Q4B 附件14 细菌内毒素测试

Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions Annex 14: Bacterial Endotoxins TestGeneral ChapterThis draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.For questions regarding this draft document contact (CDER) Robert King 301-796-1242, or (CBER) Christopher Joneckis 301-827-0373.5 10 15 20 25 30 Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 201023 INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL4 REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE6789 E VALUATION AND R ECOMMENDATION OFP HARMACOPOEIAL T EXTS FOR U SE IN THE ICH R EGIONS11 ON12 B ACTERIAL E NDOTOXINS T EST G ENERAL C HAPTER13 Q4B A NNEX 1414161718 Current Step 2 Version 119 dated 10 June 20102122232426272829 At Step 2 of the ICH Process, a consensus draft text or guideline, agreed by the appropriate ICHExpert Working Group, is transmitted by the ICH Steering Committee to the regulatory31 authorities of the three ICH regions (the European Union, Japan and the USA) for internal and32 external consultation, according to national or regional procedures.Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010333435363738394041424344 Q4B Annex 1445 Document History4647 Current Step 2 version48Code History Date Q4B Annex 14 Approval by the Steering Committee under Step 2 and release10 June 2010for public consultation.495051Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 20105253 E VALUATION AND R ECOMMENDATION OF54 P HARMACOPOEIAL T EXTS FOR U SE IN THE ICH R EGIONS55 ON56 B ACTERIAL E NDOTOXINS T EST G ENERAL C HAPTER57 Q4B Annex 145859 Draft ICH Consensus Guideline60 Released for Consultation on 10 June 2010, at Step 2 of the ICH Process616263 TABLE OF CONTENTS646566 1. INTRODUCTION (1)6768 2. Q4B OUTCOME (1)6970 3. TIMING OF ANNEX IMPLEMENTATION (1)7172 4. CONSIDERATIONS FOR IMPLEMENTATION (1)7374 4.1 General Consideration (1)7576 4.2 FDA Consideration (2)7778 4.3 EU Consideration (2)7980 4.4 MHLW Consideration (2)8182 4.5 Health Canada Consideration (2)8384 5. REFERENCES USED FOR THE Q4B EVALUATION (2)85Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 20108687 E VALUATION AND R ECOMMENDATION OF88 P HARMACOPOEIAL T EXTS FOR U SE IN THE ICH R EGIONS89 ON90 B ACTERIAL ENDOTOXINS T EST G ENERAL C HAPTER9192 Q4B Annex 14939495 1. INTRODUCTION9697 This annex is the result of the Q4B process for the Bacterial Endotoxins Test General Chapter.9899 The proposed texts were submitted by the Pharmacopoeial Discussion Group (PDG).100101 2. Q4B OUTCOME102103104 2.1. Analytical Procedures105106 The ICH Steering Committee, based on the evaluation by the Q4B Expert Working 107 Group (EWG), recommends that the analytical procedures described in the official108 pharmacopoeial texts, Ph.Eur. 2.6.14. Bacterial Endotoxins, JP 4.01 Bacterial109 Endotoxins Test, and USP General Chapter <85> Bacterial Endotoxins Test, can be 110 used as interchangeable in the ICH regions subject to the following conditions:111112 2.1.1 Any of the three techniques can be used for the test. In the event of doubt or113 dispute, the gel-clot limit test should be used to make the final decision on114 compliance for the product being tested.115116 2.1.2 The Endotoxin Reference Standard should be calibrated to the current WHO117 (World Health Organization) International Standard for Endotoxin.118119 2.2 Acceptance Criteria120 The evaluated texts did not contain acceptance criteria. Endotoxin limits should be121 specified in the application dossier unless otherwise specified in an individual monograph. 122123 3. TIMING OF ANNEX IMPLEMENTATION124125 When this annex is implemented (incorporated into the regulatory process at ICH Step 5) in a 126 region, it can be used in that region. Timing might differ for each region.127128 4. CONSIDERATIONS FOR IMPLEMENTATION129130 4.1 General Consideration131132 When sponsors or manufacturers change their existing methods to the implemented133 Q4B-evaluated pharmacopoeial texts that are referenced in Section 2.1 of this annex, 134 any change notification, variation, and/or prior approval procedures should be handled 135 in accordance with established regional regulatory mechanisms pertaining to compendial 136 changes.137Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010138139 Bacterial Endotoxins Test General Chapter140141142 4.2 FDA Consideration143144 Based on the recommendation above, and with reference to the conditions set forth in 145 this annex, the pharmacopoeial texts referenced in Section 2.1 of this annex can be 146 considered interchangeable. However, FDA might request that a company147 demonstrate that the chosen method is acceptable and suitable for a specific material 148 or product, irrespective of the origin of the method.149150 4.3 European Union Consideration151152 For the European Union, regulatory authorities can accept the reference in a marketing 153 authorisation application, renewal or variation application citing the use of the154 corresponding text from another pharmacopoeia as referenced in Section 2.1, in155 accordance with the conditions set out in this annex, as fulfilling the requirements for 156 compliance with the Ph. Eur. Chapter 2.6.14. on the basis of the declaration of157 interchangeability made above.158159 4.4 MHLW Consideration160161 The pharmacopoeial texts referenced in Section 2.1 of this annex can be used as162 interchangeable in accordance with the conditions set out in this annex. Details of 163 implementation requirements will be provided in the notification by MHLW when this 164 annex is implemented.165166 4.5 Health Canada Consideration167168 In Canada any of the pharmacopoeial texts cited in Section 2.1 of this annex and used 169 in accordance with the conditions set out in this annex can be considered170 interchangeable.171172173 5. REFERENCES USED FOR THE Q4B EVALUATION174175 5.1 The PDG Stage 5B sign-off document (Rev. 1 – Correction 1): Japanese176 Pharmacopoeial Forum, Volume 18, number 4 (December 2009).177178 5.2 The pharmacopoeial references for the Bacterial Endotoxins Test General Chapter for 179 this annex are:180181 5.2.1 European Pharmacopoeia (Ph. Eur.):182 Supplement 6.6 (official January 1, 2010), Bacterial Endotoxins (reference183 01/2010:20614).184185 5.2.2 Japanese Pharmacopoeia (JP):186 General Test 4.01 Bacterial Endotoxins Test as it will appear in the JP Sixteenth 187 Edition (March 31, 2011). The draft English version of the JP text provided by 188 MHLW is appended (see Appendix A).189190 5.2.3 United States Pharmacopeia (USP):191 Text for <85> Bacterial Endotoxins Test, USP 33 Reissue (published April 2010 192 and official October 1, 2010).193Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010194195196 APPENDIX A197198 Draft JP XVI English Text Provided by MHLW199200 4. Biological Tests/Biochemical Tests/Microbial Tests201 4.01 Bacterial Endotoxins Test202203 This test is harmonized with the European Pharmacopoeia and the U. S. Pharmacopeia. 204205 Bacterial Endotoxins Test is a test to detect or quantify bacterial endotoxins of gram­206 negative bacterial origin using an amoebocyte lysate prepared from blood corpuscle207 extracts of horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). There are 208 two types of techniques for this test: the gel-clot techniques, which are based on gel209 formation by the reaction of the lysate TS with endotoxins, and the photometric210 techniques, which are based on endotoxin-induced optical changes of the lysate TS. The 211 latter include turbidimetric techniques, which are based on the change in lysate TS212 turbidity during gel formation, and chromogenic techniques, which are based on the213 development of color after cleavage of a synthetic peptide-chromogen complex.214 Proceed by any one of these techniques for the test. In the event of doubt or dispute, 215 the final decision is made based on the gel-clot techniques, unless otherwise indicated. 216 The test is carried out in a manner that avoids endotoxin contamination.217218 1. Apparatus219 Depyrogenate all glassware and other heat-stable materials in a hot-air oven using a 220 validated process. Commonly used minimum time and temperature settings are 30221 minutes at 250°C. If employing plastic apparatus, such as multi-well plates and tips for 222 micropipettes, use only that which has been shown to be free of detectable endotoxin and 223 which does not interfere with the test.224225 2. Preparation of Solutions226227 2.1 Standard Endotoxin Stock Solution228 Prepare Standard Endotoxin Stock Solution by dissolving Japanese Pharmacopoeia 229 Reference Standard Endotoxin in water for bacterial endotoxins test (BET). Endotoxin is 230 expressed in Endotoxin Units (EU). One EU is equal to one International Unit (IU) of 231 endotoxin.232233 2.2 Standard Endotoxin Solution234 After mixing Standard Endotoxin Stock Solution thoroughly, prepare appropriate serial 235 dilutions of Standard Endotoxin Solution, using water for BET. Use dilutions as soon as 236 possible to avoid loss of activity by adsorption.237238 2.3 Sample Solutions239 Unless otherwise specified, prepare sample solutions by dissolving or diluting drugs, 240 using water for BET. If necessary, adjust the pH of the solution to be examined so that 241 the pH of the mixture of the lysate TS and sample solution falls within the specified pH 242 range for the lysate to be used. This usually applies to a sample solution with a pH in theFinal STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010243 range of 6.0 to 8.0. TSs or solutions used for adjustment of pH may be prepared using 244 water for BET, and then stored in containers free of detectable endotoxin. The TSs or 245 solutions must be validated to be free of detectable endotoxin and interfering factors. 246247 3. Determination of Maximum Valid Dilution248 The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample 249 solution at which the endotoxin limit can be determined.250 Determine the MVD from the following equation:251252 Endotoxin limit × Concentration of sample solution253 MVD ＝ ―――――――――――――――――――――――254 λ255256 Endotoxin limit:257 The endotoxin limit for injections, defined on the basis of dose, equals K/M, where 258 K is a minimum pyrogenic dose of endotoxin per kg body mass (EU/kg), and M is 259 equal to the maximum bolus dose of product per kg body mass. When the product is 260 to be injected at frequent intervals or infused continuously, M is the maximum total 261 dose administered in a single hour period.262 Concentration of sample solution:263 mg/mL in the case of endotoxin limit specified by mass (EU/mg)264 mEq/mL in the case of endotoxin limit specified by equivalent (EU/mEq)265 Units/mL in the case of endotoxin limit specified by biological unit (EU/Unit)266 mL/mL in the case of endotoxin limit specified by volume (EU/mL)267 λ: the labeled lysate sensitivity in the gel-clot techniques (EU/mL) or the lowest point 268 used (EU/mL) in the standard regression curve of the turbidimetric or chromogenic 269 techniques270271 4. Gel-clot techniques272 The gel-clot techniques detect or quantify endotoxins based on clotting of the lysate TS 273 in the presence of endotoxin. To ensure both the precision and validity of the test,274 perform the tests for confirming the labeled lysate sensitivity (4.1.1) and for interfering 275 factors (4.1.2) as described under Preparatory testing (4.1).276 4.1 Preparatory testing277 4.1.1 Test for confirmation of labeled lysate sensitivity278 The labeled sensitivity of lysate is defined as the lowest concentration of endotoxin 279 that is needed to cause the lysate TS to clot under the conditions specified for the lysate 280 to be used.281 The test for confirmation of the labeled lysate sensitivity is to be carried out when each 282 new lot of lysate is used or when there is any change in the experimental conditions283 which may affect the outcome of the test.284 Prepare standard solutions having four concentrations equivalent to 2 λ, λ, 0.5 λ and 285 0.25 λ by diluting the Standard Endotoxin Stock Solution with water for BET. Mix a 286 volume of the lysate TS with an equal volume of one of the standard solutions (usually, 287 0.1 mL aliquots) in each test tube. When single test vials or ampoules containing288 lyophilized lysate are used, add solutions directly to the vial or ampoule.289 Keep the tubes (or containers such as vials or ampoules) containing the reaction290 mixture usually at 37 ± 1°C for 60 ± 2 minutes, avoiding vibration. To test the integrity 291 of the gel after incubation, invert each tube or container through approximately 180° inFinal STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010292 one smooth motion. If a firm gel has formed that remains in place upon inversion, record 293 the result as positive. A result is negative if either a firm gel is not formed, or if a fragile 294 gel has formed but flows out upon inversion.295 Making the standard solutions of four concentrations one set, test four replicates of the 296 set.297 The test is valid when 0.25 λ of the standard solution shows a negative result in each 298 set of tests. If the test is not valid, repeat the test after verifying the test conditions.299 The endpoint is the last positive test in the series of decreasing concentrations of300 endotoxin. Calculate the geometric mean endpoint concentration of the four replicate 301 series using the following formula:302303 Geometric Mean Endpoint Concentration ＝ antilog (∑e/f )304 ∑e ＝ the sum of the log endpoint concentrations of the dilution series used305 f＝ the number of replicates306 If the geometric mean endpoint concentration is not less than 0.5 λ and not more than 2 307 λ, the labeled sensitivity is confirmed, and is used in tests performed with this lysate. 308 4.1.2 Test for interfering factors309 This test is performed to check for the presence of enhancing or inhibiting factors for 310 the reaction in sample solutions.311 Prepare the solutions A, B, C and D according to Table 4.01-1, and test solutions A 312 and B and solutions C and D in quadruplicate and in duplicate, respectively. Concerning 313 the incubation temperature, incubation time, and procedure for the confirmation of gel 314 formation, follow the procedure described in 4.1.1.315 The geometric mean endpoint concentrations of B and C solutions are determined by 316 using the formula described in 4.1.1.317 This test must be repeated when there is any change in the experimental conditions which 318 may affect the outcome of the test.319320 Table 4.01-1Solution Endotoxin concentration/Solution to whichendotoxin is addedDiluentDilutionfactorEndotoxinconcentrationNumber ofreplicatesA*1 0/Sample solution — — — 4B*2 2λ/Sample solution Samplesolution12482λ1λ0.5λ0.25λ4C*3 2λ/Water for BET WaterforBET12482λ1λ0.5λ0.25λ2D*4 0/Water for BET — — — 2 321 *1 Negative control. Sample solution only.322 *2 Sample solutions added with standard endotoxin (for testing interfering factors). 323 *3 Standard endotoxin solutions for confirmation of the labeled lysate sensitivity. 324 *4 Negative control. Water for BET only.Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010325 The test is valid if solutions A and D show no reaction and the result for solution C 326 confirms the labeled lysate sensitivity.327 If the geometric mean endpoint concentration of solution B is not less than 0.5 λ and 328 not greater than 2 λ, the sample solution being examined does not contain interfering 329 factors and complies with the test for interfering factors. Otherwise the sample solution 330 interferes with the test.331 If the sample under test does not comply with the test at a dilution less than the MVD, 332 repeat the test using a greater dilution, not exceeding the MVD. The use of a more333 sensitive lysate permits a greater dilution of the sample to be examined. Furthermore, 334 interference of the sample solution or diluted sample solution may be eliminated by335 suitable treatment, such as filtration, neutralization, dialysis or heat treatment. To336 establish that the treatment chosen effectively eliminates interference without loss of 337 endotoxins, perform the assay described above using the preparation to be examined to 338 which Standard Endotoxin has been added and which has then been submitted to the 339 chosen treatment.340 4.2 Limit test341 This method tests whether or not a sample contains endotoxins greater than the342 endotoxin limit specified in the individual monograph based on the gel formation in the 343 presence of endotoxins at a concentration of more than the labeled lysate sensitivity. 344 4.2.1 Procedure345 Prepare solutions A, B, C and D according to Table 4.01-2. Making these four346 solutions one set, test two replicates of the set.347 In preparing solutions A and B, use the sample solutions complying with 4.1.2.348 Concerning the test conditions including the incubation temperature, incubation time, and 349 procedure for the confirmation of gel formation, follow the procedure described in 4.1.1. 350351 Table 4.01-2SolutionEndotoxin concentration/Solution to which endotoxin is addedNumber ofreplicatesA*1 0/Sample solution 2B*2 2λ/Sample solution 2C*3 2λ/Water for BET 2D*4 0/Water for BET 2 352 *1 Sample solution for the limit test. The solution may be diluted not to exceed the MVD. 353 *2 Positive control. Sample solution at the same dilution as solution A, containing354 standard endotoxin at a concentration of 2λ.355 *3 Positive control. Standard endotoxin solution containing standard endotoxin at a356 concentration of 2λ.357 *4 Negative control. Water for BET only.358 4.2.2 Interpretation359 The test is valid when both replicates of solutions B and C are positive and those of 360 solution D are negative.361 When a negative result is found for both replicates of solution A, the sample complies 362 with the Bacterial Endotoxins Test.363 When a positive result is found for both replicates of solution A, the sample does not 364 comply with the test.Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010365 When a positive result is found for one replicate of solution A and a negative result is 366 found for the other, repeat the test. In the repeat test, the sample complies with the test if 367 a negative result is found for both replicates of solution A. The sample does not comply 368 with the test if a positive result is found for one or both replicates of solution A.369 However, if the sample does not comply with the test at a dilution less than the MVD, 370 the test may be repeated using a greater dilution, not exceeding the MVD.371 4.3 Quantitative Test372 This method measures endotoxin concentrations of samples by determining an373 endpoint of gel formation.374 4.3.1 Procedure375 Prepare solutions A, B, C and D according to Table 4.01-3. Making these four376 solutions one set, test two replicates of the set. When preparing solutions A and B, use 377 sample solutions complying with 4.1.2. Concerning the test conditions, follow the378 procedure described in 4.1.1.379380 Table 4.01-3Solution Endotoxin concentration/Solution to whichendotoxin is addedDiluentDilutionFactorEndotoxinconcentrationNumber ofreplicatesA*1 0/Sample solution WaterforBET1248————2B*2 2λ/Sample solution — 1 2λ 2C*3 2λ/Water for BET WaterforBET12482λ1λ0.5λ0.25λ2D*4 0/Water for BET — — — 2 381 *1 Sample solutions for the Quantitative test. The dilution range of the dilution series may 382 be changed as appropriate, but not exceeding the MVD.383 *2 Positive control. Sample solution at the same dilution as the solution A diluted at the 384 lowest dilution factor, containing standard endotoxin at a concentration of 2λ.385 *3 Standard endotoxin solutions for confirmation of the labeled lysate sensitivity.386 *4 Negative control. Water for BET only.387388 4.3.2 Calculation and interpretation389 The test is valid when the following three conditions are met: (a) both replicates of the 390 negative control solution D are negative, (b) both replicates of the positive product391 control solution B are positive and (c) the geometric mean endpoint concentration of 392 solution C is in the range of 0.5 λ to 2 λ.393 The endpoint is defined as the maximum dilution showing the last positive test in the 394 dilution series of solution A, and the endotoxin concentration of the sample solution is 395 calculated by multiplying the endpoint dilution factor by λ.396 If none of the dilutions of solution A is positive, report the endotoxin concentration of 397 the sample solution as less than λ × the lowest dilution factor of the sample solution.Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010398 If all dilutions are positive, the endotoxin concentration of the sample solution is399 reported as equal to or greater than the greatest dilution factor of solution A multiplied by 400 λ.401 Calculate the endotoxin concentration (in EU per mL, in EU per mg or mEq or in EU 402 per Unit) of the sample, based on the endotoxin concentration of the sample solution. The 403 sample complies with the Bacterial Endotoxins Test if the endotoxin concentration of the 404 sample in both replicates meets the requirement for the endotoxin limit (in EU per mL, in 405 EU per mg or mEq or in EU per Unit) specified in the individual monograph.406407 5. Photometric quantitative techniques408 5.1 Turbidimetric technique409 This technique measures the endotoxin concentrations of samples based on the410 measurement of turbidity change accompanying gel formation of the lysate TS. This411 technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric.412 The endpoint-turbidimetric technique is based on the quantitative relationship between 413 the concentration of endotoxins and the turbidity of the reaction mixture at a specified 414 reaction time.415 The kinetic-turbidimetric technique is based on the quantitative relationship between 416 the concentration of endotoxins and either the time needed to reach a predetermined417 turbidity of the reaction mixture or the rate of turbidity development.418 The test is usually carried out at 37 ± 1°C, and turbidity is expressed in terms of either 419 absorbance or transmission.420 5.2 Chromogenic technique421 This technique measures the endotoxin concentrations of sample solutions based on the 422 measurement of chromophore released from a synthetic chromogenic substrate by the 423 reaction of endotoxins with the lysate TS. This technique is classified as either endpoint­424 chromogenic or kinetic-chromogenic.425 The endpoint-chromogenic technique is based on the quantitative relationship between 426 the concentration of endotoxins and the release of chromophore at the end of an427 incubation period.428 The kinetic-chromogenic technique is based on the quantitative relationship between 429 the concentration of endotoxins and either the time needed to reach a predetermined430 absorbance (or transmittance) of the reaction mixture or the rate of color development. 431 The test is usually carried out at 37 ± 1°C.432 5.3 Preparatory testing433 To assure the precision and validity of the turbidimetric or chromogenic techniques, 434 perform both Test for assurance of criteria for the standard curve (5.3.1) and Test for 435 interfering factors (5.3.2), as indicated below.436 5.3.1 Test for assurance of criteria for the standard curve437 The test is to be carried out when each new lot of lysate is used or when there is any 438 change in the experimental conditions which may affect the outcome of the test.439 Using the Standard Endotoxin Solution, prepare at least three endotoxin concentrations 440 to generate the standard curve within the range of endotoxin concentrations indicated by 441 the instructions for the lysate used. Perform the test using at least three replicates of each 442 standard endotoxin concentration according to the optimal conditions for the lysate used 443 (with regard to volume ratios, incubation time, temperature, pH, etc.). If the desired range 444 is greater than two logs, additional standards should be included to bracket each log445 increase in the range of the standard curve.Final STEP 2 - Annex 14 Bacterial Endotoxins Test for Tallinn June 9, 2010446 If the absolute value of the correlation coefficient, | r |, is greater than or equal to 0.980 447 for the range of endotoxin concentrations set up, the criteria for the standard curve are 448 valid and the curve complies with the test.449 If the standard curve does not comply with the test, repeat the test after verifying the 450 test conditions.451 5.3.2 Test for interfering factors452 Prepare solutions A, B, C and D according to Table 4.01-4. Perform the test on these 453 solutions following the optimal conditions for the lysate used (with regard to volume of 454 sample solution and lysate TS, volume ratio of sample solution to lysate TS, incubation 455 time, etc.).456 The test for interfering factors must be repeated when any condition changes, which is 457 likely to influence the result of the test.458459460 Table 4.01-4SolutionEndotoxin concentration Solution to whichendotoxin is addedNumber of testtubes or wellsA*1 0 Sample solution Not less than 2B*2 Middle concentration of thestandard curveSample solution Not less than 2 C*3 At least 3 concentrations Water for BET Each not less than 2D*4 0 Water for BET Not less than 2 461 *1 Sample solution only (for assaying endotoxin concentration in the sample solution). 462 The sample solution may be diluted not to exceed the MVD.463 *2 Sample solution at the same dilution as solution A, containing added standard464 endotoxin at a concentration equal to or near the middle of the standard curve.465 *3 Standard endotoxin solutions at the concentrations used in 5.3.1 (for the standard466 curve).467 *4 Negative control. Water for BET only.468 The test is valid when the following conditions are met.469 1: The absolute value of the correlation coefficient of the standard curve generated 470 using solution C is greater than or equal to 0.980.471 2: The result with solution D does not exceed the limit of the blank value required in 472 the description of the lysate employed, or it is less than the endotoxin detection limit 473 of the lysate employed.474 Calculate the recovery of the endotoxin added to solution B from the concentration 475 found in solution B after subtracting the endotoxin concentration found in solution A. 476 When the recovery of the endotoxin added to solution B is within 50% to 200%, the 477 sample solution under test is considered to be free of interfering factors and the solution 478 complies with the test.479 When the endotoxin recovery is out of the specified range, the sample solution under 480 test is considered to contain interfering factors. If the sample under test does not comply 481 with the test, repeat the test using a greater dilution, not exceeding the MVD.482 Furthermore, interference of the sample solution or diluted sample solution not to exceed 483 the MVD may be eliminated by suitable treatment, such as filtration, neutralization,484 dialysis or heat treatment. To establish that the treatment chosen effectively eliminates 485 interference without loss of endotoxins, perform the assay described above using the。