EP5110 细菌内毒素测试使用指南中英文11.docx

1、EP5110 细菌内毒素测试使用指南中英文11EP5.1.10 细菌内毒素测试使用指南（中英文1/2）2015-12-28 22:58:13|分类：EP07/2016: 501105.1.10. GUIDELINES FOR USING THE TEST FOR BACTERIAL ENDOTOXINS细菌内毒素测试使用指南1. INTRODUCTION概述Endotoxins from gram-negative bacteria are the most common cause of toxic reactions resulting from contamination of phar

2、maceutical products with pyrogens; their common pyrogenic activity is much higher than that of other known pyrogenic substances. These endotoxins are lipopolysaccharides. Although there are a small number of pyrogens that possess a different structure, the conclusion is generally justified that the

3、absence of bacterial endotoxins in a substance or product implies the absence of pyrogenic components, provided the presence of non-endotoxin pyrogenic substances can be ruled out. The monocyte-activation test (2.6.30) is a suitable method to use to rule out the presence of non-endotoxin pyrogens in

4、 substances or products.革兰氏阳性菌产生的内毒素是最常见的有毒性反应原因，其原因是药品受到热源污染。其常见热源活性大大高于其它已知热源物质。这些内毒素为脂多糖。尽管有少量热源具有不同的结构，如果排除了非内毒素热源物质的存在的话，通常该原料药或制剂中不存在细菌内毒素的话就能下结论说其中不存在热源成分。单核细胞激活测试（2.6.30）是用来排除原料药或制剂中非内毒素热源存在的适当方法。The presence of endotoxins in a substance or product may be masked by factors interfering with t

5、he reaction between the endotoxins, the test reagents and the amoebocyte lysate. Also, the ability to detect endotoxins may be affected by storage conditions or storage time. Hence, the analyst who wishes to implement a test for bacterial endotoxins or to replace the pyrogen test by a test for bacte

6、rial endotoxins has to demonstrate that a valid test can be carried out on the substance or product concerned: this may entail a procedure for removing interference.原料药或制剂中含有的内毒素可能会被干扰因子通过与内毒素、测试用试剂和阿米巴样细胞鲎试剂的反应所掩蔽。存贮条件和存贮时间也可能影响其检出内毒素的能力。因此，期望进行细菌内毒素测试，或采用细菌内毒素测试取代热源测试的化验员必须证明对相关的原料药和制剂能进行有效的测试：可以通

7、过消除干扰因素来使得一个测试方法有效。As indicated in general chapter 2.6.14. Bacterial endotoxins, information must be available on the following 2 aspects before a test on a sample can be regarded as valid.正如通论2.6.14“细菌内毒素”中所指出，在对一个样品进行测试前必须获得以下两方面资料，方可认为测试有效：-The suitability of the material to be used for the test

8、has to be established. The absence of endotoxins in the water for BET (water for bacterial endotoxins test) and in the other reagents an consumables must be assured and the sensitivity of the amoebocyte lysate must be checked to confirm the sensitivity declared by the manufacturer.-要检查用于测试的物料的适用性。必须

9、确保BET用水和其它消耗性试剂中没有内毒素，必须检查阿米巴样细胞鲎试剂以确认其达到生产商所声称的灵敏度。-As the substance or product to be examined may interfere with the test, the sensitivity of the amoebocyte lysate is determined in the presence and in the absence of the substance or product to be examined. There must be no difference between the 2

10、 sensitivity values.-由于受测原料药或制剂可能会对测试产生干扰，阿米巴样细胞鲎试剂的灵敏度要在受测原料药或制剂存在时与不存在时分别测试。两者所得灵敏度值应该没有差异。General chapter 2.6.14. Bacterial endotoxins indicates methods for removing interfering factors; in the case of interference, another test must be carried out after such a method has been applied to check wh

11、ether the interference has indeed been neutralized or removed.通则2.6.14“细菌内毒素”给出了消除干扰因素的方法。如果有干扰存在，则在常规方法测试完后，还需要有采用另一方法来测试，以检查是否该干扰真的被中和或消除。This general chapter explains the reasons for the requirements in the test for bacterial endotoxins, then deals with reading and interpretation of the results.本

12、通则解释了细菌内毒素测试要求的理由，然后说明了结果读取和诠释要求。Replacement of the rabbit pyrogen test required in a pharmacopoeial monograph by an amoebocyte lysate test, or by other methods such as the monocyte-activation test or a test using recombinant factor C reagent as a replacement for the amoebocyte lysate, constitutes t

13、he use of an alternative method of analysis and hence requires demonstration that the method is appropriate for the given substance or product and gives a result consistent with that obtained with the prescribed method as described in the General Notices (see also section 12).采用阿米巴样细胞鲎试剂测试，或其它方法如单核细

14、胞激活测试，替代药典各论中所要求的兔热源测试，或使用重组因子C试剂来替代阿米巴样细胞鲎试剂，都被认为是使用替代分析方法，从而要求证明该方法适用于给定的原料药或制剂，证明其得到的结果与各论中所述预定方法得到的结果一致（参见第12部分）。The prescribed method for bacterial endotoxins may be stated in the monograph on a given substance or product. The use of a method other than the method prescribed in the monograph is

15、 considered as the use of an alternative method. Where no method is stated, any of methods A to F of general chapter 2.6.14. Bacterial endotoxins can be used.细菌内毒素所要求的方法可能会在指定物质或制剂各论里指定，如使用不同方法，则被认为是使用了替代方法。如果各论里没有指定方法，则在2.6.14细菌内毒素里的方法A至F均可使用。2. METHOD AND ACCEPTANCE CRITERIA测试方法和可接受标准2-1. METHODS

16、AND PRECAUTIONS TO BE TAKEN要使用的方法和预防措施The addition of endotoxins to amoebocyte lysate may result in turbidity, precipitation or gelation (gel-clot); initially only the gel-clot method was used in the Pharmacopoeia as an evaluation criterion in the test for bacterial endotoxins. The advantage was the

17、 simplicity of basing the decision to pass or fail the substance or product to be examined on the absence or presence of a gel-clot, visible with the naked eye. The quantitative methods C, D, E and F were developed later: they require more instrumentation, but they are easier to automate for regular

18、 testing of large numbers of samples of the same substance or product.向阿米巴样细胞鲎试剂中加入内毒素可能会产生混浊、沉淀或凝胶。刚开始只有凝胶方法进入药典用于细菌内毒素测试的评估标准。其优点是简单，对受试样品测试结果基于内容目视检查是否出现凝胶，来判定是否通过该测试。定量方法C、D、E和F是后来建立的，它们需要更多的仪器，但这些方法易于自动化，使日常大规模原料药或制剂样品测试变得更容易。Endotoxins may be adsorbed onto the surface of tubes or pipettes made

19、 from certain plastics or types of glass. Interference may appear due to the release of substances from plastic materials. Hence, the materials used must be checked.内毒素可以被一些型号的塑料或玻璃试管及吸管表面吸收，因此塑料材质上释放出的物质可能会产生干扰。所以必须对材质进行检查。2-2. ENDOTOXIN LIMIT CONCENTRATION内毒素限度浓度The decision to use the test for ba

20、cterial endotoxins as a limit test implies firstly that an endotoxin limit concentration must be defined for the substance or product to be examined, and secondly that the objective of the test is to know whether the endotoxin concentration in the sample to be examined is below or above this limit.

21、The quantitative methods C, D, E and F make it possible to determine the endotoxin concentration in the sample to be examined, but for compliance with the Pharmacopoeia and in routine quality control the final question is whether or not this concentration exceeds a defined limit.决定将细菌内毒素测试作为一个限度测试首先

22、表示定义该原料药或制剂中的细菌内毒素限度浓度，其次测试的目的是知道受测样品中内毒素浓度是否低于或高于此限度。定量方法C、D、E和F使得有可能确定受测样品中的内毒素浓度，但要符合药典，并且要用于日常质量控制，最后的问题是该浓度是否超出指定的限度。The dose of the substance or product to be examined must be taken into account in setting the endotoxin limit concentration: the limit is set so as to ensure that, as long as the

23、 endotoxin concentration in the substance or product remains below this limit, even the maximal dose administered by the intended route per hour does not contain sufficient endotoxin to cause a toxic reaction.在设定受检原料药和制剂的内毒素浓度限度时，必须要考虑其使用剂量。限度设定要确保只要原料药或制剂中的内毒素浓度低于此限度，即使通过既定摄入途径摄入了最大剂量，这些剂量也不会含有足够的内

24、毒素来引发毒性反应。When the endotoxin concentration in the substance or product exactly equals the endotoxin limit concentration, gelation will occur, as is the case when the endotoxin concentration is much higher, and the substance or product will fail the test, because the all-or-none character of the test

25、 makes it impossible to differentiate between a concentration exactly equal to the endotoxin limit concentration and one that is higher. It is only when no gelation occurs that the analyst may conclude that the endotoxin concentration is below the endotoxin limit.当原料药或制剂中的内毒素浓度正好等于内毒素限度浓度时，就会发生凝胶化反应

26、，该反应与内毒素浓度大大高于限度浓度时是一样的，这时原料药或制剂就被判定不合格，因为测试的特性是要么合格要么不合格，这样就没法区别浓度正好等于与大大超过两种情况。只有当没有凝胶反应发生时，化验员才可以得出结论内毒素浓度是低于内毒素的限度。For substances or products in the solid state, this endotoxin limit concentration per mass unit or per International Unit (IU) of substance or product has to be converted into a con

27、centration of endotoxin per milliliter of solution to be examined, as the test can only be carried out on a solution. The case of substances or products that already exist in the liquid state (such as infusion fluids) is discussed below.对于固体形态的原料药或制剂，原料药或制剂的每一质量单位或每国际单位（IU）的该内毒素限度浓度必须要转换成内每ml供试液里内毒素

28、浓度，因为测试只能在配制成溶液后进行。以液体形态存在的原料药或制剂的情况（例如灌洗液）讨论如下。2-3. CALCULATION OF THE ENDOTOXIN LIMIT内毒素限度计算The endotoxin limit for active substance administered parenterally, defined on the basis of dose, is equal to:静脉注射用原料药内毒素限度，根据基本摄入量以如下公式计算：K/MK = threshold progenic dose of endotoxin per kilogram of body ma

29、ss;M = maximum recommended bolus dose of product per kilogram of body massK：每KG体重内毒素热原剂量阈值M：第KG体重药品剂量最大建议快速静脉注射量When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose administered per hour.如果产品是频繁使用或持续注入，则M是每小时最大总摄入量The endotoxin limit depends o

30、n the product and its route of administration and may be stated in the monograph. Values for K are suggested in Tables 5.1.10-1.内毒素限度取决于药品及其摄入途径，可能会在各论中有表述。K值在表5.1.10-1中已经给出。For other routes, the acceptance criterion for bacterial endotoxins is generally determined on the basis of results obtained d

31、uring the development of the preparation.对于其它给药途径，细菌内毒素的可接受标准通常由制剂研发过程中获得的结果来确定。Table 5.1.10-1表5.1.10-1Route of administrationK给药途径KIntravenous5.0 IU of endotoxin per kilogram of body mass静脉5.0IU内毒素/KG体重Interavenous for radiopharmaceuticals2.5 IU of endotoxin per kilogram of body mass放射药物静脉2.5IU内毒素/

32、KG体重Intrathecal0.2 IU of endotoxin per kilogram of body mass鞘内0.2IU内毒素/KG体重Perenteral formulations administered per square meter of body surface100IU/m2非肠道给药，按每平方米体表面积100IU/m22-4. CONSIDERATIONS WHEN ESTABLISHING AN ENDOTOXIN LIMIT FOR A SPECIFIC SUBSTANCE OR PRODUCT在制订特定原料药或制剂内毒素限度时要考虑的内容The endotoxin limit for a substance or product is established with consideration of the following aspects.在制订原料药或制剂内毒素限度时要考虑以下方面：Calculated endotoxin limit.The endotoxin limit is calculated as described in section 2-3