivation of immunological veterinary medicinal products
live extraneous agents. manufactured with the substance are used.
General requirements: 3-2 Risk control
- any batch of substance (after inactivation and/or For each of the potential extraneous agents identified by the
processing, if relevant) found to contain or suspected of risk assessment, and taking into account the proposed use of
containing any living extraneous agent shall be discarded the substance, the risk must be controlled by the use of one
or used only in exceptional and justified circumstances; or a combination of the followings measures:
to be accepted for use, further processing must be applied - placing restrictions on the source of the material and
that will ensure elimination and/or inactivation of the auditing this;
extraneous agent, and it shall then be demonstrated that
- using validated inactivation procedures;
the elimination and/or inactivation has been satisfactory;
- demonstrating the ability of a production step to remove
- any batch of substance that, as conc1uded from the risk or inactivate extraneous agents;
assessment, may induce an unacceptable detectable
immune response in the target species as a consequence of - testing for extraneous agents.
contamination with inactivated extraneous agents, must 4. Control measures
not be used for the manufacture of that particular
irnmunological veterinary medicinal producto 4-1 Source
All substances of animal origin used in the manufacture
3. Risk management (inc1uding blending) of irnmunological veterinary medicinal
No single measure or combination of mea sures can guarantee products must be from a known and documented source
the safety of the use of substances of animal origin, but they (inc1uding species of origin and country of origin of source
can reduce the risk from such use. It is therefore necessary animals and tissues).
for the manufacturer of immunological veterinary medicinal 4-2 Preparation
products to take account of this when choosing a substance Substances of animal origin are prepared fram a
of animal origin to use in manufacture, and to conduct a risk homogeneous bulk designated with a batch number. A batch
assessment, taking into account the origin of the substance may contain substances derived from as many animals as
and the manufacturing steps applied to it. desired but once defined and given a batch number, the
In addition, risk management procedures must be applied. batch is not added to or contaminated in any way.
Any residual risk must be evaluated in relation to the The production method used to prepare the substance of
potential benefits derived from the use of the substance for animal origin from the raw material may contribute to the
the manufacture of the irnmunological veterinary medicinal removal and/or inactivation of extraneous agents (see section
producto 4-3).
3-1 Risk assessment 4-3 Inactivation andlor other processing steps lor
The risk assessment must take account of the animal diseases removal 01 extraneous agents
occurring in the country of origin of the animals used as a The inactivation procedure andlor other processing steps
source of the substance, the potential infectious diseases chosen shall have been validated and shown to be capable of
occurring in the source species and the likely infectivity in the reducing the titre of potential extraneous agents described
source organ or tissue. From this information, as part of the below in the substance concerned by a factor of at least 106 •
risk assessment, a list can be prepared of the extraneous If this reduction in titre cannot be shown experimentally, a
agents that may be present in the substance. maximum pre-treatment titre of the extraneous agent must
The risk of contamination of the substance and the resultant be set, taking into account the reduction in titre afIorded by
immunological veterinary medicinal product with living the inactivationlprocessing step and inc1uding a safety margin
A16 Appendix XV
Safety of 1 administration of an overdose An overdose Special requirements for live vaccines The following
of the product is administered by each recommended route laboratory tests must also be carried out with live vaccines.
of administration to animals of the categories of the target a) Spread 01 the vaccine strain
species which are expected to be the most sensitive, such as Spread of the vaccine strain from vaccinated to unvaccinated
animals of the youngest age and pregnant animals, if target animals is investigated using the recommended route
appropriate. The overdose normally consists of 10 doses of a of administration most likely to result in spread. Moreover, it
live vaccine or 2 doses of an inactivated product or an may be necessary to investigate the safety of spread to non-
immunoserum. For freeze-dried live vaccines, the 10 d9ses target species that could be highly susceptible to a live
shall be reconstituted in a suitable volume of diluent for the vaccine strain. An assessment must be made of how many
test. The animals are observed and examined at least daily animal-to-animal passages are likely to be sustainable under
for signs of local and systemic reactions. Other objective normal circumstances together with an assessment of the
criteria are recorded, such as body temperature (for '. likely consequences.
mammals) and performance measurements. The animals are
observed and examined for at least 14 days after
b) Dissemination in vaccinated animal
administration. If the vaccine is intended for use in pregnant Faeces, urine, milk, eggs, oral, nasal and other secretions
animals, carry out the test in these animal s at the time for shall be tested for the presence of the organismo Moreover,
which use is not contra-indicated, and extend the studies may be required of the dissemination of the vaccine
observation period at least until parturtition. The animals are strain in the body, with particular anention being paid to the
observed and effects on gestation or the offspring are predilection sites for replication of the organismo In the case
recorded. In exceptional circumstances, notably for of live vaccines for well-established zoonotic diseases for
immunosera, where there is evidence that an overdose is not food-producing animals, these studies are obligatory.
appropriate and an overdose test is not performed, a c1ear e) Increase in virulence
warning of the potential dangers of overdosing must be Use material from the passage level that is likely to be most
contained in the product literature. virulent for the target species between the master seed lot
Safety of the repeated administration of 1 dose and the final product. The animals used are of an age
Repeated administration of 1 dose may be required to reveal suitable for recovery of the virus and the animals in all
any adverse effects induced by such administration. These groups are of this age at the time of inoculation. The initial
tests are particularly important where the product, notably vaccination is carried out using the recommended route of
an immunoserum, may be administered on several occasions administration most likely to lead to reversion to virulence.
over a relativelyshort space of time. These tests are carried After this, not fewer than 5 further serial passages through
out on the most sensitive categories of the target species, animals of the target species are undertaken. The passages
using each recommended route of administration. are undertaken by the route of administration most likely to
The number of administrations must be not less than the lead to reversion to virulence. If the properties of the virus
maximum number recommended; for vaccines, this shall allow sequential passage to 5 groups via natural spreading,
take account of the number of administrations for primary this method may be used, otherwise passage as described in
vaccination and the 1st re-vaccination; for immunosera, it each monograph is carried out and the maximally passaged
shall take account of the number of administrations required virus that has been recovered is tested for increase in
for treatment. The interval between administrations shall be virulence. N ot fewer than 2 animals are used for each
suitable (e.g. period of risk or required for treatment) and passage. At each passage, the presence of living vaccine-
appropriate to the recommendations of use. Although, for derived organisms in the material used for passage is
convenience, as far as vaccines are concerned, a shorter demonstrated. The safety of material from the highest
interval may be used in the study than that recommended in successful passage is compared with that of unpassaged
the field, an interval of at least 14 days must be allowed material.
between administrations for the development of any For particular virus es, a monograph may require more
hypersensitivity reaction. For immunosera, however, passages in more animals if there is an indication from
administratian shall follow the recommended schedule. available data that this is relevant. At least the final passage is
The animals are observed and examined at least daily for at carried out using animals most appropriate to the potential
least 14 days after the last administration for signs of risk being assessed.
systemic and local reactions. Other objective criteria are d) Biological properties 01 the vaccine strain
recorded, such as body temperature and performance
Other tests may be necessary to determine as precisely as
measurements.
possible the intrinsic biological properties of the vaccine
Residues In the case of live vaccines for well-established strain (for example, neurotropism). For vector vaccines,
zoonotic diseases, the determination of residual vaccine evaluation is made of the risk of changing the tropism or
organisms at the injection site may be required, in addition virulence of the strain and where necessary specific tests are
to the studies of dissemination described below. carried out. Such tests are systematically carried out where
Adverse effects on immunological functions Where the the product of a foreign gene is incorporated into the strain
product might adversely affect the immune response of the as a structural protein.
animal to which the product is administered or of its e) Recombination or genomic reassortment 01 strain
progeny, suitable tests on the immunological functions are
The probability of recombination or genomic reassortment
carried out.
with field or other strains shall be considered.
Adverse effects from interactions Studies are
B. FIELD STUDIES
undertaken to show a lack of adverse effect on the safety of
the product when simultaneous administration is Results from laboratory studies shall normally be
recommended or where administration of the product is supplemented with supportive data from field studies.
recommended as part of a schedule of administration of Provided that laboratory tests have adequately assessed the
products within a short period of time. safety and efficacy of a product under experimental
A18 Appendix XV
conditions using vaccines of maximum and minimum titre or that there is protection from infection this must be
potency respectively, a single batch of product may be used demonstrated using re-isolation techniques. If more than one
to assess both safety and efficacy under field conditions. c1aim is made, supporting evidence for each c1aim is
In these cases, a typical routine batch of intermediate titre or required.
potency may be used. Vaccines
For food-producing mammals, the studies inc1ude The influence of passively acquired and matemally derived
measurement of the body temperatures of a sufficient antibodies on the efficacy of a vaccine is adequately
number of animals, before and after administration of the evaluated. Any c1aims, stated or implied, regarding onset and
product; for other mammals, such measurements are carried duration of protection shall be supported by data from trials.
out if the laboratory studies indicate that there might be a Claims related to duration ofirnmunity are supported by
problem. The size and persistence of any local reaction and evidence of protection. The test model described under
the proportion of animal s showing local or systemic reactions Irnmunogenicity and/or Potency is not necessarily used to
are recorded. Perfonnance measurements are made, where support c1aims regarding the duration of irnmunity afforded
appropriate. by a vaccine.
Perfonnance mea sures for broilers inc1ude week1y mortality, The efficacy of each of the components of multivalent and
feed conversion ratios, age at slaughter and weight, down combined vaccines shall be demonstrated using the combined
grading and rejects at the processing planto For vaccines for vaccine.
use in laying birds or in birds which may be maintained to
Irnmunosera
lay, the effect of the vaccine on laying perfonnance and
hatchability is investigated, as appropriate. Particular attention must be paid to providing supporting
data for the efficacy of the regime that is to be
c. ECOTOXICITY
recommended. For example, if it is recommended that the
An assessment is made of the potential harrnful effects of the immunoserum needs only to be administered once to achieve
product for the environment and any necessary precautionary a prophylactic or therapeutic effect then this must be
measures to reduce such risks are identified. The likely demonstrated. Any c1aims, stated or implied, regarding onset
degree of exposure of the environment to the product is
and duration of protection or therapeutic effect must be
assessed taking into account: the target species and mode of
supported by data from trials. For example, the duration of
administration; excretion of the product; disposal of unused the protection afforded by a prophylactic dos e of an
producto If these faCtors indicate that there will be significant antiserum must be studied so that appropriate guidance for
exposure of the environment to the product, the potential the user can be given on the label.
ecotoxicity is evaluated taking into account the properties of
the producto Studies of immunological compatibility are undertaken when
simultaneous administration is recommended or where it is a
part of a usual administration schedule. Wherever a product
is recommended as part of an administration scheme, the
K (Vet) 2. Evaluation of Efficacy of priming or booster effect or the contribution of the product
to the efficacy of the scheme as a whole is demonstrated.
Veterinary Vaccines and Immunosera LABORATORY TESTS
(Ph. Eur. general text 5.2. 7) In principIe, demonstration of efficacy is undertaken under
The tenn 'product' means either a vaccine or an well-controlled laboratory conditions by challenge of the
immunoserum throughout the texto target animal under the recommended conditions of use.
During development of the product, tests are carried out to In so far as possible, the conditions under which the
demonstrate that the product is efficacious when challenge is carried out shall mimic the natural conditions for
administered by each of the recommended routes and infection, for example with regard to the amount of challenge
methods of administration and using the recommended organism and the route of administration of the challenge.
schedule to animals of each species and category for which Vaccines
use of the product is to be recommended. The type of
Unless otherwise justified, challenge is carried out using a
efficacy testing to be carried out varies considerably
strain different from the one used in the production of the
depending on the particular type of producto
vaccine.
As part of tests carried out during development to establish
If possible, the irnmune mechanism (cell-mediated/humoral,
efficacy, the tests described in the Production section of a
local/general, c1asses of irnmunoglobulin) that is initiated
monograph may be carried out; the following must be taken
after the administration of the vaccine tolatget animals shall
into account.
be detennined.
The dose to be used is that quantity of the product to be
Irnmunosera
recommended for use and containing the minimum titre or
potency e~pected at the end of the period of validity. Data are provided from measurements of the antibody leve1s
achieved in the target species after administration of the
For live vaccines, use vaccine containing viruslbacteria at the
produd~ as recommended. Where suitable published data
most attenuated passage level that will be present in a batch
exist, references are provided to relevant published literature
ofvaccine.
on protective antibody leve1s and challenge studies are
For irnmunosera, if appropriate, the dos e tested also contains avoided.
minimum quantities of irnmunoglobulin or garnmaglobulin
Where challenges are required, these can be given before or
and/or total protein.
after administration of the product, in accordance with the
c
The efficacy evidence must support all the c1aims being indications and specific c1aims to be made.
made. For example, c1aims for protection against respiratory
disease must be supported at least by evidence of protection
from c1inical signs of respiratory disease. Where it is c1aimed
Appendix XV A19
INCUBATION CONDITIONS
Appendix XVI Incubate liquid media in tightly stoppered containers at
35-38 oC. Incubate solid media in microaerophilic conditions
(nitrogen containing 5-10 per cent of carbon dioxide and
B (Vet) 3. Test for Absence of sufficient humidity to prevent desiccation of the agar surface)
Mycoplasmas at 35-38 oC.
(Ph. Eur. method 2.6.7 as applied to vaccines for human use) NUTRITIVE PROPERTIES
Where the test for mycoplasmas is prescribed for a master Carry out the test for nutritive properties for each new batch of
cell bank, for a working cell bank, for a virus seed lot or for medium. Inoculate the chosen media with the appropriate test
control cells, both the culture method and the indic;ator cell micro-organisms; use not more than 100 CFU (colony-
culture method are used. Where the test for mycoplasmas is forming units) per 60 mm diameter plate containing 9 mL of
prescribed for a virus harvest, for a bulk vaccine or for the solid medium and per 100 mL container of liquid medium;
finallot (batch), the culture method is used. The indicator use a separate plate and container for each species of micro-
cell culture method may also be used, where necessary, for organismo Incubate the media and make sub cultures from
screening of media. 0.2 mL of liquid medium to solid medium at the specified
intervals (see be10w under Test for mycoplasmas in the
Nuc1eic acid amplification techniques (NAT) may be used as
product to be examined). The solid medium complies with
an alternative to one or both of the other methods after
the test if adequate growth is found for each test micro-
suitable validation.
organism (growth obtained do es not differ by a factor greater
Culture method than 5 from the value calculated with respect to the
inoculum). The liquid medium complies with the test if
CHOICE OF CULTURE MEDIA
growth on agar plates subcultured from the broth is found
The test is carried out using a sufficient number of both solid for at least 1 sub culture for each test micro-organismo
and liquid media to ensure growth in the chosen incubation
INHIBITORY SUBSTANCES
conditions of small numbers of mycoplasmas that may be
present in the product to be examined. Liquid media must The test for inhibitory substances is carried out once for a
contain phenol red. The range of media chosen is shown to given product and is repeated whenever there is a change in
have satisfactory nutritive properties for at least the micro- production method that may affect the detection of
organisms shown be1ow. The nutritive properties of each new mycoplasmas.
batch of medÍllm are verified for the appropriate micro- To demonstrate absence of inhibitory substances, carry out
organisms in the listo When testing for mycoplasmas in the the test for nutritive properties in the presence and absence
product to be examined, at least 1 of the following species of the product to be examined. If growth of a test micro-
will be inc1uded as a positive control: organism occurs more than 1 sub culture sooner in the
- Acholeplasma laidlawii (vaccines for human and veterinary absence of the product to be examined than in its presence,
use where an antibiotic has been used during production); or if plates directly inoculated with the product to be
examined have fewer than 1/5 of the number of colonies of
- Mycoplasma gallisepticum (where avían material has been
those inoculated without the product to be examined,
used during production or where the vaccine is intended
inhibitory substances are present and they must be
for use in poultry);
neutralised or their effect otherwise countered, for example
- Mycoplasma hyorhinis (non-avian veterinary vaccines); by passage in substrates not containing inhibitors or dilution
- Mycoplasma orale (vaccines for human and veterinary use); in a larger volume of medium before the test. If dilution is
- Mycoplasma pneumoniae (vaccines for human .use) or other used, larger medium volumes may be used or the inoculum
suitable species of D-glucose fermenter such as volume may be divíded among several100 mL fiasks.
Mycoplasma fermentans; The effectiveness of the neutralisation or other process is
- Mycoplasma synoviae (where avian material has been used checked by repeating the test for inhibitory substances after
during production or where the vaccine is intended for neutralisation.
use in poultry). TEST FOR MYCOPLASMAS IN THE PRODUCT TO BE
The test strains are field isolates having undergone a limited EXAMINED
number of sub cultures (not more than 15), and are stored Inoculate 10 mL of the product to be examined per 100 mL
frozen or freeze-dried. After c1oning, the strains are identified of each liquid medium. If it has been found that a significant
as being of the required species by comparison with type pH change occurs upon the addition of the product to be
cultures, for example: examined, the liquid medium is restored to its original pH
value by the addition of a solution of either sodium
A. laidlawii NCTC10116 CIP 75.27 ATCC 23206 hydroxide or hydrochloric acid. Inoculate 0.2 mL of the
M. gallisepticum NCTC10115 CIP 104967 ATCC 19610 product to be examined on each plate of each solid medium.
M. fermentans NCTC10117 CIP 105680 ATCC 19989 Incubate liquid media for 20-21 days. Incubate solid media
M. hyorhinis NCTC10130 CIP 104968 ATCC 17981 for not less than 14 days, except those corresponding to the
M. orale NCTC10112 CIP 104969 ATCC 23714 20-21 day subculture, which are incubated for 7 days. At the
M. pneumoniae NCTC 10119 CIP 103766 ATCC 15531 same time incubate an uninoculated 100 mL portion of each
M. synoviae NCTC 10124 CIP 104970 ATCC 25204 liquid medium and agar plates, as a negative control.
Acholeplasma laidlawii BRP, Mycoplasma fermentans BRP, On days 2-4 after inoculatíon, sub culture each liquid
Mycoplasma hyorhinis BRP, Mycoplasma orale BRP and medium by inoculating 0.2 mL on at least 1 plate of each
Mycoplasma synoviae BRP are suitable for use as low-passage solid medium. Repeat the procedure between the 6th and 8th
reference strains. days, again between the 13th and 15th days and again
between the 19th and 21 st days of the test. Observe the liquid
media every 2 or 3 days and if a colour change occurs,
A22 Appendix XVI
(3) Agar~ purified The indicator cells must be subcultured without an antibiotic
A highly refined agar for use in microbiology and before use in the test.
immunology, prepared by an ion-exchange procedure that TEST METHOD
results in a product having superior purity, clarity and gel 1. Seed the indicator cell culture at a suitable density (for
strength. It contains about: example, 2 x 104 to 2 x 105 cells/mL, 4 x 10 3 to 2.5
Water 12.2 per cent x 104 cells/cm2) that will yield confiuence after 3 days
Ash 1.5 per cent of growth. Inoculate 1 mL of the product to be
Acid-insoluble ash 0.2per cent examined into the cell culture vessel and incubate at
Chlorine O 35-38 oC.
Phosphate (calculated as P 205) 0:3 per cent 2. After at least 3 days of incubation, when the cells have
Total nitrogen 0.\3 per cent grown to confiuence, make a sub culture on cover slips in
Copper 8ppm suitable containers or on sorne other surface(for
Iron 170 ppm example, chambered slides) suitable for the test
Calcium 0.28 per cent procedure. Seed the cells at low density so that they
Magnesium 0.3 2 per cent reach 50 per cent confiuence after 3-5 days of
(4) Hanks' balanced salt solution (modijied) incubation. Complete confiuence impairs visualisation of
mycoplasmas after staining and must be avoided.
Sodium chloride 6.4 g
Potassium chloride 0.32 g 3. Remove the medium and rinse the indicator cells with
Magnesium sulfate heptahydrate 0.08 g phosphate buffered saline pH 7. 4 R~ then add a suitable
Magnesium chloride hexahydrate 0.08 g fixing solution (a freshly prepared mixture of 1 volume
Calcium chloride, anhydrous 0.112 g of glacial acetic acid R and 3 volumes of methanol R is
Disodium hydrogen phosphate dihydrate 0.0596 g suitable when bisbenzimide R is used for staining).
Potassium dihydrogen phosphate, anhydrous 0.048 g 4. Remove the fixing solution and wash the cells with sterile
Distilled water to 800 mL water R. Dry the slides completely if they are to be
stained more than 1 h later (particular care is needed for
(5) Brain heart infusion
staining of slides after drying owing to artefacts that may
Calf-brain infusion 200 g be produced).
Beef-heart infusion 250 g
5. Add a suitable DNA stain and allow to stand for a
Proteos e peptone 10 g
suitable time (bisbenzimide working solution R and a
Glucose monohydrate 2g
standing time of 10 min are suitable).
Sodium chloride 5g
Disodium hydrogen phosphate, anhydrous 2.5 g 6. Remove the stain and rinse the monolayer with water R.
Distilled water to 1000 mL 7. Mount each coverslip, where applicable (a mixture of
equal volumes of glycerol R and phosphate-citrate buffer
(6) PPLO broth
solution pH 5.5 R is suitable for mounting). Examine by
Beef-heart infusion 50 g fiuorescence (for bisbenzimide stain a 330 nm/380 nm
Peptone 10 g excitation filter and an LP 440 nm barrier filter are
Sodium chloride 5g suitable) at 400 x magnification or greater.
Distilled water to 1000 mL
8. Compare the microscopic appearance of the test cultures
with that of the negative and positive controls,
Indicator cell culture method
examining for extranuc1ear fiuorescence. Mycoplasmas
Cell cultures are stained with a fiuorescent dye that binds to produce pinpoints or filaments over the indicator cell
DNA. Mycoplasmas are detected by their characteristic cytoplasm. They may also produce pinpoints and
particulate or filamentous pattem of fiuorescence on the cell filaments in the intercellular spaces. Multiple
surface and, if contamination is heavy, in surrounding areas. microscopic fields are examined according to the
Mitochondria in the cytoplasm may be stained but are readily protocol established during validation.
distinguished from mycoplasmas.
INTERPRET ATION OF RESULTS
If for viral suspensions the interpretation of results is affected The product to be examined complies with the test if
by marked cytopathic effects, the virus may be neutralised fiuorescence typical of mycoplasmas is not presento The test
using a specific antiserum that has no inhibitory effects on is invalid if the positive controls do not show fiuorescence
mycoplasmas or a cell culture substrate that do es not allow typical of mycoplasmas. The test is invalid if the negative
growth of the virus may be used. To demonstrate the controls show fiuorescence typical of mycoplasmas.
absence of inhibitory effects of serum, carry out the positive
control tests in the presence and absence of the antiserum. Nuc1eic acid amplification techniques (NAT)
VERIFICATION OF THE SUBSTRATE NAT (2.6.21) may be used for detection ofmycoplasmas by
Use Vero cells or another cell culture (for example, the amplification of nucleic acids extracted from a test sample
production cell line) that is equivalent in effectiveness for with specific primers that reveal the presence of the target
detecting mycoplasmas. Test the effectiveness of the cells to nucleic acid. NAT indicate the presence of a particular
be used by applying the procedure shown below and nucleic acid sequence and not necessarily the presence of
inoculating not more than 100 CFU or CFU-like micro- viable mycoplasmas. A number of different techniques are
organisms of suitable reference strains of M. hyorhinis and M. available. This general chapter does not prescribe a particular
orale. The following strains have been found to be suitable: method for the test. The procedure applied must be
M. hyorhinis ATCC 29052 validated as described, taking account of the guidelines
M. orale NCTC 10112 CIP 104969 ATCC 23714 presented at the end of this section. Where a commercial kit
is used, certain elements of the validation may be carried out
The cells are suitable if both reference strains are detected.
A24 Appendix XVI
by the manufacturer and information provided to the user but does not necessarily represent the same matrix as the test
but it must be remembered that full information on the article.
primers may not be available and that production of the kit INTERPRETATION OF RESULTS
may be modified or discontinued. The primers used may also amplify non-mycoplasmal
NATare applied where prescribed in a monograph. They bacterial nucleic acid, leading to false positive results.
may also be used instead of the culture method and the Procedures are established at the time of validation for
indicator cell culture method after suitable validation. dealing with confirmation of positive results, where necessary.
Direct NAT Can be applied in the presence of cytotoxic The following section is published for information.
material and where a rapid method is needed.
Cell-culture enrichment lollowed by NAT The test Validation of nuc1eic acid amplification techniques
sample and a suitable cell substrate (as described under the (NAT) for the detection of mycoplasmas : guidelines
indicator cell-culture method) are cultured together for a 1 SCOPE
suitable period; the nucleic acids are then extracted from Nucleic acid amplification techniques (NAT) are either
cells and supernatant and used for detection by NAT. qualitative or quantitative tests for the presence of nucleic
VALIDATION acid. For the detection of mycoplasma contamination of
Reference standards are required at various stages during various samples such as vaccines and cell substrates,
validation and for use as controls during routine application qualitative tests are adequate and may be considered to be
of the test. The reference standards may be mycoplasmas or limit tests.
nucleic acids. These guidelines describe methods to validate qualitative
For validation of thelimit of detection, the following species nucleic acid amplification analytical procedures for assessing
represent an optimal selection in terms of the frequency of mycoplasma contamination. They may also be applicable for
occurrence as contaminants and phylogenetic relationships: real-time NAT used as limit tests for the control qf
contaminants.
- A. laidlawiij
The 2 characteristics regarded as the most important for
- M. fermentansj
validation of the analytical procedure are the specificity and
- M. hyorhinis (where cell-culture enrichment is used, a the detection limito In addition, the robustness of the
fastidious strain such as ATCC 29052 is included); analytical procedure should be evaluated.
- M. oralej For the purpose of this document, an analytical procedure is
- M. pneumoniae or M. gallisepticum; defined as the complete procedure from extraction of nucleic
- M. synoviae (where there is use of or exposure to avian acid to detection of the amplified products.
material during production); Where commercial kits are used for part or all of the
- Mycoplasma arginini; analytical procedure, documented validation points already
- Spiroplasma citri (where there is use of or exposure to covered by the kit manufacturer can replace validation by the
insect or plant material during production). user. Nevertheless, the performance of the kit with respect to
its intended use has to be demonstrated by the user
Demonstration of specificity requires the use of a suitable
(e.g. detection limit, robustness, cross-detection of other
range of bacterial species other than mycoplasmas. Bacterial
classes of bacteria).
genera with close phylogenetic relation to mycoplasmas are
most appropriate for this validation; these include Clostridium, NAT may be used as:
Lactobacillus and Streptococcus. - a complementary test (for example, for cytotoxic viral
Comparability studies for use 01 NAT as an alternative suspensions) or for in-process control purposes;
method For each mycoplasma test species: - an alternative method to replace an official method
- as an alternative to the culture method: the NAT test ,(indicator cell culture method or culture method).
system must be shown to detect 10 CFU/mL; These guidelines will thus separate these 2 objectives by
- as an alternative to the indicator cell culture method: the presenting first a guideline for the validation of the NAT
NAT test system must be shown to detect 100 CFU/mL; themselves, and second, a guideline for a comparability study
between NAT and official methods.
or an equivalent limit of detection in terms of the number of
copies of mycoplasma nucleic acid in the test sample (using 2 GUIDELINE FOR MYCOPLASMA NAT VALIDATION
suitable reference standards of mycoplasma nucleic acid). 3 parameters should be evaluated: specificity, detection limit
CONTROLS
and robustness.
Internal controls Internal controls are necessary for 2-1 Specificity Specificity is the ability to unequivocally
routine verification of absence of inhibition. The internal assess target nucleic acid in the presence of components that
control may contain the primer binding-site, or sorne other may be expected to be presento
suitable sequence may be used. It is preferably added to the The specificity of NAT is dependent on the choice of
test materiaÍ before isolating the nucleic acid and therefore primers, the choice of probe (for analysis of the final
acts as an overall control (extraction, reverse transcription, product) ~nd the stringency of the test conditions (for both
amplification, detection). the amplification and detection steps).
External controls The external positive control contains a The ability of the NAT to detect a large panel of
defined number of target-sequence copies or CFUs from 1 mycopl~sma species will depend on the choice of primers,
or more suitable species of mycoplasma chosen from those probes and method parameters. This ability should be
used during validation of the test conditions. 1 of the demonstrated using characterised reference panels
positive controls is set close to the positive cut-off point to (e.g. reference strains provided by the EDQM). Since NAT
demonstrate that the expected sensitivity is achieved. systems are usually based on a mix of primers, the theoretical
The external negative control contains no target sequence analysis of primers and probes by comparison with databases
Appendix XVI A25
is not recommended, because interpretation of the results (i.e. the highest dilution giving a positive signal). The range
may be quite complex and may not reflect the experimental of dilutions can then be chosen around the predetermined
results. preliminary cut-off point. The concentration of mycoplasmas
Moreover, as it is likely that the primers will detect other (CFUs or copies) that can be detected in 95 per cent of test
bacterial species, the potential cross-detection should be runs can then be ca1culated using an appropriate statistical
documented in the validation study. Bacterial genera such as evaluation.
gram-positive bacteria with close phylogenetic relation to These results may also serve to evaluate the variability of the
mycoplasmas are most appropriate for this validation; ~these analytical procedure.
include Clostridium) Lactobacillus and Streptococcus. However, 2-3 Robustness The robustness of an analytical procedure
this is not an exhaustive list and species to be tested will is a measure of its capacity to remain unaffected by small
depend on the theoretical ability (based on primers/vrobes but deliberate variations in method parameters, and provides
sequences) of the NAT system to detect such other species. an indication of its reliability during normal usage.
Based on the results from this validation of the specificity, if The evaluation of robustness should be considered during
a gap in the specificity of the method is identified (such as the development phase. It should show the reliability of the
detection of non-mycoplasmal bacterial nucleic acid), an analytical procedure with respect to deliberate variations in
appropriate strategy must be proposed in the validation study method parameters. For NAT, small variations in the
to allow interpreÚltion of positive results on a routine basis. method parameters can be crucial. However, the robustness
F or example, a second test may be performed using an of the method can be demonstrated during its development
altemative method without this specificity gap or using an when small variations in the concentrations of reagents
official method. (e.g. MgCI2, primers or deoxyribonucleotides) are tested.
2-2 Detection limito The detection limit of an individual Modifications of extraction kits or extraction procedures as
analytical procedure is the lowest amount of target nucleic well as different thermal cycler types may also be evaluated.
acid in a sample that can be detected but not necessarily Finally, robustness of the method can be evaluated through
quantitated as an exact value. collaborative studies.
For establishment of the detection limit, a positive cut-off 3 GUIDELINE FOR COMPARABILITY STUDY
point should be determined for the nucleic acid amplification NAT may be used instead of official methods (indicator cell
analytical procedure. The positive cut-off point (as defined in culture method and/or culture method). In this case a
general chapter 2.6.21) is the minimum number of target comparability study should be carried out. This comparability
sequence copies per volume of sample that can be detected study should include mainly a comparison of the respective
in 95 per cent of test runs. This positive cut-off point is detection limits of the altemative method and official
influenced by the distribution of mycoplasmal genomes in the methods. However, specificity (mycoplasma panel detected,
individual samples being tested and by factors such as putative false positive results) should also be considered.
enzyme efficiency, and can result in different 95 per cent cut-
For the detection limit, acceptability criteria are defined as
off values for individual analytical test runS.
follows:
To determine the positive cut-off point, a dilution series of
- if the alternative method is proposed to replace the culture
characterised and calibrated (either in CFUs or nucleic acid
method, the NAT system must be shown to detect 10
copies) in-house working strains or EDQM standards should
CFU/mL for each mycoplasma test species described in
be tested on different days to examine variation between test
paragraph 2-2;
runs.
- if the alternative method is proposed to replace the
For validation of the limit of detection, the following species
indicator cell culture method, the NAT system must be
represent an optimal selection in terms of the frequency of
shown to detect 100 CFU/mL for each mycoplasma test
occurrence as contaminants and phylogenetic relationships:
species described in paragraph 2-2
- A. laidlawii;
For both cases, suitable standards calibrated for the number
- M. fermentans; of nucleic acid copies and the number of CFUs may be used
- M. hyorhinis; for establishing that these acceptability criteria are reached.
- M. orale; The relation between CFUs and nucleic acid copies for the
- M. pneumoniae or M. gallisepticum; reference preparations should be previously established to
compare the performance of the altemative NAT method
- M. synoviae (where there is use of or exposure to avian
with the performance of the official methods.
material during production);
1 of the following 2 strategies can be used to perform this
- M. arginini;
comparability study:
- S. citri (where there is use of or exposure to insect or
- perform the NAT alternative method in parallel with the
plant material during production).
official method(s) to evaluate simultaneously the detection
For each strain, at least 3 independent 10-fold dilution series limit of both methods using the same samples of
should be tested, with a sufficient number of replicates at calibrated strains;
each dilution to give a total number of 24 test results for
- compare the performance of the NAT altemative method
each dilution, to enable a statistical analysis of the results.
using previously obtained data from official method
For example, a laboratory may test 3 dilution series on validation. In this case, calibration of standards used for
different days with 8 replicates for each dilution, 4 dilution both validations as well as their stabilities should be
series on different days with 6 replicates for each dilution, or documented carefully.
6 dilution series on different days with 4 replicates for each
Comparability study reports should describe all the validation
dilution. In order to keep the number of dilutions at a
elements described in section 2 (specificity, limit of detection
manageable level, a preliminary test should be performed to
and variability, as well as robustness) in order to assess all
obtain a preliminary value for the positive cut-off point
A26 Appendix XVI
the advantages andlor disadvantages of the altemative NAT - group 2: 0.2 mL onto me chorio-allantoic membrane of
method compared to official methods. each 9- to 11-day-old embryonated egg;
- group 3: 0.2 mL into me yolk sac of each 5- to 6-day-old
embryonated egg.
Candle me eggs in groups 1 and 2 dai1y for 7 days and the
8 (Vet) 4. Avian Viral Vaccines: Tests for eggs in group 3 daily for 12 days. Discard embryos mat die
Extraneous Agents in Seed Lots during me first 24 h as non-specific deaths; the test is not
valid unless at least 6 embryos in each group survive beyond
(Ph. Eur. method 2.6.24) me first 24 h after inoculation. Examine macroscopically for
abnormalities all embryos that die more man 24 h after
General provisions
inoculation, or mat survive the incubation periodo Examine
a) In me following tests, chickens and/or chicken material also me chorio-allantoic membranes of these eggs for any
such as eggs and cell cultures shall be derived from abnormality and test me allantoic fluids for the presence of
chicken flocks free from specified pathogens (SPF) haemagglutinating agents.
(5.2.2).
Carry out a further embryo passage. Pool separately material
b) Cell cultures for the testing of extraneous agents comply from live and from the dead and abnormal embryos.
wim the requirements for the master cell seed of chapter Inoculate each pool into 10 eggs for each route as described
5.2.4. Cell cultures for the production of veterinary vaccines) above, chorio-allantoic membrane material being inoculated
wim the exception of me karyotype test and me onto chorio-allantoic membranes, allantoic fluids into the
tumorigenicity test, which do not have to be carried out. allantoic cavity and embryo material into the yolk saco
c) In tests using cell cultures, precise specifications are For eggs inoculated by the allantoic and chorio-allantoic
given for the number of replicates, monolayer surface routes, candle the eggs daily for 7 days, proceeding and
areas and minimum survival rate of the cultures. examining the material as described above. For eggs
Alternative numbers of replicates and cell surface areas inoculated by the yolk sac route, candle me eggs daily for
are possible as well, provided mat a minimum of 2 12 days, proceeding and examining me material as described
replicates are used, the total surface area and the total above.
volume of test substance applied are not less than mat The seed lot complies with me test if no test embryo shows
prescribed here and the survival rate requirements are macroscopic abnormalities or dies from causes attributable to
adapted accordingly. the seed lot and if examination of me chorio-allantoic
d) For a freeze-dried preparation, reconstitute using a membranes and testing of the allantoic fluids show no
suitable liquido Unless otherwise stated or justified, me evidence of me presence of any extraneous agent.
test substance must contain a quantity of virus
equivalent to at least 10 doses of vaccine in 0.1 mL of 2. Test in chicken kidney cells
inoculum. Prepare 7 monolayers of chicken kidney cells, each
e) If the virus of the seed lot would interfere with the monolayer having an area of about 25 cm2 • Maintain 2
conduct and sensitivity of the test, neutralise the virus in monolayers as negative controls and treat these in the same
the preparation wim a monospecific antiserum. way as the 5 monolayers inoculated wim the test substance,
f) Monospecific antiserum and serum of avian origin used as described below. Remove the culture medium when me
for cell culture or any other purpose, in any of these cells reach confluence. Inoculate 0.1 mL of the test substance
tests, shall be free from antibodies against and free from onto each of the 5 monolayers. Allow adsorption for 1 h, add
inhibitory effects on me organisms listed hereafter under culture medium and incubate the cultures for a total of at
7 Antibody specifications for sera used in extraneous least 21 days, subculturing at 4- to 7-day intervals. Each
agents testing. passage is made with pooled cells and fluids from all 5
monolayers after carrying out a freeze-thaw cyc1e. Inoculate
g) Where specified in a monograph or otherwise justified, if
0.1 mL of pooled material onto each of 5 recently prepared
neutralisation of the virus of the seed lot is required but
difficult to achieve, the in vitro tests described below are monolayers of about 25 cm2 each, at each passage. For the
adapted, as required, to provide me necessary guarantees last passage, grow the cells also on a suitable substrate so as
of freedom from contamination with an extraneous to obtain an area of about 10 cm2 of cells from each of the
monolayers for test A. The test is not valid if les s than
agent.
80 per cent of the monolayers survive after any passage.
h) Other types of tests than mose indicated may be used
provided they are at least as sensitive as mose indicated Examine microscopically all the cell cultures frequently
and of appropriate specificity. Nuc1eic acid amplification throughout me entire incubation period for any signs of
cytopathic effect or other evidence of the presence of
techniques (2.6.21) give specific detection for many
contaminating agents in the test substance. At the end of me
agents and can be used after validation for sensitivity
and specificity. total incubation period, carry out the following procedures.
A. Fix and stain (wim Giemsa or haematoxylin and eosin)
1. Test for extraneous agents using embryonated aboJt 10 cm2 of confluent cells from each of the 5
hens'eggs monolayers. Examine the cells microscopically for any
Use a test substance, diluted if necessary, containing a cytopathic effect, inc1usion bodies, syncytial formation,
quantity of neutralised virus equivalent to at least 10 doses of or/other evidence of the presence of contaminating
vaccine in 0.2 mL of inoculum. Suitable antibiotics may be agents from the test substance.
added. Inoculate the test substance into 3 groups of B. Drain and wash about 25 cm2 of cells from each of me
10 embryonated hens' eggs as follows: 5 monolayers. Cover mese cells wim a 0.5 per cent
- group 1: 0.2 mL into the allantoic cavity of each suspension of washed chicken erythrocytes (using at least
9- to 11-day-old embryonated egg; 1 mL of suspension for each 5 cm2 of cells). Incubate
Appendix XVI A27
the cells at 4 oC for 20 min and then wash gently in Rernove the culture medium when the cells reach confiuence.
phosphate buffered saline pH 7.4. Examine the cells Inoculate 0.1 mL of the test substance onto each of 5 of the
microscopically for haemadsorption attributable to the monolayers. Allow adsorption for 1 h, and add culture
presence of a haemadsorbing agent in the test substance. medium. Inoculate 4 of the monolayers with avían
C. Test individual samples of the fiuids from each cell reticuloendotheliosis virus as positive controls (not more than
culture using chicken erythrocytes for haemagglutination 10 CCID so in 0.1 mL). Maintain 2 non-inoculated
attributable to the presence of a haemagglutinating agent monolayers as negative controls.
in the test substance. Incubate the cells for a total of at least 10 days, subculturing
The test is not valid if there are any signs of extraneous twice at 3- to 4-day intervals. The test is not valid if fewer
agents in the negative control cultures. The seed lot complies than 3 of the 4 positive controls or fewer than 4 of the 5 test
with the test if there is no evídence of the presence 6f any monolayers or neither of the 2 negative controls survive after
extraneous agent. any passage.
For the last subculture, grow the fibroblasts on a suitable
3. Test for avian leucosis virus es substrate so as to obtain an area of about 10 cm2 of
Prepare at least 13 replicate monolayers of either DF-1 cells confiuent fibroblasts from each of the original 11 rnonolayers
or primary or secondary chick embryo fibroblasts from the for the subsequent test: test about 10 cm2 of confiuent
tissues of 9- to 11-day-old embryos that are known to be fibroblasts derived from each of the original 11 monolayers
genetically susceptible to subgroups A, B and J of avían by immunostaining for the presence of avían
leucosis viruses and that support the growth of exogenous reticuloendotheliosis virus. The test is not valid if avían
but not endogenous avían leucosis viruses (cells from CIE reticuloendotheliosis virus is detected in fewer than 3 of the 4
strain chickens are suitable). Each replicate shall have an area positive control monolayers or in any of the negative control
of about 50 cm2 • monolayers, or if the results for both of the 2 negative
Remove the culture medium when the cells reach confiuence. control monolayers are inconc1usive. If the results for more
Inoculate 0.1 mL of the test substance onto each of 5 of the than 1 of the test monolayers are inconc1usive then further
replicate monolayers. Allow adsorption for 1 h, and add subcultures of reserved portions of the fibroblast monolayers
culture medium. Inoculate 2 of the replicate monolayers with shall be made and tested until an unequivocal result is
subgroup A avían leucosis virus (not more than 10 CCID so obtained.
in 0.1 mL), 2 with subgroup B avían leucosis virus (not more The seed lot complies with the test if there is no evídence of
than 10 CCIDso in 0.1 mL) and 2 with subgroup J avían the presence of avían reticuloendotheliosis virus.
leucosis virus (not more than 10 CCID so in 0.1 mL) as
5. Test for chicken anaemia virus
positive controls. Maintain not fewer than 2 non-inoculated
replicate monolayers as negative controls. Prepare eleven 20 mL suspensions of the MDCC-MSBI cell
Incubate the cells for a total of at least 9 days, subculturing line or another cellline of equivalent sensitivíty in 25 cm2
at 3- to 4-day intervals. Retain cells from each passage level cell culture fiasks containing about 5 x lOs cells/rnL.
and harvest the cells at the end of the total incubation Inoculate 0.1 mL of the test substance into each of 5 fiasks.
periodo Wash cells from each passage level from each Inoculate 4 of the suspensions with 10 CCID so chicken
replicate and resuspend the cells at 107 cells per millilitre in anaemia virus as positive controls. Maintain not fewer than 2
barbital-buffered saline for subsequent testing by a non-inoculated suspensions. Maintain all the cell cultures for
Complement Fixation for Avían Leucosis (COFAL) test or a total of at least 24 days, subculturing 8 times at 3- to 4-day
in phosphate buffered saline for testing by Enzyme-Linked intervals. During the subculturing the presence of chicken
Immunosorbent Assay (ELISA). Then, carry out 3 cyc1es of anaemia virus may be indicated by a metabolic colour change
freezing and thawing to release any group-specific antigen in the infected cultures, the culture fiuids becoming red in
and perform a COFAL test or an EUSA test on each extract comparison with the control cultures. Examine the cells
to detect group-specific avían leucosis antigen if presento microscopically for cytopathic effect. At this time or at the
end of the incubation period, centrifuge the cells from each
The test is not valid if group-specific antigen is detected in fiask at low speed and resuspend at about 106 cells/mL and
fewer than 5 of the 6 positive control replicate monolayers or place 25 IlL in each of 10 wells of a multi-well slide.
if a positive result is obtained in any of the negative control Examine the cells by immunostaining.
monolayers, or if the results for both of the 2 negative
control monolayers are inconc1usive. If the results for more The test is not valid if chicken anaemia virus is detected in
than 1 of the test replicate monolayers are inconc1usive, then fewer than 3 of the 4 positive controls or in any of the non-
further sub cultures of reserved portions of the fibroblast inoculated controls. If the results for more than 1 of the test
monolayers shall be made and tested until an unequivocal suspensions are inconclusive, then further sub cultures of
result is obtained. If a positive result is obtained for any of reserved portions of the test suspensions shall be made and
the test monolayers, then the presence of avían leucosis virus tested until an unequivocal result is obtained.
in the test substance has been detected. The seed lot complies with the test if there is no evidence of
The seed lot complies with the test if there is no evidence of the presence of chicken anaemia virus.
the presence of any avían leucosis virus. 6. Test for extraneous agents using chicks
4. Test for avian reticuloendotheliosis virus Inoculate each of at least 10 chicks with the equivalent of
100 doses of vaccine by the intramuscular route and with the
Prepare 11 monolayers of primary or secondary chick embryo equivalent of 10 doses by eye-drop. Chicks that are 2 weeks
fibroblasts from the tissues of 9- to 11-day old chick embryos of age are used in the test except that if the seed virus is
or duck embryo fibroblasts from the tissues of 13- to 14-day- pathogenic for birds of this age, older birds may be used, if
old embryos, each monolayer having an area of about required and justified. In exceptionaI cases, for inactivated
25 cm2 • vaccines, the virus may be neutralised by specific antiserum if
the seed virus is pathogenic for birds at the age of
A28 Appendix XVI
administration. Repeat these inoculations 2 weeks latero A test for freedom from turkey lympho-proliferative
Observe the chicks for a period of 5 weeks from the day of disease virus is carried out by intraperitoneal inoculatioIl
the first inoculation. No antimicrobial agents shall be of twenty 4-week-old turkey poults. Observe the poults
administered to the chicks during the test periodo The test is for 40 days. The test is not valid if more than
not valid if fewer than 80 per cent of the chicks survive to the 20 per cent of the poults die from non-specific causes.
end of the test periodo The seed lot complies with the test if sections of spleen
Collect serum from each chick at the end of the test periodo and thymus taken from 10 poults 2 weeks after
Test each serum sample for antibodies against each of the inoculation show no macroscopic or microscopic lesions
agents listed below (with the exception of the virus type of (other than those attributable to the seed lot virus) and
the seed lot) using one of the methods indicated for testing no poult dies from causes attributable to the seed loto
for the agent. C. Additional tests for duck extraneous agents
Clinical signs of disease in the chicks during the test period If the seed virus is of duck origin or was propagated in
(other than signs attributable to the virus of the seed lot) and duck substrates, tests for antibodies against the following
the detection of antibodies in the chicks after inoculation agents are also carried out.
(with the exception of antibodies to the virus of the seed lot),
are classed as evidence of the presence of an extraneous Agent Type oftest
agent in the seed loto
Chlamydia spp. EIA
It is recommended that sera from these birds is retained so
that additional testing may be carried out if requirements Duck and goose parvoviruses SN, EIA
change. Duck enteritis virus SN
A. Standard tests
Duck hepatitis virus type 1 SN
Avian orthoreoviruses IS, EIA Duck and goose parvovirus SN, EIA
Chicken anaemia virus IS, EIA, SN Coose haemorrhagic polyomavirus test in goslings shown below
or another suitable test
Egg drop syndrome virus HI,EIA
Avian infectious bursal disease virus Serotype 1: ACP, EIA, SN Inoculate subcutaneously the equivalent of at least 10
Serotype 2: SN doses to each of ten l-day-old susceptible goslings.
Influenza A virus ACP, EIA, HI Observe the goslings for 28 days. The test is not valid if
ACP
more than 20 per cent of the goslings die from non-
Marek's disease virus
specific causes. The seed virus complies with the test if
Newcastle disease virus HI, EIA no gosling die s from causes attributable to the seed loto
Turkey rhinotracheitis virus EIA
7. Antibody specifications for sera used in
Salmonella pullorum Agg extraneous agents testing
Agg: agglutination AH batches of serum to be used in extraneous agents testing,
ACP: agar gel precipitation either to neutralise the vaccine virus (seed lot or batch of
EIA: enzyme immunoassay (e.g. ELISA) finished product) or as a supplement for culture media used
HI: haemagglutination inhibition for tissue culture propagation, shall be shown to be free from
IS: immunostaining (e.g. fluorescent antibody) antibodies against and free from inhibitory-effects on the
SN: serum neutralisation following micro-organisms by suitably sensitive tests.
Avian adenoviruses
B. Additional tests for turkey extraneous agents
Avian encephalomyelitis virus
If the seed virus is of turkey origin or was propagated in
Avian int;ectious bronchitis viruses
turkey substrates, tests for antibodies against the
following agents are also carried out. Avian infectious bursal disease virus types 1 and 2
Avian infectious haemorrhagic enteritis virus
Agent Type oftest
Avian rnfectious laryngotracheitis virus
Chlamydia spp. EIA Avian leucosis viruses
Avian infectious haemorrhagic enteritis virus ACP Avian nephritis virus
Avian paramyxovirus 3 HI Avian paramyxoviruses 1 to 9
Avian orthoreoviruses
Avian infectious bursal disease virus type 2 SN
Avian reticuloendotheliosis virus
Appendix XVI A29
The batch of vaccine complies with the test if there is no monolayers. Inoculate 0.1 mL ofthe pooled material onto
evidence of the presence of Marek's disease virus or any each of 5,4 and 2 recently prepared monolayers ofVero
other extraneous agent. cells, each monolayer having an area of about 25 cm2 as
before. The test is not valid if fewer than 4 of the 5 test
5. Tests for turkey rhinotracheitis virus monolayers or fewer than 3 of the 4 positive control s or
A. In chicken embryo fibroblasts neither of the 2 negative controls survive after any passage.
NOTE: this test can be combined with Test 2 by using For the last sub culture, grow the cells on a suitable substrate
the same test monolayers and negative controls, for all so as to obtain an area of about 10 cm2 of confluent cells
stages up to the final specific test for turkey from each of the original 11 monolayers for the subsequent
rhinotracheitis virus on cens prepared from the last test: test about 10 cm2 of confluent cells derived from each
subculture. of the original 11 monolayers by immunostaining for the
Prepare 11 monolayers of primary or secondary chick embryo presence of turkey rhinotracheitis virus. The test is not valid
fibroblasts from the tissues of 9- to 11-day-old embryos, each if turkey rhinotracheitis virus is detected in fewer than 3 of
monolayer having an area of about 25 cm2 • Remove the the 4 positive control monolayers or in any of the negative
culture medium when the cells reach confluence. Inoculate control monolayers, or if the results for both of the 2
0.1 mL of test vacs;ine onto each of 5 of the monolayers (test negative control monolayers are inconc1usive. If the results
monolayers). Allow adsorption for 1 h, and add culture for more than 1 of the test monolayers are inconc1usive then
medium. Inoculate 4 of the monolayers with a suitable strain further sub cultures of reserved portions of the monolayers
of turkey rhinotracheitis virus as positive controls (not more shall be made and tested until an unequivocal result is
than 10 CCID so in 0.1 mL). Maintain 2 non-inoculated obtained.
monolayers as negative controls. The batch of vaccine complies with the test if there is no
Incubate the cultures for a total of at least 21 days, evidence of the presence of turkey rhinotracheitis virus or any
subculturing at 4- to 5-day intervals. Each passage is made as other extraneous agent.
follows: carry out a freeze-thaw cyc1e; prepare separate pools
6. Test for chicken anaemia virus
of the cells plus fluid from the test monolayers, from the
positive control monolayers and from the negative control Prepare eleven 20 mL suspensions of the MDCC-MSBI cell
monolayers; inoculate 0.1 mL of the pooled material onto line or another cellline of equivalent sensitivity in 25 cm2
each of 5, 4 and 2 recently prepared monolayers of chicken flasks containing about 5 x 105 cells/mL. Inoculate 0.1 mL
embryo fibroblasts cens, each monolayer having an area of of test vaccine into each of 5 of these flasks. Inoculate 4
about 25 cm2 as before. The test is not valid if fewer than 4 other suspensions with 10 CCID so chicken anaemia virus as
of the 5 test monolayers or fewer than 3 of the 4 positive positive controls. Maintain not fewer than 2 non-inoculated
control s or neither of the 2 negative control monolayers suspensions. Maintain all the cell cultures for a total of at
survive after any passage. least 24 days, subculturing 8 times at 3- to 4-day intervals.
For the last sub culture, grow the cells on a suitable substrate During the subculturing the presence of chicken anaemia
so as to obtain an area of about 10 cm2 of confluent cens virus may be indicated by a metabolic colour change in the
from each of the original 11 monolayers for the subsequent infected cultures, the culture fluids becoming red in
test: test about 10 cm2 of confluent cens derived from each comparison with the control cultures. Examine the cens
of the original 11 monolayers by immunostaining for the microscopicany for cytopathic effect. At this time or at the
presence of turkey rhinotracheitis virus. The test is not valid end of the incubation period, centrifuge the cells from each
if turkey rhinotracheitis virus is detected in fewer than 3 of flask at low speed, resuspend at about 106 cells per millilitre
the 4 positive control monolayers or in any of the negative and place 25 ¡lL in each of 10 wells of a multi-well slide.
control monolayers, or if the results for both of the 2 Examine the cells by immunostaining.
negative control monolayers are inconc1usive. If the results The test is not val id if chicken anaemia virus is detected in
for both of the 2 test monolayers are inconc1usive then fewer than 3 of the 4 positive controls or in any of the non-
further subc:ultures of reserved portions of the fibroblasts inoculated controls. If the results for more than 1 of the test
shall be made and tested until an unequivocal result is suspensions are inconc1usive then further sub cultures of
obtained. reserved portions of the test suspensions shall be made and
The batch of vaccine complies with the test if there is no tested until an unequivocal result is obtained.
evidence of the presence of turkey rhinotracheitis virus or any The batch of vaccine complies with the test if there is no
other extraneous agent. evidence of the presence of chicken anaemia virus.
E. In Vero cens
7. Test for duck enteritis virus
Prepare 11 monolayers of Vero cells, each monolayer having
This test is carried out for vaccines prepared on duck or
an area of about 25 cm2 • Remove the culture medium when
goose substrates.
the cells reach confluence. Inoculate 0.1 mL oftest vaccine
onto each of 5 ofthe monolayers (test monolayers). Allow Prepare 11 monolayers of primary or secondary Muscovy
adsorption for 1 h, and add culture medium. Inoculate 4 of duck embryo liver cells, from the tissues of 21- or 22-day-old
the monolayers with a suitable strain of turkey rhinotracheitis embryos, each monolayer having an area of about 25 cm2 •
virus (not more than 10 CCID so in 0.1 mL) to serve as Remove the culture medium when the cells reach confluence.
positive controls. Maintain 2 non-inoculated monolayers as Inoculate 0.1 mL of test vaccine onto each of 5 of the
negative controls. monolayers (test monolayers). Allow adsorption for 1 h and
add culture medium. Inoculate 4 of the monolayers with a
Incubate the cultures for a total of at least 21 days,
suitable strain of duck enteritis virus (not more than 10
subculturing at 4- to 5-day intervals. Each passage is made as
CCID so in 0.1 mL) to serve as positive controls. Maintain
follows: carry out a freeze-thaw cyc1e. Prepare separate pools
2 non-inoculated monolayers as negative controls.
of the cens plus fluid from the test monolayers, from the
positive control monolayers and from the negative control
Appendix XXI A33
Appendix XXI
B (Vet). Approved Synonyms
Where the English tide at the head of a monograph in the European Pharmacopoeia is different from that at the head of the
text incorporated into the British Pharmacopoeia or the British Pharmacopoeia (Veterinary), an Approved Synonym (or
Approved Synonyms) is declared in accordance with s~ction 65(8) of the Medicines Act 1968.
In accordance with the General Notice on Titles, the name or names given in the right-hand column of the list be10w are
Approved Synonyms for the name at the head of the monograph of the European Pharmacopoeia given in the left-hand
column. Where there is more than one entry in the tight-hand column, the first entry is used as the title of the monograph in
the British Pharmacopoeia or the British Pharmacop'oeia (Veterinary) and the remaining entries are included as subsidiary titles.
Approved Synonyms and subsidiary titles have the same significance as the main title and are thus official titles.
Names made by changing the order of the words in an Approved Synonym, with the addition of a preposition when necessary,
are also Approved Synonyms.
Where square brackets are used in a title these may be replaced by round brackets, and vice versa. The words 'per cent' may be
replaced by the symbol '%'.
Where the word 'Injection' appears in the title or synonym of a monograph in the European Pharmacopoeia, the abbreviation
'Inj.' is declared to be an Approved Synonym for that part of the title.
A consolidated list of aH Approved Synonyms is included in Appendix XXI B of the British Pharmacopoeia.
Index
Page numbers in bold type relate to monograph titIes.
Canine Adenovirus Vaccine (Live), see Cloprostenol Sodium, 51, S8 Cloxacillin Sodium Intramammary
Canine Adenovirus Vaccine J Living Closantel Sodium Dihydrate, 52 Infusion (Lactating Cow), Ampicillin
Canine Adenovirus Vaccine, Living, 220 Closantel Sodium Dihydrate for Sodium and, 143
Canine Distemper Vaccine (Live), see Veterinary Use, see Closantel Sodium Cobalt Depot-Tablets, xxiii
Canine Distemper VaccineJ Living Dihydrate Cobalt Oxide, 54
Canine Distemper Vaccine, Living, 222 Clostridium Botulinum Vaccine, 227 Coccidiosis Vaccine (Live) for
Canine Leptospirosis Vaccine Clostridium Botulinum Vaccine for Chickens, 235
(Inactivated), 275 Veterinary Use, see Clostridium Code of Practice, x, xxi
Canine Parainfiuenza Virus Vaccine Botulinum Vaccine Cold-water Vibriosis Vaccine for
(Live),223 Clostridium Chauvoei Vaccine, 227 Salmonids, Inactivated, 315
Canine Parvovirosis Vaccine Clostridium Chauvoei Vaccine for Colouring Agents, Permitted
(Inactivated), see Canine Parvovirus Veterinary Use, see Clostridium Alternatives, 11
VaccineJ Inactivated Chauvoei Vaccine Competent authority, Definition of, 5
Canine Parvovirosis Vaccine (Live), see Clostridium Novyi Alpha Antitoxin, 188 Complementary Medicines, Crude
Canine Parvovirus Vaccine J Living Clostridium N ovyi Alpha Antitoxin for Drugs; Traditional Herbal and, Status
Canine Parvovirus Vaccine, Inactivated, Veterinary Use, see Clostridium Novyi of,17
224 Alpha Antitoxin Compound Pyrethrum Spray, xxiii
Canine Parvovirus Vaccine, Living, 225 Clostridium Novyi Type B Vaccine, 228 Constant Weight, Definition of, 6
Capital Initial Letters, Significance of, 8 Clostridium Novyi (Type B) Vaccine for Contagious Pustular Dermatitis Vaccine,
Carprofen,46 Veterinary Use, see Clostridium Novyi Living, 238
Carprofen for Veterinary Use, see Type B Vaccine Content, Expression of, 23
Carprofen Clostridium Perfringens Antitoxins, 189 Contents of the General Notices, 1
Catechu,48 Clostridium Perfringens Beta Conventional terms, 20
Catechu, Pale, 48 Antitoxin, 189 Coronavirus Diarrhoea Vaccine
Catechu Tincture, xxiii Clostridium Perfringens Beta Antitoxin (Inactivated), Calf, 216
Catgut in Distributor, Sterile, 325 for Veterinary Use, see Clostridium Corresponds, definition of, 5
Caution Statements, 7 Peifringens Beta Antitoxin Co-trimazine Injection, 155
CCID so , Definition of, 29 Clostridium Perfringens Epsilon Co-trimazine Oral Suspension, xxiii
Cefalonium, 48, S7 Antitoxin, 191 Co-trimazine Tablets, 156
Cefalonium Eye Ointment, xxiii Clostridium Perfringens Epsilon Co-trimazine Veterinary Oral Powder,
Cefalonium Intramammary Infusion Antitoxin for Veterinary Use, see 156
(Dry Cow), 150 Clostridium Perjringens Epsilon Antitoxin CRS, 15
Cell Cultures for the Production of Clostridium Perfringens Type B Vaccine, Crude Drugs, Macroscopical
Veterinary Vaccines, A12 see Clostridium Peifringens Vaccines characteristics of, 17
Celsius, Definition of, 31 Clostridium Perfringens Type C Vaccine, Crude Drugs; Traditional Herbal and
Centigrade, Definition of, 31 see Clostridium Perjringens Vaccines Complementary Medicines,
Centrifugation, Definition of, 31 Clostridium Perfringens Type D Status of, 17
Changes, Editorial, xx Vaccine, see Clostridium Peifringens Cutaneous Application of the BP (Vet),
Characteristics, Status of, 11 Vaccines Liquid Preparations for, 128
Characters, 25 Clostridium Perfringens Vaccine for Cutaneous Application, Powders for, 135
Chemical Abstracts Service Registry Veterinary U se, see Clostridium Cutaneous Application, Veterinary
Number, Status, 5 Peifringens Vaccines Liquids for, 127
Chemical Formulae, 5 Clostridium Perfringens Vaccines, 230
Chemical Reference Substances, 15 Clostridium Septicum Vaccine, 232
Chicken Anaemia Vaccine (Live) ,
Infectious, 270
Clostridium Septicum Vaccine for
Veterinary U se, see Clostridium
D
Dalmation Insect Flowers, see Pyrethrum
Chicken anaemia virus, test for, A27 Septicum Vaccine
Flower
Chicken Flocks Free from Specified Clostridium Tetani Antitoxin, 192
Date, Effective dates, vi
Pathogens for the Production and Clostridium Tetani Vaccine for Equidae,
Declaration of Interests, x
Quality Control of Vaccines, A9 see Clostridium Tetani Vaccines
Decoquinate, 55, S9
Chickens, Salmonella Enteritidis Vaccine Clostridium Tetani Vaccines, 233
Decoquinate Premix, 157
(Inactivated) forJ 309 Cloxacillin Benzathine, 54, S8
Definition, 24
Chickens, Salmonella Typhimurium Cloxacillin Benzathine Intramammary
Definition of Terms, 5
Vaccine (Inactivated) for, 310 Infusion (Dry Cow), 154
Definition, Status of, K_
Chlamydiosis Vaccine (Inactivated), Cloxacillin Benzathine Intramammary
Deltamethrin, 55, S9
Feline, 250 Infusion (Dry Cow), Ampicillin
Deltamethrin Pour-on, 157
Chloramphenicol, S7 Trihydrate and, 145
Dembrexine Hydrochloride
Chlortetracycline Soluble Powder, see Cloxacillin Intramammary Infusion
Monohydrate, 57
Chlortetracycline Veterinary Oral Powder (DC), Ampicillin and, 145
Dembrexine Hydrochloride Monohydrate
Chlortetracycline Tablets, 152 Cloxacillin Intramammary Infusion
for Veterinary U se, see Dembrexine
Chlortetracycline Veterinary Oral (DC), see Cloxacillin Benzathine'¡,
Hydrochloride Monohydrate
Powder, 151 Intramammary Infusion (Dry Cow)
Detomidine Hydrochloride, 58
Choice of vaccine strain, Choice of Cloxacillin Intramammary Infusion
Detomidine Hydrochloride for Veterinary
vaccine composition, 24 (LC), Ampicillin and, 143
Use, see Detomidine Hydrochloride
Chromatographic tests, xx Cloxacillin Intramammary Infusion
Diagnostic Preparations, 319
C.I.P. - Collection de Bactéries de (LC), see Cloxacillin Sodium
Diclazuril, 59
l'Institut Pasteur, address of, 30 Intramammary Infusion (Lactating Cow)
Diclazuril for Veterinary U se, see
Clazuril,49 Cloxacillin Sodium, S8
Diclazuril
Clazuril for Veterinary Use, see Clazuril Cloxacillin Sodium Intramammary
Difioxacin Hydrochloride Trihydrate, 60
Cloprostenol Injection, 153 Infusion (Lactating Cow), 154
page numbers in bold type relate to monograph tides Index A41
Microkatal, Definition of, 31 Orbifloxacin for Veterinary U se, see Porcine Influenza Vaccine (Inactivated),
Mink Distemper Vaccine, Living, Ferret Orbijloxacin see Swine Influenza Vaccine, Inactivated
and,257 Orf Vaccine, see Contagious Pustular Porcine Parvovirus Vaccine,
Mole, Definition of, 31 Dermatitis Vaccine, Living Inactivated, 299
Molecular Formula, Status of, 8 Ovine Enzootic Abortion Vaccine, Porcine Progressive Atrophic Rhinitis
Molecular Weight, Status of, 8 Inactivated, 292 Vaccine, Inactivated, 300
Monograph Revisions, xx Oxfendazole, 92, S15 Potassium Selenate, 95
Morantel Hydrogen Tartrate for Oxfendazole for Veterinary U se, see Potency, Estimated, 14
Veterinary Use, see Morantel Tartrate Oxfendazole Potency of Antibiotics, 14
Morantel Tartrate, 85 Oxfendazole Oral Suspension, 173 Potency, Stated, 14
Moxidectin, 87 Oxyc1ozanide, 93, S 16 Potential Adulteration, 24
Moxidectin Injection, 171 Oxyc1ozanide Oral Suspension, 173 Powders for Cutaneous Application, 135
Mycoplasma Gallisepticum Vaccine Oxytetracyc1ine Injection, xxiii Powders, Topical, 135
(Inactivated), 284 Oxytetracyc1ine Intramammary Infusion ppm, Definition of, 7
Mycoplasmas, Test for Absence of, A21 (Lactating Cow), xxiii Preface, vii
Myxomatosis Vaccine (Live) for Oxytetracyc1ine Veterinary Oral Premixes, 135
Rabbits, 285 Powder, 174 Premixes for Medicated Feeding Stuffs
for Veterinary Use, see Premixes
Premixes of the BP (Vet), 136
N p Preparations for Oral Use, Liquid, 132
Procaine Benzylpenicillin Injection, 176
Nandrolone Laurate, 89, S15 Pale Catechu, 48
Procaine Benzylpenicillin Intramammary
Nandrolone Laurate Injection, 172 Panels of Experts, ix
Infusions, xxiii
NCIMB - National Collection of Parainfluenza Virus Vaccine (Live),
Production statements, 24
Industrial and Marine Bacteria, Canine, 223
Production, Status of, 9
address of, 30 Parainfluenza V;irus Vaccine, Living,
Protected from Light, 12
NCPF - National Collection of Bovine,211
Protected from light, Definition of, 27
Pathogenic Fungi, address of, 30 Parametric Release, 4
Pulpy Kidney Vaccine, see Clostridium
NCTC - National Collection of Type Paramyxovirus 3 Vaccine, Inactivated
Perfringens Vaccines
Cultures, address of, 30 Avian, 209
Pustular Dermatitis Vaccine, Living
NCYC - National Collection of Yeast Parenteral Preparations, 132
Contagious, 238
Cultures, address of, 30 Parenteral Preparations of the
Pyrethrum Dusting Powder, xxiii
Neonatal Piglet Colibacillosis Vaccine BP (Vet), 134
Pyrethrum Extract, 177
(Inactivated), see Porcine E. Coli Parenteral Preparations, 134, see also
Pyrethrum Flower, 95
Vaccine, Inactivated name of substance
Pyrethrum Spray, Compound xxiii
Neonatal Ruminant Colibacillosis Parvovirus Vaccine, Inactivated,
Vaccine (Inactivated), see Ruminant Canine, 224
E. Coli Vaccine, Inactivated Parvovirus Vaccine, Living, Canine, 225
Newcastle Disease and Avian Infectious Parvovirus Vaccine, Porcine, Q
Bronchitis Vaccine, Living, 286 Inactivated, 299 Quality systems, 20
Newcastle Disease Vaccine, Pastes, Veterinary Oral, 135
Inactivated, 288 Pasteurella Vaccine (lnactivated) for
Newcastle Disease Vaccine (Live), see
Newcastle Disease Vaccine, Living
Sheep, 292
Patents, vi
R
Rabbit Haemorrhagic Disease Vaccine
Newcastle Disease Vaccine, Living, 290 PD so, Definition of, 29
(Inactivated), 306
Nitroxinil, 90, S 15 Pentobarbital, S 16
Rabies Vaccine for Foxes, Living, 302
Nitroxinil Injection, 172 Pentobarbital Injection, 175
Rabies Vaccine (Inactivated) far
Nitroxynil Injection, see Nitroxinil PFU, 29
Veterinary U se, see Rabies Veterinary
Injection Pharmaceutical Use, Substances for, 37
Vaccine, inactivated
Nitroxynil, see Nitroxinil Phenylbutazone, S 16
Rabies Vaccine (Live, Oral) for Foxes,
Non-absorbable Strands in Distributor, Phenylbutazone Tablets, 175
see Rabies Vaccine for Foxes, Living
Sterile, 326 Ph. Eur., Definition of, 3
Rabies Veterinary Vaccine,
Notices, vi Piperazine Adipate Tablets, xxiii
Inactivated, 303
Piperazine Citrate Tablets, 176
Radian, Definition of, 31
Piperonyl Butoxide, 94
o Polyamide 6 Suture in Distributor,
Sterile, 328
Reagents, Description of, 7
Recently Prepared, Definition of, 10
Official, Definition of, 3 Reference Materials and Spectra, 12
Official Standard s, 4 Polyamide 6/6 Suture in Distributor,
Reference Preparation, Intemational, 14
Official Titles, 7 Sterile, 328
Reference spectra, 12
Omissions, xx Poly(ethylene terephthalate) Suture in
Reference spectra, concordance of, 12
Omissions, List of, xxiii Distributor, Sterile, 328
Reference Spectra, Preparation of
Oral Liquids, 132 Polystyrene, S3-S4
Infrared, S2
Oral Liquids of the BP (Vet), 132 Porcine Actinobacillosis Vaccine,
Reference Substances, xxiv
Oral Liquids, 132, see also name of Inactivated, 294
Reference Substances and Reference
substance Porcine E. Coli Vaccine, Inactivated, 297
Preparations, 15
Oral Powders of the BP (Vet), 135 Porcine Enzootic Pneumonia Vaccine
Relative Atomic and Molecular
Oral Powders (Vet), Definition of, 135 (Inactivated), 296
Masses,23
Oral Powder, 135, see also na me of Porcine Escherichia Coli Vaccine,
Requirements, Pharmacopoeial, xxi
substance Inactivated, see Porcine E. Coli Vaccine,
Requirements, Release, 4
Orbifloxacin, 91 Inactivated
A44 Index
v Tumorigenicity, A13
Vibriosis Vaccine for Salmonids,
Vaccine Strain, Choice of, 9 Inactivated, 317
Vaccines, Bacterial, 194 Vibriosis Vaccine for Salmonids,
Vaccines for Veterinary U se, see Inactivated, Cold-water, 315
Vetennary Vaccines Viral Hepatitis Type 1 Vaccine (Live),
Vaccines, List of, 194 Duck, 240
Vaccines, Vector, 194 Viral Rhinotracheitis Vaccine,
Vaccines, Veterinary, General Inactivated, Feline, 255
Monograph, 194 Viral Rhinotracheitis Vaccine, Living,
Vaccines, Viral, 194 Feline, 256
Validation of pharmacopoeial Viral Tenosynovitis Vaccine (Live),
methods,20 Avian, 210
Valnemulin Hydrochloride, 119 Viral Vaccines, 194
Valnemulin Hydrochloride for Veterinary Visual Comparative Tests, 12
Use, see Valnemulin Hydrochlonde
Vector Vaccines, 194
Vedaprofen, 120
Vedaprofen for Veterinary Use, see
w
Water Bath, Definition of, 7
Vedaprofen, 120 Water, Potable, 10
Veterinary Immunosera, 185 Water-bath,22
Veterinary Liquid Preparations for Websites, xxii
Cutaneous Application, 127 Weights and Measures, Expression of, 6
Veterinary Oral Pastes, 135 Weights, Atomic, 6
Veterinary Oral Powders, 135 Working Parties, ix
Veterinary Vaccines, 194, A9
Terminology Used, A9
Veterinary Vaccines, Absence of
Contaminating Virus es, A13-14
x
Veterinary Vaccines and Xylazine Hydrochloride, 121
Immunosera, A18 Xylazine Hydrochloride for Veterinary
Evaluation of, Efficacy of, A18 U se, see Xylazine Hydrochlonde