EP5.1.10 细菌内毒素测试使用指南

EP5.1.10 细菌内毒素测试使用指南（中英文1/2）  

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07/2016: 50110

5.1.10. GUIDELINES FOR USING THE TESTFOR BACTERIAL ENDOTOXINS 细菌内毒素测试使用指南

1. INTRODUCTION 概述

Endotoxins from gram-negative bacteriaare the most common cause of toxic reactions resulting from contamination ofpharmaceutical products with pyrogens; their common pyrogenic activity is muchhigher than that of other known pyrogenic substances. These endotoxins arelipopolysaccharides. Although there are a small number of pyrogens that possessa different structure, the conclusion is generally justified that the absenceof bacterial endotoxins in a substance or product implies the absence ofpyrogenic components, provided the presence of non-endotoxin pyrogenicsubstances can be ruled out. The monocyte-activation test (2.6.30) is asuitable method to use to rule out the presence of non-endotoxin pyrogens insubstances 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 asubstance or product may be masked by factors interfering with the reactionbetween the endotoxins, the test reagents and the amoebocyte lysate. Also, theability to detect endotoxins may be affected by storage conditions or storagetime. Hence, the analyst who wishes to implement a test for bacterialendotoxins or to replace the pyrogen test by a test for bacterial endotoxinshas to demonstrate that a valid test can be carried out on the substance orproduct 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 aspectsbefore 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 beused for the test has to be established. The absence of endotoxins in the waterfor BET (water for bacterial endotoxins test) and in the other reagents anconsumables must be assured and the sensitivity of the amoebocyte lysate mustbe 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 beexamined may interfere with the test, the sensitivity of the amoebocyte lysateis determined in the presence and in the absence of the substance or product tobe examined. There must be no difference between the 2 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. Bacterialendotoxins indicates methods for removing interfering factors; in the case ofinterference, another test must be carried out after such a method has beenapplied to check whether the interference has indeed been neutralized orremoved.

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 thereasons for the requirements in the test for bacterial endotoxins, then dealswith reading and interpretation of the results.

本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 testrequired in a pharmacopoeial monograph by an amoebocyte lysate test, or byother methods such as the monocyte-activation test or a test using recombinantfactor C reagent as a replacement for the amoebocyte lysate, constitutes theuse of an alternative method of analysis and hence requires demonstration thatthe method is appropriate for the given substance or product and gives a resultconsistent with that obtained with the prescribed method as described in theGeneral 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 bacterialendotoxins may be stated in the monograph on a given substance or product. Theuse of a method other than the method prescribed in the monograph is consideredas the use of an alternative method. Where no method is stated, any of methodsA 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 AND PRECAUTIONS TO BE TAKEN 要使用的方法和预防措施

The addition of endotoxins to amoebocytelysate may result in turbidity, precipitation or gelation (gel-clot); initiallyonly the gel-clot method was used in the Pharmacopoeia as an evaluationcriterion in the test for bacterial endotoxins. The advantage was thesimplicity of basing the decision to pass or fail the substance or product tobe examined on the absence or presence of a gel-clot, visible with the nakedeye. The quantitative methods C, D, E and F were developed later: they requiremore instrumentation, but they are easier to automate for regular testing oflarge 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 thesurface of tubes or pipettes made from certain plastics or types of glass.Interference may appear due to the release of substances from plasticmaterials. 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 forbacterial endotoxins as a limit test implies firstly that an endotoxin limitconcentration must be defined for the substance or product to be examined, andsecondly that the objective of the test is to know whether the endotoxinconcentration in the sample to be examined is below or above this limit. Thequantitative methods C, D, E and F make it possible to determine the endotoxinconcentration in the sample to be examined, but for compliance with thePharmacopoeia and in routine quality control the final question is whether ornot 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 tobe examined must be taken into account in setting the endotoxin limitconcentration: the limit is set so as to ensure that, as long as the endotoxinconcentration in the substance or product remains below this limit, even themaximal dose administered by the intended route per hour does not containsufficient 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毒素来引发毒性反应。

When the endotoxin concentration in thesubstance or product exactly equals the endotoxin limit concentration, gelationwill occur, as is the case when the endotoxin concentration is much higher, andthe substance or product will fail the test, because the all-or-none characterof the test makes it impossible to differentiate between a concentrationexactly equal to the endotoxin limit concentration and one that is higher. Itis only when no gelation occurs that the analyst may conclude that theendotoxin 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，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 solidstate, this endotoxin limit concentration per mass unit or per InternationalUnit (IU) of substance or product has to be converted into a concentration ofendotoxin per milliliter of solution to be examined, as the test can only becarried out on a solution. The case of substances or products that alreadyexist 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 substanceadministered 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/M

K = threshold progenic dose of endotoxinper kilogram of body mass;

M = maximum recommended bolus dose ofproduct per kilogram of body mass

K：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 atfrequent intervals or infused continuously, M is the maximum total doseadministered 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 on theproduct 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 acceptancecriterion for bacterial endotoxins is generally determined on the basis ofresults obtained during 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5.1.10-1

Route of administration	K
给药途径	K
Intravenous	5.0 IU of endotoxin per kilogram of body mass
静脉	5.0IU内毒素/KG体重
Interavenous for radiopharmaceuticals	2.5 IU of endotoxin per kilogram of body mass
放射药物静脉	2.5IU内毒素/KG体重
Intrathecal	0.2 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 surface	100IU/m2
非肠道给药，按每平方米体表面积	100IU/m2

2-4. CONSIDERATIONS WHEN ESTABLISHING ANENDOTOXIN 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 orproduct 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 asdescribed in section 2-3. This represents a safety limit not to be exceeded ifthe product is to be administered to humans.

计算出的内毒素限度：内毒素限度按2-3部分所述进行计算。这个值表示的是人类服用时不能超过的一个安全限度。

Limit prescribed in an individualsubstance monograph.

The limit stated in an individualsubstance monograph frequently reflects what is achievable in a controlledproduction environment. The limit prescribed in a monograph can therefore belower than the calculated endotoxin limit. However a manufacturer may specify alimit that is more stringent than that stated in the monograph.

在单个物质各论中所述的限度：这个值通常反映的是在受控生产环境下可以达到的限度。在各论里所述的限度可能低于计算出的内毒素限度。但是，生产商可以使用比各论里更为严格的限度。

Process capability.

The capability of the process to reduceor remove bacterial endotoxins during manufacture might result in lowerendotoxin limits for specific processes.

工艺能力：生产过程中工艺降低或清除细菌内毒素的能力。特定的工艺有可能达到更低的内毒素限度。

Additional safety requirements.

Precautions are taken in considerationof patient (such as pediatric use, malnourished or cachectic patients, etc.),specific local requirements (e.g. countries might wish to operate with a loweraverage body weight of 60kg instead of 70kg frequently employed in Europe) orany additional safety margins requested by the competent authority.

其它的安全要求：要考虑患者因素（例如儿科用药、营养不良或恶病体质患者等）、特定地方要求（例如，希望使用较低的平均体重60kg替代欧洲常用的70kg的国家）或任何监管当局其它安全边际要求。

Formulation of the product.

The limit must take into considerationany theoretical bacterial endotoxin load introduced by any other componentsused for reconstitution and/or dilution of the product (e.g. water forinjections) or introduced by stating materials and/or raw material.

药品配方：限度必须考虑产品重建和/或稀释所用其它组件（例如注射用水），或起始物料和/或原料引入的所有理论存在的细菌内毒素。

2-5. MAXIMUM VALID DILUTION 最大有效稀释倍数

Which dilution of the substance orproduct is to be used in the test to obtain maximal assurance that a negativeresult means that the endotoxin concentration of the substance or product isless than the endotoxin limit and that a positive result means that the lysatedetected an endotoxin concentration equal to or greater than the endotoxinlimit? This dilution depends on the endotoxin limit and on the sensitivity ofthe lysate; it is called the maximum valid dilution (MVD) and its value may becalculated using the following expression:

在测试中，要最大地保证在得到阴性结果时即表示样品中内毒素浓度低于内毒素限度，而阳性结果即表示鲎试剂检出的内毒素浓度等于或高于内毒素限度，样品配制时该使用什么样的稀释倍数呢？该稀释倍数取决于内毒素限度，以及鲎试剂的灵敏度，被称为最大有效稀释倍数（MVD），其值可以使用以下公式计算：

Endotoxin limit X concentration of test solution

λ

Concentration of test solution:

-     In mg/mL. if the endotoxin limit isspecified by mass (IU/mg);

-     In Units/mL if the endotoxin limit isspecified by unit of biological activity (IU/Unit)

-     In mL/mL if the endotoxin limit isspecified by volume (IU/mL).

λ= the labeled lysate sensitivity in thegel-clot technique (IU/mL) or the lowest concentration used in the standardcurve of the turbidimetric or chromogenic techniques.

内毒素限度 X 供试液浓度

λ

样品溶液浓度

-     mg/mL，如果内毒素限度以质量为单位（IU/mg）

-     Units/mL，如果内毒素限度以生物效价为单位（IU/单位）

-     mL/mL，如果内毒素限度以体积为单位（IU/ml）

λ= 凝胶技术标识的鲎试剂灵敏度(IU/mL)，或浊度或比色技术标准曲线中所用的最低浓度

When the value of the MVD is not a wholenumber, a convenient whole number smaller than the MVD may be used for routinepurposes (which means preparing a solution of the substance or product that isless diluted than the MVD indicates). In this case, a negative result indicatesthat the endotoxin concentration of the substance or product lies below thelimit value. However, when the endotoxin concentration of the substance orproduct in such a test is less than the endotoxin limit but high enough to makethe reaction with the lysate result in a clot, the test may be positive underthese conditions. Hence, when a test with this convenient dilution factor ispositive, the substance or product is diluted to the MVD and the test isrepeated. In any case of doubt or dispute, the MVD must be used.

如果MVD值不是整数，可以使用小于MVD的整数，以方便日常检测（表示制备一份原料药或制剂溶液，稀释倍数略低于MVD所示数）。在此情况下，阴性结果表示原料药或制剂内毒素浓度低于限度值。但是，如果此测试中原料药或制剂内毒素浓度低于内毒素限度，但又足够之高足以与鲎试剂反应引起凝胶反应，则这种情况下测试可能显示为阳性。因此，如果采用方便起见所用的稀释因子测试得到阳性，则应采用准确的MVD稀释因子重复测试。如有争议或质疑，应以MVD为准。

This stresses the importance of theconfirmation of the sensitivity of the lysate.

这就强调了确认鲎试剂灵敏度的重要性。

Example 举例

A 50mg/mL solution of phenytoin sodium(intended for intravenous injection) has to be tested. Determine the MVD, giventhe following variables:

要测试50mg/mL苯妥英钠（用于静脉注射），按以下给定的变量确定MVD：

M = maximum human dose = 15mg perkilogram of body mass

C = 50 mg/mL

K = 5 IU of endotoxin per kilogram ofbody mass

λ= =0.4 IU of endotoxin per milliliter

M = 最大人用剂量=15mg/kg体重

C = 50 mg/mL

K = 5 IU 内毒素/kg体重

λ= =0.4 IU内毒素/ml

MVD =	5 x 50	x	1	=41.67
	15		0.4

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EP 5.1.10 细菌内毒素测试使用指南（中英文2/2）  

   

For routine tests on this product, itmay be expedient to dilute 1 mL of the solution to be examined to 20 mL (MVD/2rounded to the next lower whole number). However, if this test result ispositive the analyst will have to dilute 1 mL to 41.67mL and repeat the test. Adilution to 41.67 mL is also necessary when the test is performed to settle adispute.

对于此药品的日常检查，可以取1ml供试液稀释至20ml（MVD/2四舍五入至最接近的整数）。但是，如果该测试结果为阳性，则化验室要稀释1ml至41.67ml，重复测试。如果有争议，仲裁测试也必须使用41.67ml的稀释。

3. RISK ASSESSMENT 风险评估

As stated in section 1 of this generalchapter, the conclusion is generally justified that the absence of bacterialendotoxins in a substance or product implies the absence of pyrogeniccomponents, provided the presence of non-endotoxin pyrogenic substances can beruled out. To rule out the presence of non-endotoxin pyrogens in substance orproducts, the use of the monocyte-activation test (2.6.30) is recommended atrelease or during development of the production process; if any changes aremade to the production process that could influence the quality of the productregarding pyrogenicity, the monocyte-activation test is repeated. Examples ofsuch changes include the use of different raw materials, a different productionsite and different process parameters.

正如本通论第1部分所述，如果可以排除非内毒素热源物质的存在，则原料药或制剂中不存在细菌内毒素通常可以论述得到结论不存在热源成分。为了排除原料药或制剂中存在非内内毒素热源，推荐在放行或生产工艺研发时使用单核细胞激活测试（2.6.30）。如果对生产工艺进行了变更，可能会影响产品热源方面的质量，则需要重新进行单核细胞激活测试。这类变更的例子包括使用了不同的原料、不同的生产场所和不同的工艺参数。

The decision to use the test forbacterial endotoxins as the sole pyrogenicity test is to be made after carefulevaluation of the risk of the substance or product containing non-endotoxinpyrogens. The risk assessment is made with consideration given to any factorthat could result in the inclusion of pyrogens not detected by the test forbacterial endotoxins. The items below constitute a non-exhaustive list offactors to be considered in the risk assessment.

使用细菌内毒素测试作为单一热源测试的决策要在对含有非内毒素热源的原料药或制剂进行谨慎的风险评估之后方可做出。风险评估要考虑可能会导致产品含有不被细菌内毒素测试检出的热源。以下所列项目是风险评估中要考虑的因素清单，并非完整清单，

Production process (chemical synthesis, fermentation,biotechnological method).

For products of fermentation, the expressionsystem is to be considered (prokaryotic, eukaryotic) and, for a prokaryoticexpression system, whether gram-positive or gram-negative bacteria are used.Also the culture media components are examined with consideration given totheir origin (synthetic, animal, plant).

生产工艺（化学合成、发酵、生物技术方法）：对于发酵产品，要考虑表达系统（原核、真核），对于原核表达系统，要看是否使用革兰氏阳性或革兰氏阴性菌。对于发培养基成分，要检查其来源（合成、动物来源、植物来源）。

Bioburden.

The potential presence of gram-positivebacteria and fungi as contaminants of the active substance, excipients orstarting materials and raw materials used in the production of the medicinalproduct, and the origin of the raw materials (synthetic, animal, plant) have tobe taken into consideration. The quality of the water plays an important roleon the overall evaluation.

生物负载：要考虑革兰氏阳性菌和霉菌可能会污染原料药、辅料，或药品生产用的起始物料和原料药，以及原料来源（合成、动物来源、植物来源）。水的质量在总体评估中起着尤为重要的作用。

Capability of the downstream process.

It must be verified whether bacterialendotoxin removal steps are part of the downstream process.

下游工艺能力：要确认下游工艺中是否有细菌内毒素清除步骤。

Safety.

The target population and the route ofadministration (e.g. intravenous, intrathecal) have to be taken into account inthe risk assessment.

安全性：在风险评估中要考虑目标种群和给药途径（例如，静脉注射、鞘内）。

Stability of the detectable endotoxins.

It has to be considered that the abilityto detect endotoxins can be affected by interaction with certain components,storage conditions or storage time, temperature and handling of the testsample. Procedures that demonstrate stability of the detectable endotoxincontent have to be established for storing, handling and mixing of samples.

可检出内毒素的稳定性：要考虑检出内毒素的能力会受到特定组份的相互反应、存贮容器或存贮时长、温度和测试样品的处理方式的影响。证明可检出内毒素含量的稳定性的程序必须指定其存贮情况、处理情况和样品混和情况。

4. REFERENCE MATERIAL 对照物

Endotoxin standard BRP is intended foruse as the reference preparation. It has been assayed against the WHOInternational Standard for Endotoxin and its potency is expressed inInternational Units of endotoxin per vial. The International Unit of endotoxinis defined as the specific activity of a defined mass of the InternationalStandard.

内毒素标准BRP是用作对照的物品。它根据WHO国际内毒素标准进行了标定，其效价表述为国际单位内毒素/瓶。内毒素的国际单位定义为指定质量的国际标准物的活性。

For routine purposes, anotherpreparation of endotoxin may be used; provided it has been assayed against theInternational Standard for Endotoxin or the BRP and its potency is expressed inInternational Units of endotoxin.

日常工作中，也可以使用另一个内毒素对照品，但需要已根据国际标准内毒素或BRP进行了标定，且其效价表述为国际单位内毒素。

NOTE: 1 International Unit (IU) ofendotoxin is equal to 1 Endotoxin Unit (E.U.)

注：1国际单位（IU）内毒素等于1内毒素单位（EU）。

5. WATER FOR BET 细菌内毒素测试用水

Water for BET is sterile water that isfree of detectable levels of endotoxin. Usually it is commercially availableand certified.

BET测试用水为无菌水，内毒素低于可检测水平。通常可能商业采购获取并有证书认可。

General chapter 2.6.14. Bacterialendotoxins indicates that methods other than triple distillation may be used toprepare water for BET. Reverse osmosis has been used with good results; someanalysts may prefer to distill the water more than 3 times. Whatever method isused, the resultant product must be free of detectable bacterial endotoxins.

通则2.6.14“细菌内毒素”说明三次蒸馏以外的方法也可以用于制备BET检测用水。反渗透水的使用情况良好，一些化验员可能倾向于使用蒸馏3次以上的水。不管使用哪种方法，所用的水中细菌内毒素必须低于可检测水平。

6. pH OF THE MIXTURE 混合物的PH值

In the test for bacterial endotoxins,optimum gel-clot occurs for a mixture at pH 6.0-8.0. However, the addition of thelysate to the sample may result in a lowering of the pH.

在细菌内毒素的测试中，混合物理想凝胶pH值为6.0-8.0.当然，将鲎试剂加入样品后可能会使得pH值降低。

7. VALIDATION OF THE LYSATE 鲎试剂的验证

It is important to follow themanufacturer’s instructions for the preparation of the solutions of the lysate.

要根据生产商的指示对制备鲎试剂溶液。

The positive end-point dilution factorsin gel-clot methods A and B are converted to logarithms. The reason is that ifthe frequency distribution of these logarithmic values is plotted, it usuallyapproaches a normal distribution curve much more closely than the frequencydistribution of the dilution factors themselves; in fact it is so similar thatit is acceptable to use the normal frequency distribution as a mathematicalmodel and to calculate confidence limits with Student’s t-test.

将凝胶方法A和B中的阳性终点稀释因子转换成log对数。原因是如果这些对数值的频数分布画出后，通常会比稀释因子本身的频数分布更接近正态分布曲线。由于其非常的相似，因此可以使用正态分布作为数学模型，并采用t检验来计算置信限度。

8. PRELIMINARY TEST FOR INTERFEREINGFACTORS 干扰因素初步测试

Some substances or products cannot betested directly for the presence of bacterial endotoxins because they are notmiscible with the reagents, they cannot be adjusted to pH 6.0-8.0 or theyinhibit or activate enzymatic reaction (such as β-D-glucans).

有些物质或制剂不能直接测试内毒素，因为它们无法与试剂混合，它们不能调节pH值至6.0-8.0，或它们的性质或活化酶反应（例如β-D-葡聚糖）。

Therefore a preliminary test is requiredto check for the presence of interfering factors; when these are found theanalyst must demonstrate that the procedure to remove them has been effectiveand that by applying this procedure, any bacterial endotoxins present have notbeen removed.

因此，需要进行初步试验来检查是否存在干扰因素。如果化验员发现了干扰因素，则必须证明测试程序能有效清除它们，并且通过执行这些程序，不会清除任何细菌内毒素。

The object of the preliminary test is totest the null hypothesis that the sensitivity of the lysate in the presence ofthe substance or product to be examined does not differ significantly from thesensitivity of the lysate in the absence of the product. A simple criterion isused in methods A and B: the null hypothesis is accepted when the sensitivityof the lysate in the presence of the product is at least 0.5 times and not morethan twice the sensitivity of the lysate by itself.

初步试验的目的是测试零假设（原假设），即鲎试剂在有原料药或制剂存在时的灵敏度，与没有原料药或制剂存在时灵敏度并无显著区别。在方法A和B中使用了一个简单的标准：当鲎试剂在有产品存在时，其灵敏度至少达到0.5倍，且不超过2倍鲎试剂自身的灵敏度，则零假定成立。

The test for interfering factors ingel-clot methods A and B requires the use of a sample of the substance orproduct in which no endotoxins are detectable. This presents a theoreticalproblem when an entirely new product has to be tested. Hence, a differentapproach was designed for quantitative methods C, D, E and F.

在凝胶方法A和B中干扰因素测试要求使用原料药或制剂的样品，其中不允许检出内毒素，如果要测试的是一个全新的产品，这时理论上是行的通的。因此，在定量方法C、D、E和F中，设计了不同的方法。

Note that methods D and E, which used achromogenic peptide, require reagents that are absent in methods A, B, C and F,and hence compliance of methods A, B, C or F with the requirements forinterfering factors cannot be extrapolated to method D or method E withoutfurther testing.

注意，方法D和E使用了色谱多肽，要使用方法A、B、C和F中不曾使用的试剂，因此在没有进一步测试时，方法A、B、C或F符合干扰因素要求的结论就不能外推至方法或方法E。

9. REMOVAL OF INTERFERING FACTORS 干扰因素的消除

The procedures to remove interferingfactors must not increase or decrease (for example, by adsorption) the amountof endotoxin in the substance or product to be examined. The correct way ofchecking this is to apply the procedures to a spiked sample of the substance orproduct to be examined, that is, a sample to which a known amount of endotoxinhas been added, and then to measure the recovery of the endotoxin after theremoval process has been conducted.

消除干扰因素的程序一定不能增加或降低受检原料药或制剂中的内毒素数量（例如，由于吸附）。正确的检查方法是使用受检原料药或制剂的加标样品来测试，也就是说，将已知数量的内毒素加入一个样品，然后在实施了清除过程后，检测内毒素的回收率。

Methods C and D. 方法C和D

If the nature of the product to beexamined results in an interference that cannot be removed by classical methods(e.g. dilution or centriguation), it may be possible to determine the standardcurve in the same type of substance or product freed from endotoxins byappropriate treatment or by dilution of the substance or product. Theendotoxins test is then carried out by comparison with this standard curve.

如果受检产品的性质会导致一种干扰，而不能采用传统的方法消除（例如，稀释或离心），有可能通过对原料药制剂进行适当的处理或稀释，使用无内毒素的同类原料药或制剂来确立一个标准曲线。然后通过与此标准曲线比较来进行内毒素测试。

Ultrafiltration with cellulosetriacetate asymmetric membrane filters has been found to be suitable in mostcases. The filters must be properly validated, because under some circumstancecellulose derivatives (β-D-glucans) can cause false positiveresults.

目前已经发现三醋酸纤维素非对称膜过滤超滤方法在多数情况下是适用的。过滤器必须经过适当验证，因为在有些情形下，纤维素衍生物（β-D-葡聚糖）可能会导致假阳性结果。

Another option to remove interferingfactors is a 2-step procedure in which 1) endotoxin within the interferingsample is fixed on a solid phase, and 2) after removal of the interferingsubstance (e.g. by washing ) the endotoxin is detected unimpaired undersuitable testing conditions.

另一个清除干扰因素的办法是分两步实现的，第一步将干扰样品中的内毒素固定在一个固定相上，第二步清除干扰物质（例如，通过洗涤）后，在稳定的测试条件下进行不受影响的测试。

10. THE PURPOSE OF THE CONTROLS 控制的目的

The purpose of the control made up withwater for BET and the reference preparation of endotoxin at twice theconcentration of the labeled lysate sensitivity is to verify the activity ofthe lysate at the time and under the conditions of the test (for method A andB). The purpose of the negative control is to verify the absence of t adetectable concentration of endotoxin in the water of BET.

通过对BET用水和内毒素对照品在标示鲎试剂灵敏度2倍浓度的控制所要达到的目的是确认鲎试剂在测试的条件下测试的时间内的活性（对于方法A和B）。阴性控制的目的是确认在BET用水中没有可检出的内毒素浓度水平。

The positive control, which contains theproduct to be examined at the concentration used in the test, is intended toshow the absence of inhibiting factors at the time and under the conditions ofthe test.

阳性控制，其中含有用于测试相同浓度的受检样品，是为了显示在测试条件下在测试时间内没有抑制因子。

11. READING AND INTERPRETATION OFRESULTS 结果读数和诠释

Minute amounts of the bacterialendotoxin in the water for BET, or in any other reagent or material to whichthe lysate is exposed during the test, may escape detection as long as they donot reach the sensitivity limit of the lysate. However, they may raise theamount of bacterial endotoxin in the solution containing the substance orproduct to be examined to just above the sensitivity limit and cause a positivereaction.

在BET用水中，或在其它试剂或鲎试剂在测试中会暴露的物料中的微量细菌内毒素，只要其未达到鲎试剂的灵敏限，就可能会在检测中有被测到。但是，他们可能会抬高细菌内毒素在受检原料药或制剂溶液中的数量，正好超出灵敏限导致阳性反应。

The risk of this happening may bereduced by testing the water for BET and the other reagents and materials withthe most sensitive lysate available, or at least one that is more sensitivethan the one used in the test on the product. Even then, the risk of such a‘false positive result’ cannot be ruled out completely.

通过使用可能找到的最灵敏（或者至少比产品测试所用鲎试剂更灵敏）的鲎试剂对水和其它试剂及物料进行BET测试，可以降低上述情况发生的风险。即使是这样，发生“假阳性结果”的风险仍不能完全排除。

12. REPLACEMENT OF METHODS PRESCRIBED INMONOGRAPHS 各论中所述方法的取代方法

12-1. REPLACEMENT BY ANOTHER PH. EUR.METHOD 使用另一欧洲药典方法取代

As stated in the General Notice, thetest methods given in monographs and general chapters have been validated inaccordance with accepted scientific practice and current recommendations onanalytical validation. The methods described in general chapters 2.6.14.Bacterial endotoxins and 2.6.30. Monocyte-activation test therefore do not haveto be re-validated per se, other than in consideration of their use for aspecific substance or product in a specific analytical environment.

正如凡例中声明，各论和通论中给定的检测方法是根据可接受的科学实践和现行分析验证建议经过了验证。在通论2.6.14“细菌内毒素”和2.6.30“单核细胞激活测试”因而不必要进行再验证，其在特定原料药或制剂在特定分析环境中使用时适用性需要考虑者以外。

The procedure and the materials andreagents used in the method must be validated as described for the testconcerned. The absence of interfering factors (and, if necessary, the procedurefor removing them) is verified on samples of at least 3 production batches.

测试程序和方法中所用物料和试剂必须针对所涉及的测试所述进行验证。至少要使用3个生产批次来确认没有干扰因素存在（并且必要时，要确认消除这些因素的程序）。

The necessary information is sought frommanufacturers; companies are invited to provide any validation data that theyhave concerning the applicability of the replacement test to the substances andproducts of interest; such data includes details of sample preparation and ofany procedures necessary to eliminate interfering factors.

应从生产商处获取必要的信息。期望公司能提交其所关注的感兴趣的原料药和制剂的替代方法适用性的验证数据，该类数据包括样品制备的详细信息，以及消除干扰因素所需的程序详细资料。

As stated in general chapter 2.6.30.Monocyte-activation test, the monocyte-activation test is primarily intended asa replacement of the rabbit pyrogen test. Guidelines on which methods to use(A, B, or C) and on how to validate the monocyte-activation test are describedin general chapter 2.6.30. Monocyte-activation test.

正如通论2.6.30“单核细胞激活测试”所述，单核细胞测试基本目的是替代兔热源测试。在通论2.6.30“单核细胞激活测试”中描述了使用哪种方法（A、B或C）以及如何验证单核细胞激活测试指南。

12-2. REPLACEMENT BY AN ALTERNATIVEMETHOD NOT DESCRIBED IN THE PH.EUR. 使用非欧洲药典方法取代

The use of alternative reagents such asrecombinant factor C as a replacement to the amoebocyte lysate eliminates theuse of a reagent extracted from live animals.

使用其它试剂，例如，重组因子C，替代阿米巴样细胞鲎试剂以避免使用活体动物上提取的试剂。

Replacement of a rabbit pyrogen test ora bacterial endotoxin test prescribed in a monograph by a test usingrecombinant factor C reagent or any other reagent as a replacement of theamoebocyte lysate is to be regarded as the use of an alternative method in thereplacement of a pharmacopoeial test, as described in the General Notices.

使用重组因子C试剂或任何其它试剂替代阿米巴样细胞鲎试剂，用于各论所述的兔热源测试或细菌内毒素测试，会被认为是使用了其它方法取代药典测试，如通论所述。

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