6508 (81) / Biological Tests USP 43

exceeds the critical value in Table

Table A2-1. Test for Outlier Measurements

Insamples from a normal population, gaps equalto or largerthan the following values of G" Gz, and G3 occurwith a probability P =0.01, when outlier
measurements can occuronlyat one end; or with P =0.02, when they mayoccur at eitherend.
N 3 4 5 6 7
G, 0.889 0.698 0.637
N 8 9 10 - -
Gz :~~h§~~ 0.597
N 11 12 13 - -
G3 ~q:q~.~ QZ~~

EXAMPLE
Estimated potencies of sample in log scale =1.561, 1.444, 1.517, 1.535.
Check the lowest potency for outlier:
G, = (1.517 - 1.444)/(1.561 - 1.444) =0.624 < 0.889

Therefore, 1.444 is not an outlier.

Check the highest potency for outlier:
G, = (1.561 - 1.535)/{1.561 - 1.444) = 0.222 < 0.889
Therefore, 1.561 is not an outlier.
Outlier potencies should be marked as outlier values and excluded from the assay calculations. NMT 1 potency can be
excluded as an outlier.

(85) BACTERIAL ENDOTOXINS TEST

·Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopoeia
and/or the Japanese Pharmacopoeia. Those portions that are not harmonized are marked with symbols (+ .) to specify this fact.,
The BacterialEndotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte
lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
There are three techniques for this test: the gel-clot technique, which is based on gel formation; the turbidimetric technique,
based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on
the development of color after cleavage of a synthetic peptide-chromogen complex. Proceed by any of the three techniques
for the test. In the event of doubt or dispute, the final decision is made based upon the gel-clot limit test unless otherwise
indicated in the monograph for the product being tested. The test is carried out in a manner that avoids endotoxin
contamination.

APPARATUS
Depyrogenate all glassware and other heat-stable materials in a hot air oven using a validated process.· 1 • A commonly
used minimum time and temperature is 30 min at 250 0 • If employing plastic apparatus, such as microplates and pipet tips for
automatic pipetters, use apparatus that is shown to be free of detectable endotoxin and does not interfere in the test.
[NOTE-In this chapter, the term "tube" includes any other receptacle such as a microtiter welL]

REAGENTS AND TEST SOLUTIONS

Amoebocyte Lysate

A lyophilized product obtained from the lysate of amoebocytes (white blood cells) fromthe horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus). This reagent refers only to a product manufactured in accordance with the regulations
of the competent authority. [NOTE-Amoebocyte Lysate reacts to some p-glucans in addition to endotoxins. Amoebocyte Lysate
preparations that do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from

.1 For a validitytest of the procedure for inactivating endotoxins, see Dry-Heat Sterilization under SterilityAssurance (1211). Use Lysate TS having a
sensitivity of not less than 0.15 Endotoxin Unit per rnt.,

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 USP 43 Biological Tests / (85) 6509

Amoebocyte Lysate or by inhibiting the G factor reacting system of Amoebocyte Lysate and may be used for endotoxin testing
in the presence of glucans.]

Water for Bacterial Endotoxins Test (BET)

Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed, at the
detection limit of the reagent.

Lysate TS

Dissolve Amoebocyte Lysate in Waterfor BET, or in a buffer recommended by the lysate manufacturer, by gentle stirring. Store
the reconstituted lysate, refrigerated or frozen, according to the specifications of the manufacturer.

PREPARATION OF SOLUTIONS

Standard Endotoxin Stock Solution

A StandardEndotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the
current WHO International Standard for Endotoxin. Follow the specifications in the package leaflet and on the label for
preparation and storage of the StandardEndotoxin Stock Solution. Endotoxin is expressed in Endotoxin Units (EU). [NoTE-One
USP Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin.]

Standard Endotoxin Solutions

After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions of StandardEndotoxin
Solution, using Waterfor BET. Use dilutions as soon as possible to avoid loss of activity by adsorption.

Sample Solutions

Prepare the Sample Solutions by dissolving or diluting drugs using Waterfor BET. Some substances or preparations may be
more appropriately dissolved, or diluted in other aqueous solutions. If necessary, adjust the pH of the solution to be examined
(or dilution thereof) so that the pH of the mixture of the lysate and Sample Solution falls within the pH range specified by the
lysate manufacturer, usually 6.0-8.0. The pH may be adjusted by use of an acid, base, or suitable buffer as recommended by
the lysate manufacturer. Acids and bases may be prepared from concentrates or solids with Waterfor BET in containers free of
detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.

DETERMINATION OF MAXIMUM VALID DILUTION (MVD)

The maximum valid dilution is the maximum allowable dilution of a specimen at which the endotoxin limit can be
determined. Determine the MVD from the following equation:

MVD =(endotoxin limit x concentration of Sample Solution)/('A.)

Endotoxin Limit

The endotoxin limit for parenteral drugs, defined on the basis of dose, equals K/M • 2 ., where K is a threshold pyrogenic
dose of endotoxin per kg of body weight, and M is equal to the maximum recommended bolus dose of product per kg of body
weight. When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose
administered in a single hour period. The endotoxin limit for parenteral drugs is specified in the individual monograph in units
such as EU/mL, EU/mg, EU/Unit of biological activity, etc.

Concentration of Sample Solution

mg/mL: in the case of endotoxin limit specified by weight (EU/mg);

Units/mL: in the case of endotoxin limit specified by unit of biological activity (EU/Unit);
mL/mL: when the endotoxin limit is specified by volume (EU/mL).
'A.: the labeled sensitivity in the Gel-Clot Technique (EU/mL) or the lowest concentration used in the standard curve for the
Turbidimetric Technique or Chromogenic Technique.

• 2 K is 5 USP-EU/kg of body weight for any route of administration other than intrathecal (for which K is 0.2 USP-EU/kg of body weight). For
radiopharmaceutical products not administered intrathecally, the endotoxin limitis calculated as 175 EU/V, where V is the maximum recommended
dose in mL. Forintrathecally administered radiopharmaceuticals, the endotoxin limit is obtained by the formula 14 EU/ V. Forformulations (usually
anticancer products) administered on a per square meter of body surface,the formula is K/M, where K=100 EU/m 2 and M is the maximum dose/rn-.•

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 6510 (85)/Bro~gkaIT~~ USP 43

GEL-CLOT TECHNIQUE

The gel-clot technique is used for detecting or quantifying endotoxins based on clotting of the lysate reagent in the presence
of endotoxin. The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the
labeled sensitivity of the lysate reagent. To ensure both the precision and validity of the test, perform the tests for confirming
the labeled lysate sensitivity and for interfering factors as described in Preparatory Testing, immediately below.

Preparatory Testing
TEST FOR CONFIRMATION OF LABELED LYSATE SENSITIVITY

Confirm in four replicates the labeled sensitivity, A, expressed in EU/mL of the lysate prior to use in the test. The test for
confirmation of lysate sensitivity is to be carried out when a new batch of lysate is used or when there is any change in the test
conditions that may affect the outcome of the test. Prepare standard solutions having at least four concentrations equivalent
to 2A, A, 0.5A, and 0.25A by diluting the USP Endotoxin RS with Waterfor BET.
Mix a volume of the Lysate TS with an equal volume (such as O.l-mL aliquots) of one of the Standard Endotoxin Solutions in
each test tube. When single test vials or ampuls containing lyophilized lysate are used, add solutions directly to the vial or ampul.
Incubate the reaction mixture for a constant period according to the directions of the lysate manufacturer (usually at 37 ± 10
for 60 ± 2 min), avoiding vibration. To test the integrity of the gel, take each tube in turn directly from the incubator, and invert
it through about 180 0 in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result
as positive. A result is negative if an intact gel is not formed. The test is considered valid when the lowest concentration of the
standard solutions shows a negative result in all replicate t e s t s . ' ,
The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots the
lysate. Determine the geometric mean endpoint by calculating the mean of the logarithms of the endpoint concentrations of
the four replicate series and then taking the antilogarithm of the mean value, as indicated in the following formula:

geometric mean endpoint concentration = antilog (Ze/t)

where Ze is the sum of the log endpoint concentrations of the dilution series used, and f is the number of replicate test tubes.
The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL). If this is not less than 0.5A and
not more than 2A, the labeled sensitivity is confirmed and is used in tests performed with this lysate.

TEST FOR INTERFERING FACTORS

Usually prepare solutions (A-D) as shown in Table 7, and perform the inhibition/enhancement test on the Sample Solutions
at a dilution less than the MVD, not containing any detectable endotoxins, operating as described for Test for Confirmation of
Labeled Lysate Sensitivity. The geometric mean endpoint concentrations of Solutions Band C are determined using the formula
described in the Test for Confirmation of Labeled Lysate Sensitivity. The test for interfering factors must be repeated when any
condition changes that is likely to influence the result of the test.

Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques
Endotoxin Concentration/
Solution to Which Endotoxin Dilution Endotoxin Number of
Solution Is Added Diluent Factor Concentration Replicates
Aa None/Sample Solution - - - 4
Bb 2'A./ Sample Solution Sample Solution 1 2"- 4
2 1).. 4
4 0.5"- 4
8 0.25"- 4
CC 2A/Waterfor BET Waterfor BET 1 2"- 2
2 1).. 2
4 0.5"- 2
8 0.25"- ,2
Od None/Water for BET - - - 2

a Solution A: A Sample Solutionof the preparation under test that isfree of detectable endotoxins.
b Solution B: Testfor interference.
C Solution C:Controlfor labeled lysatesensitivity.

d Solution 0: Negative control of Waterfor BET.

The test is considered valid when all replicates of Solutions A and 0 show no reaction and the result of SolutionC conflrms
the labeled sensitivity. ' .. .<

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 USP 43 Biological Tests / (85) 6511

Ifthe sensitivity of the lysate determined in the presence of Solution 8 is not less than 0.5A. and not greater than 2A., the
Sample Solution does not contain factorsthat interfereunder the experimental conditionsused. Otherwise, the Sample Solution
to be examined interferes with the test.
Ifthe sample under test does not complywith the test at a dilution less than the MVD, repeat the test using a greater dilution,
not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the sample to be examined, and this
may contribute to the elimination of interference. . .
Interference may be overcome by suitable treatment such as filtration, neutralization, dialysis, or heating. To establish that
the chosen treatment effectively eliminates interferencewithout loss of endotoxins, performthe assaydescribed above using
the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted to the
chosen treatment.

Limit Test
PROCEDURE
Prepare Solutions A, 8, C/ and D as shown in Table 2, and performthe test on these solutions following the procedure above
for Preparatory Testing, Test for Confirmation of Labeled Lysate Sensitivity.
Table 2. Preparation of Solutions for the Gel-Clot Limit Test
Endotoxin Concentration/
Solution to Which
Solution' Endotoxin Is Added Number of Replicates
A None/Diluted Sample Solution 2
B 2A./Diluted Sample Solution 2
C 2A./Water for BET 2
D None/Waterfor BET 2

• Prepare Solution A and the positiveproduct control Solution B using a dilution not greater than the MVD and treatments as described for the Test for Interfering
Factors in Preparatory Testing. The positivecontrol Solutions Band C contain the Standard Endotoxin Solution at a concentration corresponding to twice the labeled
lysate sensitivity. The negative control Solution D consists of Waterfor BET.

INTERPRETATION
The test isconsideredvalid when both replicates of Solutions 8 and (are positive and those of Solution D are negative.Whena
negative result isfound for both replicates of Solution A, the preparation under test complies with the test. When a positive
result is found for both replicates of Solution A, the preparation under test does not complywith the test.
When a positive result isfound for one replicateof Solution A and a negative result isfound for the other, repeat the test. In
the repeat test, the preparation under test complieswith the test ifa negative result isfound for both replicates of Solution A.
The preparation does not complywith the test ifa positive result isfound for one or both replicates of Solution A. However, if
the preparation does not complywith the test at a dilutionless than the MVD, the test may be repeated using a greater dilution,
not exceeding the MVD.

Quantitative Test
PROCEDURE
The test quantifies bacterial endotoxins in Sample Solutions by titration to an endpoint. Prepare Solutions A, 8, (, and D as
shown in Table 3, and test these solutions by following the procedure in Preparatory Testing, Test for Confirmation of Labeled
Lysate Sensitivity.

Table 3. Preparation of Solutions for the Gel-Clot Assay

Endotoxin Concentration/
Solution to Which Endotoxin Dilution Fac- Endotoxin Number of
Solution Is Added ' Diluent tor Concentration Replicates
A" None/ Sample Solution Waterfor BET 1 - 2
2 - 2
4 - 2
8 - 2
Bb 2A/Sample Solution - 1 21.. 2
c- 2A/Waterfor BET Waterfor BET 1 21.. 2
2 tx 2
4 0.51.. 2
8 0.251.. 2

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6512 .(85) / Biological Tests USP43

Table 3. Preparation of Solutions for the Gel-Clot Assay (continued)

Endotoxin Concentration/
Solution to Which Endotoxin Dilution Fac- Endotoxin Number of
Solution Is Added Diluent tor Concentration Replicates
Dd None/Water for BET - - - 2

a Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test for Interfering Factors was completed. Subsequent dilution of
the Sample Solution must not exceed the MVD. Use Waterfor BETto make a dilutionseriesof four tubes containing the Sample Solution under test at concentrations
of 1, Yz, lA, and Va relative to the concentration used in the Test for InterferingFactors. Other dilutions up to the MVD may be used as appropriate.
b Solution B: Solution A containing standard endotoxin at a concentration of 21.. (positiveproduct control).
C Solution C:Two replicates of four tubes of Waterfor BET containing the standard endotoxin at concentrations of 21.., A., 0.51.., and 0.251../ respectively.

d Solution D: Waterfor BET (negative control).

CALCULATION AND INTERPRETATION

The test isconsidered valid when the following three conditionsare met: (1) Both replicates of negative control Solution 0
are negative; (2) Both replicates of positive product control Solution B are positive; and (3) The geometric mean endpoint
concentration of Solution C is in the range of O.SA. to 2A..
To determine the endotoxin concentration of Solution A, calculate the endpoint concentration for each replicate by
multiplying each endpoint dilution factor by A.. The endotoxin concentration in the Sample Solution is the endpoint
concentration of the replicates. If the test is conducted with a diluted Sample Solution, calculatethe concentration of endotoxin
in the original Sample Solution by multiplying by the dilution factor. If none of the dilutions of the Sample Solution is positive
in a valid assay, report the endotoxin concentration as less than A. (ifthe diluted sample was tested, report as less than A. times
the lowest dilution factor of the sample). Ifall dilutionsare positive, the endotoxin concentration is reported as equal to or
greater than the greatest dilution factor multiplied by A. (e.g., initial dilution factor times eight times A. in Table 3).
The preparation under test meets the requirementsof the test ifthe concentration of endotoxin in both replicates islessthan
that specified in the individual monograph.

PHOTOMETRIC QUANTITATIVE TECHNIQUES

Turbidimetric Technique
This technique isa photometric assaymeasuring increases in reactant turbidity. On the basis of the particular assay principle
employed, this technique may be classified as either an endpoint-turbidimetric assayor a kinetic-turbidimetric assay. The
endpoint-turbidimetric assay is based on the quantitative relationship between the concentration of endotoxins and the
turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric
assayis a method to measureeither the time (onset time) needed to reach a predetermined absorbance or transmission of the
reaction mixture, or the rate of turbidity development. The test iscarried out at the incubation temperature recommended by
the lysate manufacturer (which is usually 37 ± 1°).

Chromogenic Technique
This technique is an assay to measure the chromophore released from a suitable chromogenic peptide by the reaction of
endotoxins with lysate. On the basisof the particularassay principle employed, this technique may be classified as either an
endpoint-chromogenic assayor a kinetic-chromogenic assay. The endpoint-chromogenic assay is based on the quantitative
relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. The
kinetic-chromogenic assay is a method to measure either the time (onset time) needed to reach a predetermined absorbance
of the reaction mixture, or the rate of color development. The test is carried out at the incubation temperature recommended
by the lysate manufacturer(which is usually 37 ± 1°).

Preparatory Testing
To assurethe precision or validity of the turbidimetricand chromogenictechniques, preparatorytests are conducted to verify
that the criteria for the standard curve are valid and that the sample solutiondoes not interfere with the test. Validation for the
test method is required when conditions that are likely to influence the test resultchange.
ASSURANCE OF CRITERIA FOR THE STANDARD CURVE
The test must be carriedout for each lot of lysate reagent. Using the StandardEndotoxin Solution, prepare at least three
endotoxin concentrationswithin the range indicated by the lysate manufacturer to generate the standard curve. Perform the
assay using at least three replicates of each standard endotoxin concentration according to the manufacturer'sinstructions for
the lysate (volume ratios, incubation time, temperature, pH, etc.). If the desired range is greater than two logs in the kinetic
methods, additional standards should be includedto bracket each log increase in the range of the standard curve.The absolute
value of the correlation coefficient, r, must be greater than or equal to 0.980 for the range of endotoxin concentrationsset up.

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USP 43 Biological Tests / (85) 6513

TEST FOR INTERFERING FACTORS

Select an endotoxin concentration at or near the middle of the endotoxin standard curve. Prepare Solutions A, 8, C, and 0
as shown in Table 4. Perform the test on Solutions A, 8, C, and 0 at least in duplicate, accordingto the instructions for the lysate
employed,for example, concerning volumeof Sample Solution and Lysate TS, volume ratio of Sample Solution to Lysate TS,
incubation time, etc.
Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques
Solution to Which
Solution Endotoxin Concentration Endotoxin Is Added Number of Replicates
Ai None Sample Solution Not less than 2
Bb Middle concentration of the standard curve Sample Solution Not less than 2
At least three concentratIons (lowest concentration is designat-
CC ed A) Waterfor BET Each not less than 2
Dd None Waterfor BET Not less than 2

a Solution A: The Sample Solution may be diluted not to exceed MVD.

b Solution 8: The preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the standard
curve.
C Solution C:The standard endotoxin at the concentrations used in the validation of the method described for Assurance of Criteria for the StandardCurve under
Preparatory Testing (positive controls).
d Solution D: Waterfor BET (negative control).

The test is considered valid when the following conditions are met.
1. The absolute value of the correlation coefficient of the standard curve generated using Solution C isgreater than or equal
to 0.980.
2. The resultwith Solution 0 does not exceed the limitof the blank value required in the description of the lysate reagent
employed, or it is less than the endotoxin detection limit of the lysatereagent employed.
Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution, if
any (Solution A, Table 4), from that containing the added endotoxin (Solution B, Table 4). In order to be considered free of
factors that interferewith the assay under the conditions of the test, the measured concentration of the endotoxin added to
the Sample Solution must be within 50%-200% ofthe knownadded endotoxin concentration aftersubtraction of anyendotoxin
detected in the solution without added endotoxin.
Whenthe endotoxin recovery isout of the specified range, the Sample Solution under test isconsideredto contain interfering
factors. Then, repeat the test using a greater dilution, not exceeding the MVD. Furthermore, interference of the Sample
Solution or diluted Sample Solution not to exceed the MVD may be eliminated by suitablevalidatedtreatment such as filtration,
neutralization, dialysis, or heat treatment. Toestablish that the chosen treatment effectively eliminates interferencewithout loss
of endotoxins, perform the assay described above, using the preparation to be examined to which Standard Endotoxin has
been added and which has then been submitted to the chosen treatment.

Test Procedure
Follow the procedure described for Test for Interfering Factors under Preparatory Testing, immediately above.

Calculation
Calculate the endotoxin concentration of each of the replicates of Solution A, using the standard curve generated by the
positive control Solution C. The test is considered valid when the following three requirementsare met.
1. The results of the control Solution Ccomplywith the requirements for validation defined for Assurance of Criteria for the
StandardCurve under Preparatory Testing.
2. The endotoxin recovery, calculatedfrom the concentration found in Solution B after subtracting the concentration of
endotoxin found in Solution A, is within the range of 50%-200%.
3. The result of the negative control Solution 0 does not exceed the limitof the blankvalue required in the description of
the lysate employed, or it is less than the endotoxin detection limitof the lysate reagent employed.

Interpretation
In photometric assays, the preparation under test complieswith the test ifthe mean endotoxin concentration of the
replicates of Solution A, after correction for dilution and concentration, is less than the endotoxin limit for the product.
USP Reference Standards (11)
USP Endotoxin RS

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 6514 (87) / Biological Tests USP 43

(87) BIOLOGICAL REACTIVITY TESTS, IN VITRO

The following tests are designed to determine the biological reactivity of mammalian cell culturesfollowing contact with the
elastomeric plastics and other polymeric materials with director indirectpatient contact or of specific extracts preparedfrom
the materials under test. It isessential that the tests be performedon the specified surfacearea. When the surface area of the
specimen cannot be determined, use 0.1 g of elastomeror 0.2 g of plastic or other material for everyml of extraction fluid.
Exercise care in the preparation of the materials to prevent contaminationwith microorganisms and other foreign matter.
Three tests are described (Le., the Agar Diffusion Test, the DirectContact Test, and the Elution Test).' The decision as to which
type of test or the number of tests to be performedto assess the potential biological response of a specific sampleor extract
depends upon the material, the final product, and its intended use. Other factors that may also affectthe suitability of a
sample for a specific use are the polymeric composition; processing and cleaning procedures; contacting media; inks;
adhesives; absorption, adsorption, and permeability of preservatives; and conditions of storage. Evaluation of such factors
should be made by appropriate additionalspecific tests before determining that a product made from a specific material is
suitablefor its intended use. Materials that fail the in vitro tests are candidatesfor the in vivo tests described in Biological
Reactivity Tests, In Vivo (88).
PROCEDURES
• TEST CONTROL
Positivecontrol: Polyurethane film containingzincdiethyldithiocarbamate (ZDEC)2 or zinc dibutyldithiocarbamate (ZDBC)
Cell culture preparation: Prepare multiple culturesof l-929 (ATCC cell line CCl 1, NCTC clone 929; alternative cell lines
obtained from a standard repository may be used with suitablevalidation) mammalian fibroblast cells in
serum-supplementedminimum essential medium havinga seeding densityofabout lOs cells per mL. Incubatethe cultures
at 37 ± 1° in a humidified incubator for NlT 24 h in a 5 ± 1% carbon dioxide atmosphere untila monolayer, with greater
than 80% confluence, is obtained. Examine the prepared culturesunder a microscope to ensure uniform, near-confluent
monolayers. [NOTE-The reproducibility of the in vitro biological reactivity tests depends upon obtaining uniform cell
culture density.]
Extraction solvents: Sodium Chloride Injection [see. monograph-use Sodium Chloride Injection containing 0.9% of sodium
chloride(NaCI)]. Alternatively, serum-free mammalian cell culture media or serum-supplemented mammalian cell culture
media may be used. Serumsupplementation is used when extraction is done at 37° for 24 h.
• ApPARATUS
Autoclave: Employ an autoclavecapable of maintaining a temperature of 121 ± 2°,equipped with a thermometer, a pressure
gauge, a vent cock, a rackadequate to accommodate the test containersabovethe water level, and a water cooling system
that will allowfor coolingof the test containersto about 20°, but not below20°, immediatelyfollowing the heating cycle.
Oven: Use an oven, preferably a mechanical convection model, that will maintain operating temperatures in the range of
50°_70° within ±2°.
Incubator: Use an incubatorcapable of maintaining a temperature of 37 ± 1°and a humidified atmosphere of5 ± 1% carbon
dioxide in air.
Extraction containers: Use only containers,such as ampuls or screw-cap culture test tubes, or their equivalent, of Type I
glass. If used, culture test tubes, or their equivalent, are closedwith a screwcap having a suitable elastomeric liner. The
exposed surfaceof the elastomeric lineriscompletelyprotected with an inert soliddisk50-75 IJm in thickness. Asuitable
disk can be fabricated from polytef.
Preparation of apparatus: Cleanse all glassware thoroughlywith chromicacidcleansing mixtureand, ifnecessary, with hot
nitric acidfollowed by prolonged rinsing with Sterile Water for Injection. Sterilize and dry by a suitableprocessfor containers
and devices usedfor extraction,transfer, or administration oftest material. If ethylene oxide isused as the sterilizing agent,
allow NlT 48 h for complete degassing.
• PROCEDURE .
Preparation of sample for extracts: Prepare as directed in the Procedure in (88).
Preparation of extracts: Prepare as directed for Preparation of extracts in (88) using either Sodium Chloride Injection [0.9%
sodium chloride(NaCI)] or serum-free mammalian cell culture media as Extraction solvents. [NOTE-If extractionisdone at
37° for 24 h in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any
instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence.]
• AGAR DIFFUSION TEST
This test isdesigned for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from
mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens. Extracts of
materials that are to be tested are applied to a piece of filter paper.
Sample preparation: Use extracts prepared as directed, or use portions of the test specimens havingflat surfaces NlT
100 rnrn- in surfacearea.
Positive control preparation: Proceed as directed for Sample preparation.
Negative control. preparation: Proceed as directed for Sample preparation.
Procedure: Using 7 ml of cell suspension prepared as directed in Cell culture preparation, prepare the monolayers in plates
having a 60-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with
serum-supplemented culture medium containing NMT 2% of agar. [NoTE-The quality of the agar must be adequate to

1 Furtherdetailsare given inthe following publicationsof the American SocietyforTestingand Materials, 1916 Race St., Philadelphia, PA19103: Standard
test method for agar diffusion cell culture screening for cytotoxicity, ASTM Designation F895-84; Standard practicefor direct contact cellculture
evaluation of materialsfor medical devices, ASTM Designation F813-83.
2 ZDEC and ZDBC polyurethanes are available from the Food and Drug SafetyCenter, Hatano Research Institute, Ochiai 729-5, Hadanoshi, Kanagawa
257, Japan.

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USP 43 Biological Tests / (87) 6515

support cell growth. The agar layermust be thin enough to permitdiffusion of leached chemicals.] Place the flat surfaces
of Sample preparation, Positive controlpreparation, and Negative controlpreparation or their extracts in an appropriate
extracting medium, in duplicate cultures in contact with the solidified agar surface. Use no more than three specimens
per prepared plate. Incubate all culturesfor NLT 24 h at 37 ± 1 preferably in a humidified incubator containing 5 ± 1%
0
,

of carbon dioxide. Examine each culture around each sample, negative control,and positive control under a microscope,
using a suitablestain, ifdesired. .
Interpretation of results: The biological reactivity (cellular degeneration and malformation) is described and rated on a
scale of 0-4 (see Table 7). Measure the responses of the cell cultures to the Sample preparation, the Positive control
preparation, and the Negative controlpreparation. The cell culture test system issuitableifthe observed responses to the
Negative controlpreparation is grade 0 (no reactivity) and to the Positive controlpreparation is at least grade 3 (moderate).
Thesample meets the requirementsof the test ifthe responseto the Sample preparation isnot greater than grade 2 (mildly
reactive). Repeatthe procedure ifthe suitability of the system is not confirmed.
Table 1. Reactivity Grades for Agar Diffusion Test and Direct Contact Test
Grade Reactivity Description of Reactivity Zone
0 None No detectable zone around or under specimen
1 Slight Some malformed or degenerated cells under specimen
2 Mild Zone limited to area under specimen and less than 0.45 cm beyond specimen
3 Moderate Zone extends 0.45-1 .0 cm beyond specimen
4 Severe Zone extends greater than 1.0 cm beyond specimen

• DIRECT CONTACT TEST

This test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of
leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very
low- or high-density materials that could cause mechanical damage to the cells.
Sample preparation: Use portions of the test specimen havingflat surfaces NLT 100 rnm' in surfacearea.
Positive control preparation: Proceed as directed for Sample preparation.
Negative control preparation: Proceed as directed for Sample preparation.
Procedure: Using 2 mL of cell suspension prepared as directed in Cellculturepreparation, prepare the monolayers in plates
havinga 35-mm diameter: Following incubation,aspiratethe culture mediumfrom the cultures, and replace it with 0.8 mL
of fresh culture medium. Place a single Sample preparation, a Positive controlpreparation, and a Negativecontrol
preparation in each of the duplicate cultures. Incubate all culturesfor NLT 24 h at 37 ± 1 in a humidified incubator
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containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample preparation, a Positive control
preparation, and a Negative controlpreparation, under a microscope, using a suitablestain, ifdesired.
Interpretation of results: Proceed as directed for Interpretation of results in Agar Diffusion Test. The sample meets the
requirements of the test ifthe responseto the Sample preparation is not greater than grade 2 (mildly reactive). Repeatthe
procedure ifthe suitability of the system is not confirmed.
• ELUTION TEST
This test isdesignedfor the evaluation ofextractsof polymeric materials. The procedureallows for extraction of the specimens
at physiological or nonphysiological temperatures for varying time intervals. It is appropriate for high-density materials
and for dose-response evaluations.
Sample preparation: Prepare as directed in Preparation of extracts, using either Sodium Chloride Injection [0.9% sodium
chloride (NaCl)] or serum-free mammalian cell culture media as Extraction solvents. If the sizeof the sample cannot be
readily measured, a massof NLT 0.1 g of elastomeric material or 0.2 g of plastic or polymeric material per mL of extraction
medium may be used. Alternatively, use serum-supplemented mammalian cell culture media as the extracting medium
to simulatemore closely physiological conditions. Preparethe extracts by heating for 24 h in an incubator containing 5
± 1% of carbon dioxide. Maintain the extraction temperature at 37 ± 1 because higher temperatures may cause
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,

denaturation of serum proteins.

Positive control preparation: Proceed as directed for Sample preparation.
Negative control preparation: Proceed as directed for Sample preparation.
Procedure: Using 2 mL of cell suspension prepared as directed in Cellculturepreparation, prepare the monolayers in plates
having a 35-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with
extracts of the Sample preparation, Positive controlpreparation, or Negative controlpreparation. The serum-supplemented
and serum-free cellculture media extracts are tested in duplicate without dilution (100%). The Sodium Chloride Injection
extract is diluted with serum-supplemented cell culture medium and tested in duplicate at 25% extract concentration.
Incubateall culturesfor 48 h at 37 ± 1 in a humidified incubator preferably containing5 ± 1% of carbon dioxide. Examine
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each culture at 48 h, under a microscope, using a suitablestain, if desired.

Interpretation of results: Proceed as directed for Interpretation of results in Agar Diffusion Test but use Table 2. The sample
meets the requirements of the test ifthe response to the Sample preparation is not greater than grade 2 (mildly reactive).
Repeatthe procedure ifthe SUitability of the systemisnot confirmed. Fordose-response evaluations, repeat the procedure,
using quantitative dilutions of the sample extract.
Table 2. Reactivity Grades for Elution Test
Grade Reactivity Conditions of All Cultures
o None Discrete intracytoplasmic granules; no cell lysis

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