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GeneralChapters:<51>ANTIMICROBIALEFFECTIVENESSTESTING

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ANTIMICROBIALEFFECTIVENESSTESTING

Antimicrobialpreservativesaresubstancesaddedtononsteriledosageformstoprotectthemfrommicrobiologicalgrowthor
frommicroorganismsthatareintroducedinadvertentlyduringorsubsequenttothemanufacturingprocess.Inthecaseof
sterilearticlespackagedinmultipledosecontainers,antimicrobialpreservativesareaddedtoinhibitthegrowthof
microorganismsthatmaybeintroducedfromrepeatedlywithdrawingindividualdoses.
Antimicrobialpreservativesshouldnotbeusedasasubstituteforgoodmanufacturingpracticesorsolelytoreducethe
viablemicrobialpopulationofanonsterileproductorcontrolthepresterilizationbioburdenofmultidoseformulationsduring
manufacturing.AntimicrobialpreservativesincompendialdosageformsmeettherequirementsforAddedSubstancesunder
IngredientsandProcessesintheGeneralNotices.
Allusefulantimicrobialagentsaretoxicsubstances.Formaximumprotectionofpatients,theconcentrationofthe
preservativeshowntobeeffectiveinthefinalpackagedproductshouldbebelowalevelthatmaybetoxictohuman
beings.
Theconcentrationofanaddedantimicrobialpreservativecanbekeptataminimumiftheactiveingredientsofthe
formulationpossessanintrinsicantimicrobialactivity.Antimicrobialeffectiveness,whetherinherentintheproductor
whetherproducedbecauseoftheadditionofanantimicrobialpreservative,mustbedemonstratedforallinjections
packagedinmultipledosecontainersorforotherproductscontainingantimicrobialpreservatives.Antimicrobial
effectivenessmustbedemonstratedformultipledosetopicalandoraldosageformsandforotherdosageformssuchas
ophthalmic,otic,nasal,irrigation,anddialysisfluids(seePharmaceuticalDosageForms 1151 ).
Thischapterprovidesteststodemonstratetheeffectivenessofantimicrobialprotection.Addedantimicrobialpreservatives
mustbedeclaredonthelabel.Thetestsandcriteriaforeffectivenessapplytoaproductintheoriginal,unopenedcontainer
inwhichitwasdistributedbythemanufacturer.

PRODUCTCATEGORIES
Forthepurposeoftesting,compendialarticleshavebeendividedintofourcategories(seeTable1).Thecriteriaof
antimicrobialeffectivenessfortheseproductsareafunctionoftherouteofadministration.
Table1.CompendialProductCategories

Category
ProductDescription
1
Injections,otherparenteralsincluding
emulsions,oticproducts,sterilenasal
products,andophthalmicproductsmade
withaqueousbasesorvehicles.
2

Topicallyusedproductsmadewithaqueous
basesorvehicles,nonsterilenasalproducts,
andemulsions,includingthoseapplied
tomucousmembranes.

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Oralproductsotherthanantacids,madewith
aqueousbasesorvehicles.

Antacidsmadewithanaqueousbase.
TESTORGANISMS

Useculturesofthefollowingmicroorganisms 1:Candidaalbicans(ATCCNo.10231),Aspergillusniger(ATCCNo.16404),
Escherichiacoli(ATCCNo.8739),Pseudomonasaeruginosa(ATCCNo.9027),andStaphylococcusaureus(ATCCNo.
6538).TheviablemicroorganismsusedinthetestmustnotbemorethanfivepassagesremovedfromtheoriginalATCC
culture.Forpurposesofthetest,onepassageisdefinedasthetransferoforganismsfromanestablishedculturetofresh
medium.Alltransfersarecounted.Inthecaseoforganismsmaintainedbyseedlottechniques,eachcycleoffreezing,
thawing,andrevivalinfreshmediumistakenasonetransfer.Aseedstocktechniqueshouldbeusedforlongtermstorage
ofcultures.CulturesreceivedfromtheATCCshouldberesuscitatedaccordingtodirections.Ifgrowninbroth,thecellsare
pelletedbycentrifugation.Resuspendin1/20ththevolumeoffreshmaintenancebroth,andaddanequalvolumeof20%
(v/vinwater)sterileglycerol.Cellsgrownonagarmaybescrapedfromthesurfaceintothe10%glycerolbroth.Dispense
smallaliquotsofthesuspensionintosterilevials.Storethevialsinliquidnitrogenorinamechanicalfreezeratnomore
than 50 .Whenafreshseedstockvialisrequired,itmayberemovedandusedtoinoculateaseriesofworkingcultures.
Theseworkingculturesmaythenbeusedperiodically(eachdayinthecaseofbacteriaandyeast)tostarttheinoculum
culture.

MEDIA
Allmediausedinthetestmustbetestedforgrowthpromotion.UsethemicroorganismsindicatedaboveunderTest
Organisms.

PREPARATIONOFINOCULUM
Preparatorytothetest,inoculatethesurfaceofasuitablevolumeofsolidagarmediumfromarecentlyrevivedstock
cultureofeachofthespecifiedmicroorganisms.ThecultureconditionsfortheinoculumculturearedescribedinTable2in
whichthesuitablemediaareSoybeanCaseinDigestorSabouraudDextroseAgarMedium(seeMicrobialLimitsTesting
61 ).
ToharvestthebacterialandC.albicanscultures,usesterilesalineTS,washingthesurfacegrowth,collectingitina
suitablevessel,andaddingsufficientsterilesalineTStoobtainamicrobialcountofabout1108colonyformingunits
(cfu)permL.ToharvestthecellsofA.niger,usesterilesalineTScontaining0.05%ofpolysorbate80,andaddsufficient
sterilesalineTStoobtainacountofabout1108cfupermL.
Alternatively,thestockcultureorganismsmaybegrowninasuitableliquidmedium(i.e.,SoybeanCaseinDigestBrothor
SabouraudDextroseBroth)andthecellsharvestedbycentrifugation,thenwashedandresuspendedinsterilesalineTSto
obtainamicrobialcountofabout1108cfupermL.[NOTETheestimateofinoculumconcentrationmaybeperformedby
turbidimetricmeasurementsforthechallengemicroorganisms.Refrigeratethesuspensionifitisnotusedwithin2hours.]
DeterminethenumberofcfupermLineachsuspension,usingtheconditionsofmediaandmicrobialrecoveryincubation
timeslistedinTable2toconfirmtheinitialcfupermLestimate.Thisvalueservestocalibratethesizeofinoculumusedin
thetest.Thebacterialandyeastsuspensionsaretobeusedwithin24hoursofharvest,butthefungalpreparationmaybe
storedunderrefrigerationforupto7days.

PROCEDURE
Thetestcanbeconductedeitherinfiveoriginalcontainersifsufficientvolumeofproductisavailableineachcontainerand
theproductcontainercanbeenteredaseptically(i.e.,needleandsyringethroughanelastomericrubberstopper),orinfive
sterile,cappedbacteriologicalcontainersofsuitablesizeintowhichasufficientvolumeofproducthasbeentransferred.
Inoculateeachcontainerwithoneofthepreparedandstandardizedinoculum,andmix.Thevolumeofthesuspension
inoculumusedisbetween0.5%and1.0%ofthevolumeoftheproduct.Theconcentrationoftestmicroorganismsthatis
addedtotheproduct(Categories1,2,and3)aresuchthatthefinalconcentrationofthetestpreparationafterinoculationis
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between1105and1106cfupermLoftheproduct.ForCategory4products(antacids)thefinalconcentrationofthe
testpreparationafterinoculationisbetween1103and1104cfupermLoftheproduct.
Theinitialconcentrationofviablemicroorganismsineachtestpreparationisestimatedbasedontheconcentrationof
microorganismsineachofthestandardizedinoculumasdeterminedbytheplatecountmethod.
Incubatetheinoculatedcontainersat22.52.5 .SampleeachcontainerattheappropriateintervalsspecifiedinTable3.
Recordanychangesobservedinappearanceattheseintervals.Determinebytheplatecountprocedurethenumberofcfu
presentineachtestpreparationfortheapplicableintervals(seeProcedureunderMicrobialLimitTests 61 ).Incorporate
aninactivator(neutralizer)ofthespecificantimicrobialintheplatecountorintheappropriatedilutionpreparedforplating.
Theseconditionsaredeterminedinthevalidationstudyforthatsamplebasedupontheconditionsofmediaandmicrobial
recoveryincubationtimeslistedinTable2.UsingthecalculatedconcentrationsofcfupermLpresentatthestartofthe
test,calculatethechangeinlog10valuesoftheconcentrationofcfupermLforeachmicroorganismattheapplicabletest
intervals,andexpressthechangesintermsoflogreductions.
Table2.CultureConditionsforInoculumPreparation

Inoculum
Incubation
Time

Microbial
Recovery
IncubationTime

32.52.5

18to24
hours

3to5days

SoybeanCaseinDigest
Broth
SoybeanCaseinDigest
Agar
Staphylococcusaureus SoybeanCaseinDigest
(ATCCNo.6538)
Broth
SoybeanCaseinDigest
Agar
Candidaalbicans
SabouraudDextroseAgar
(ATCCNo.10231)
SabouraudDextroseBroth

32.52.5

18to24
hours

3to5days

32.52.5

18to24
hours

3to5days

22.52.5

44to52
hours

3to5days

Aspergillusniger
(ATCCNo.16404)

22.52.5

6to10days

3to7days

Organism
Escherichiacoli
(ATCCNo.8739)

SuitableMedium
SoybeanCaseinDigest
Broth
SoybeanCaseinDigest
Agar

Pseudomonas
aeruginosa
(ATCCNo.9027)

SabouraudDextroseAgar
SabouraudDextroseBroth

Incubation
Temperature

CRITERIAFORANTIMICROBIALEFFECTIVENESS
TherequirementsforantimicrobialeffectivenessaremetifthecriteriaspecifiedunderTable3aremet(seeSignificant
FiguresandTolerancesunderGeneralNotices).Noincreaseisdefinedasnotmorethan0.5log10unithigherthanthe
previousvaluemeasured.
Table3.CriteriaforTestedMicroorganisms

ForCategory1Products
Bacteria: Notlessthan1.0logreductionfromtheinitialcalculatedcountat7days,notlessthan3.0
logreductionfromtheinitialcountat14days,andnoincreasefromthe14days'countat
28days.
Yeast
Noincreasefromtheinitialcalculatedcountat7,14,and28days.
and
Molds:
ForCategory2Products
Bacteria: Notlessthan2.0logreductionfromtheinitialcountat14days,andnoincreasefromthe
14days'countat28days.
Yeast
Noincreasefromtheinitialcalculatedcountat14and28days.
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and
Molds:
ForCategory3Products
Bacteria: Notlessthan1.0logreductionfromtheinitialcountat14days,andnoincreasefromthe
14days'countat28days.
Yeast
Noincreasefromtheinitialcalculatedcountat14and28days.
and
Molds:
ForCategory4Products
Bacteria, Noincreasefromtheinitialcalculatedcountat14and28days.
Yeast,
and
Molds:
1AvailablefromAmericanTypeCultureCollection,10801UniversityBoulevard,Manassas,VA201102209(http://www.atcc.org).

AuxiliaryInformationStaffLiaison:RadhakrishnaSTirumalai,Scientist
ExpertCommittee:(MSA05)MicrobiologyandSterilityAssurance
USP29NF24Page2499
PhoneNumber:13018168339

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