〈85〉 BACTERIAL ENDOTOXINS TEST

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2 <strong>〈85〉</strong> Bacterial Endotoxins Test / Biological Tests USP 36Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot TechniquesEndotoxin Concentration/Solution to Which Endotoxin Dilution Endotoxin Number ofSolution Is Added Diluent Factor Concentration ReplicatesA a None/Sample Solution — — — 4B b 2λ/Sample Solution Sample Solution 1 2λ 42 1λ 44 0.5λ 48 0.25λ 4C c 2λ/Water for BET Water for BET 1 2λ 22 1λ 24 0.5λ 28 0.25λ 2D d None/Water for BET — — — 2a Solution A: A Sample Solution of the preparation under test that is free of detectable endotoxins.b Solution B: Test for interference.c Solution C: Control for labeled lysate sensitivity.d Solution D: Negative control of Water for BET.ensure both the precision and validity of the test, perform operating as described for Test for Confirmation of Labeledthe tests for confirming the labeled lysate sensitivity and for Lysate Sensitivity. The geometric mean endpoint concentrainterferingfactors as described in Preparatory Testing, imme- tions of Solutions B and C are determined using the formuladiately below.described in the Test for Confirmation of Labeled Lysate Sensitivity.The test for interfering factors must be repeated whenPreparatory Testingany condition changes that is likely to influence the result ofthe test.The test is considered valid when all replicates of Solu-Test for Confirmation of Labeled Lysate Sensitivity— tions A and D show no reaction and the result of Solution CConfirm in four replicates the labeled sensitivity, λ, ex- confirms the labeled sensitivity.pressed in EU/mL of the lysate prior to use in the test. The If the sensitivity of the lysate determined in the presencetest for confirmation of lysate sensitivity is to be carried out of Solution B is not less than 0.5λ and not greater than 2λ,when a new batch of lysate is used or when there is any the Sample Solution does not contain factors that interferechange in the test conditions that may affect the outcome under the experimental conditions used. Otherwise, theof the test. Prepare standard solutions having at least four Sample Solution to be examined interferes with the test.concentrations equivalent to 2λ, λ, 0.5λ, and 0.25λ by di- If the sample under test does not comply with the test atluting the USP Endotoxin RS with Water for BET.a dilution less than the MVD, repeat the test using a greaterMix a volume of the Lysate TS with an equal volume dilution, not exceeding the MVD. The use of a more sensi-(such as 0.1-mL aliquots) of one of the Standard Endotoxin tive lysate permits a greater dilution of the sample to beSolutions in each test tube. When single test vials or ampuls examined, and this may contribute to the elimination ofcontaining lyophilized lysate are used, add solutions directly interference.to the vial or ampul. Incubate the reaction mixture for aInterference may be overcome by suitable treatment suchconstant period according to the directions of the lysate as filtration, neutralization, dialysis, or heating. To establishmanufacturer (usually at 37 ± 1° for 60 ± 2 min), avoiding that the chosen treatment effectively eliminates interferencevibration. To test the integrity of the gel, take each tube in without loss of endotoxins, perform the assay describedturn directly from the incubator, and invert it through about above using the preparation to be examined to which Stan-180° in one smooth motion. If a firm gel has formed that dard Endotoxin has been added and which has then beenremains in place upon inversion, record the result as posi- submitted to the chosen treatment.tive. A result is negative if an intact gel is not formed. Thetest is considered valid when the lowest concentration ofthe standard solutions shows a negative result in all replicateLimit Testtests.The endpoint is the smallest concentration in the series of Procedure—Prepare Solutions A, B, C, and D as shown indecreasing concentrations of standard endotoxin that clots Table 2, and perform the test on these solutions followingthe lysate. Determine the geometric mean endpoint by cal- the procedure above for Preparatory Testing, Test for Confirculatingthe mean of the logarithms of the endpoint con- mation of Labeled Lysate Sensitivity.centrations of the four replicate series and then taking theantilogarithm of the mean value, as indicated in the follow- Table 2. Preparation of Solutions for the Gel-Clot Limit Testing formula:Endotoxin Concentration/geometric mean endpoint concentration = antilog (Σe/f)Solution to WhichNumber ofSolution* Endotoxin Is Added Replicateswhere Σe is the sum of the log endpoint concentrations of A None/Diluted Sample Solution 2the dilution series used, and f is the number of replicate test B 2λ/Diluted Sample Solution 2tubes. The geometric mean endpoint concentration is the* Prepare Solution A and the positive product control Solution B usingmeasured sensitivity of the lysate (in EU/mL). If this is nota dilution not greater than the MVD and treatments as described forless than 0.5λ and not more than 2λ, the labeled sensitivitythe Test for Interfering Factors in Preparatory Testing. The positive conisconfirmed and is used in tests performed with this lysate.trol Solutions B and C contain the Standard Endotoxin Solution at aTest for Interfering Factors—Usually prepare solutions concentration corresponding to twice the labeled lysate sensitivity.(A–D) as shown in Table 1, and perform the inhibition/en- The negative control Solution D consists of Water for BET.hancement test on the Sample Solutions at a dilution lessthan the MVD, not containing any detectable endotoxins,
USP 36 Biological Tests / <strong>〈85〉</strong> Bacterial Endotoxins Test 3Table 2. Preparation of Solutions for the Gel-Clot Limit positive; and (3) The geometric mean endpoint concentra-Test (Continued) tion of Solution C is in the range of 0.5λ to 2λ.To determine the endotoxin concentration of Solution A,Endotoxin Concentration/calculate the endpoint concentration for each replicate bySolution to WhichNumber ofmultiplying each endpoint dilution factor by λ. The endo-Solution* Endotoxin Is Added Replicatestoxin concentration in the Sample Solution is the endpointC 2λ/Water for BET 2 concentration of the replicates. If the test is conducted withD None/Water for BET 2 a diluted Sample Solution, calculate the concentration of en-* Prepare Solution A and the positive product control Solution B using dotoxin in the original Sample Solution by multiplying by thea dilution not greater than the MVD and treatments as described for dilution factor. If none of the dilutions of the Sample SolutheTest for Interfering Factors in Preparatory Testing. The positive con- tion is positive in a valid assay, report the endotoxin concentrolSolutions B and C contain the Standard Endotoxin Solution at a tration as less than λ (if the diluted sample was tested, reconcentrationcorresponding to twice the labeled lysate sensitivity. port as less than λ times the lowest dilution factor of theThe negative control Solution D consists of Water for BET.sample). If all dilutions are positive, the endotoxin concentrationis reported as equal to or greater than the greatestdilution factor multiplied by λ (e.g., initial dilution factorInterpretation—The test is considered valid when both times eight times λ in Table 3).replicates of Solutions B and C are positive and those of Solutestif the concentration of endotoxin in both replicates isThe preparation under test meets the requirements of thetion D are negative. When a negative result is found forboth replicates of Solution A, the preparation under test less than that specified in the individual monograph.complies with the test. When a positive result is found forboth replicates of Solution A, the preparation under testdoes not comply with the test.PHOTOMETRIC QUANTITATIVE TECHNIQUESWhen a positive result is found for one replicate of SolutionA and a negative result is found for the other, repeatthe test. In the repeat test, the preparation under test com-Turbidimetric Techniqueplies with the test if a negative result is found for both replicatesof Solution A. The preparation does not comply with This technique is a photometric assay measuring increasesthe test if a positive result is found for one or both replicates in reactant turbidity. On the basis of the particular assayof Solution A. However, if the preparation does not comply principle employed, this technique may be classified as eiwiththe test at a dilution less than the MVD, the test may ther an endpoint-turbidimetric assay or a kinetic-turbidimetberepeated using a greater dilution, not exceeding the ric assay. The endpoint-turbidimetric assay is based on theMVD.quantitative relationship between the concentration of endotoxinsand the turbidity (absorbance or transmission) ofQuantitative Testthe reaction mixture at the end of an incubation period.The kinetic-turbidimetric assay is a method to measure eitherthe time (onset time) needed to reach a predeterminedProcedure—The test quantifies bacterial endotoxins in absorbance or transmission of the reaction mixture, or theSample Solutions by titration to an endpoint. Prepare Solu- rate of turbidity development. The test is carried out at thetions A, B, C, and D as shown in Table 3, and test these incubation temperature recommended by the lysate manusolutionsby following the procedure in Preparatory Testing, facturer (which is usually 37 ± 1°).Test for Confirmation of Labeled Lysate Sensitivity.Calculation and Interpretation—The test is consideredvalid when the following three conditions are met: (1) BothChromogenic Techniquereplicates of negative control Solution D are negative; (2)Both replicates of positive product control Solution B areThis technique is an assay to measure the chromophorereleased from a suitable chromogenic peptide by the reac-Table 3. Preparation of Solutions for the Gel-Clot AssayEndotoxin Concentration/Solution to Which Endotoxin Dilution Endotoxin Number ofSolution Is Added Diluent Factor Concentration ReplicatesA a None/Sample Solution Water for BET 1 — 22 — 24 — 28 — 2B b 2λ/Sample Solution — 1 2λ 2C c 2λ/Water for BET Water for BET 1 2λ 22 1λ 24 0.5λ 28 0.25λ 2D d None/Water for BET — — — 2a 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 Water for BET to make a dilution series of four tubes containing theSample Solution under test at concentrations of 1, 1 /2, 1 /4, and 1 /8 relative to the concentration used in the Test for Interfering Factors. Other dilutionsup to the MVD may be used as appropriate.b Solution B: Solution A containing standard endotoxin at a concentration of 2λ (positive product control).c Solution C: Two replicates of four tubes of Water for BET containing the standard endotoxin at concentrations of 2λ, λ, 0.5λ, and 0.25λ,respectively.d Solution D: Water for BET (negative control).
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〈85〉 BACTERIAL ENDOTOXINS TEST

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