欧洲药典EP8.0 2.6.1无菌检验 sterility中英文翻译.doc

2.6.1. STERILITY 2.6.1 无菌检查法The test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test. 本检查方法适用于按照药典要求应当无菌的原料、制剂或其他物质。但是，如果按照本无菌检查法的结果符合要求，仅表明在该检查条件下未发现微生物污染。PRECAUTIONS AGAINST MICROBIAL CONTAMINATION 微生物污染防范The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls. 无菌检测试验应在无菌的条件下进行。为了达到这样的条件，检测环境应当与无菌检测的操作要求相适应。避免污染的防范措施应当不对本检查方法进行检测的微生物造成影响（应并不影响用本检查法检测的微生物）。通过对工作区域的适当取样以及进行适当的控制来对无菌检查的工作环境进行例行监测。CULTURE MEDIA AND INCUBATION TEMPERATURES 培养基和培养温度Media for the test may be prepared as described below, or equivalent commercial media may be used provided that they comply with the growth promotion test. 应按下面描述的方法制备无菌检查的培养介质，如果满足生长促进试验要求，与本处所述培养基相当的商业化培养基也可以采用（也可采用与本处）。The following culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria. Soya -bean casein digest medium is suitable for the culture of both fungi and aerobic bacteria. 下述的培养基已被证明（经证明）适用于无菌检查。硫乙醇酸盐流体培养基主要用于厌氧菌培养，但是，也适用于需氧菌检测。大豆酪蛋白消化物培养基适用于真菌和需氧菌培养。Fluid thioglycollate medium硫乙醇酸盐流体培养基L-Cystine 0.5 gL-胱氨酸 0.5g Agar 0.75 g琼脂 0.75gSodium chloride 2.5 g氯化钠 2.5gGlucose monohydrate/anhydrous 5.5 g/5.0 g葡萄糖一水合物/无水葡萄糖5.5 g/5.0 gYeast extract (water-soluble) 5.0 g酵母提取物（水溶性） 5.0gPancreatic digest of casein 15.0 g酪蛋白胰酶消化物 15.0gSodium thioglycollate or 0.5 g 硫乙醇酸钠 0.5gThioglycollic acid 0.3 mL硫乙醇酸 0.3mlResazurin sodium solution (1 g/L of resazurin1.0 mL sodium), freshly prepared 刃天青钠溶液（刃天青钠1 g/L），新鲜配制Water R 1000 mL水 R 1000mlpH after sterilisation 7.1 0.2 灭菌后的pH 7.1 0.2Mix the L-cystine, agar, sodium chloride, glucose, water-soluble yeast extract and pancreatic digest of casein with the water R and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycollic acid in the solution and, if necessary, add 1 M sodium hydroxide so that, after sterilisation, the solution will have a pH of 7.10.2. If filtration is necessary, heat the solution again without boiling and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix and place the medium in suitable vessels which provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergon e a colour change indicative of oxygen uptake at the end of the incubation period. Sterilise using a validated process. If the medium is stored, store at a temperature between 2 C and 25 C in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink colour, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink colour disappears and cooling quickly, taking care to prevent the introduction of non -sterile air into the container. Do not use the medium for a longer storage period than has been validated. 将L-胱氨酸、琼脂、氯化钠、葡萄糖、水溶性酵母提取物以及酪蛋白胰酶消化物与水R混合，加热至溶解。将硫乙醇酸钠或硫乙醇酸用上述溶液溶解，必要时用1M氢氧化钠调节pH值，使灭菌后培养基溶液的pH值为7.10.2。如需要过滤处理，将溶液在此加热（加热此溶液），但不得煮到沸腾，乘热采用经润湿的滤纸进行过滤。加入刃天青钠溶液，混合均匀，将制备的培养基装入合适的容器中。在该容器中，培养基的表面和高度应具有恰当的比例，以便在灭菌结束后指示氧气摄入的颜色变化不超过培养基的上半部分。采用经验证的工艺灭菌。如果需要保存，将培养基装入无菌、气密容器并在2-25C 之间储存。如果培养基的上面超过1/3的部分已经出现粉红色，将装有培养基的容器采用水浴或自由流动蒸气加热，直到粉红颜色消失，之后快速冷却，注意预防非无菌的气体被引入装培养基的容器，以此进行培养基再生处理。如果培养基保存的时间超过经验证的保存期限，不得使用。（禁止使用超过验证储存期限的培养基）Fluid thioglycollate medium is to be incubated at 30-35 C. For products containing a mercurial preservative that cannot be tested by the membrane-filtration method, fluid thioglycollate medium incubated at 20-25 C may be used instead of soya-bean casein digest medium provided that it has been validated as described in growth promotion test. 硫乙醇酸盐流体培养基用于（应）在30-35C条件下培养。对于含有汞类防腐剂无法采用薄膜过滤法进行检查的产品，如果已按照生长促进试验所述方法验证，硫乙醇酸盐流体培养基可代替替代重复大豆酪蛋白消化物培养基在20-25C条件下进行培养。Where prescribed or justified and authorised, the following alternative thioglycollate medium may be used. Prepare a mixture having the same composition as that of the fluid thioglycollate medium, but omitting the agar and the resazurin sodium solution, sterilise as directed above. The pH after sterilisation is 7.1 0.2. Heat in a water-bath prior to use and incubate at 30-35 C under anaerobic conditions. 按照规定或者证明合理并获得主管机构许可时（如果有经批准的规定或正当理由），也可以使用以下替代硫乙醇酸盐流体培养基。配制与硫乙醇酸盐流体培养基成分相同的混合物，但是不包括琼脂和刃天青钠溶液，按照上面说明的方法进行灭菌。灭菌后培养基的pH值为7.1 0.2，使用前采用水浴加热，在厌氧及30-35C条件下培养。Soya-bean casein digest medium大豆-酪蛋白消化物培养基Pancreatic digest of casein 17.0 g 酪蛋白胰酶消化物 17.0gPapaic digest of soya-bean meal 3.0 g 大豆粉木瓜蛋白酶消化物 3.0gSodium chloride 5.0 g 氯化钠 5.0gDipotassium hydrogen phosphate 2.5 g 磷酸氢二钾 2.5gGlucose monohydrate/anhydrous 2.5 g/2.3 g 葡萄糖一水合物/无水葡萄糖2.5 g/2.3 gWater R 1000 mL 水 R 1000mlpH after sterilisation 7.3 0.2 灭菌后pH 7.3 0.2Dissolve the solids in water R, warming slightly to effect solution. Cool the solution to room temperature. Add 1M sodium hydroxide, if necessary, so that after sterilisation the solution will have a pH of 7.3 0.2. Filter, if necessary, to clarify, distribute into suitable vessels and sterilise using a validated process. Store at a temperature between 2 C and 25 C in a sterile well-closed container, unless it is intended for immediate use. Do not use the medium for a longer storage period than has been validated. 将上述固体用水R溶解，微微加热直到溶解，再放至室温。必要时用1M氢氧化钠调节pH值，使灭菌后培养基溶液的pH值为7.30.2。如需要，过滤使培养基溶液澄清，再将其装入合适的容器中并采用经验证的工艺灭菌。除非立即使用，应将培养基装入无菌、气密容器并在2-25C 之间储存。如果培养基保存的时间超过经验证的保存期限，不得使用。（禁止使用超过验证储存期限的培养基） Soya-bean casein digest medium is to be incubated at 20-25 C. The media used comply with the following tests, carried out before or in parallel with the test on the product to be examined. 大豆-酪蛋白消化物培养基用于在20-25C条件下培养。使用的培养基应满足以下试验要求，相关检查可以在使用前或者和待测样品同时进行。Sterility. Incubate portions of the media for 14 days. No growth of micro-organisms occurs.无菌 取部分培养基培养14天，不得出现微生物生长。 Growth promotion test of aerobes, anaerobes and fungi. 需氧菌、厌氧菌和真菌的生长促进试验Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. Suitable strains of micro-organisms are indicated in Table 2.6.1.-1. 对每一批配好待用的培养基以及每一批采用脱水培养基或成分配制的培养基进行检测。适合的微生物菌株见表2.6.1.-1.Table 2.6.1.-1 Strains of the test micro-organisms suitable for use in the growth promotion test and the method validation表2.6.1.-1.生长促进试验及方法验证中使用的试验微生物菌株Aerobic bacteria需氧菌Staphylococcus aureus 金黄色葡萄球菌 ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276Bacillus subtilis枯草芽孢杆菌ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134Pseudomonas aeruginosa 铜绿假单胞菌ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275Anaerobic bacterium 厌氧菌Clostridium sporogenes 生孢梭菌ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293Fungi 真菌Candida albicans白色念珠菌ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594Aspergillus brasiliensis 黑曲霉菌ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455Inoculate portions of fluid thioglycollate medium with a small number (not more than 100 CFU) of the following micro -organisms, using a separate portion of medium for each of the following species of micro-organism: Clostridium sporogenes, Pseudomonas aeruginosa,Staphylococcus aureus. Inoculate portions of soya-bean casein digest medium with a small number (not more than 100 CFU) of the following micro-organisms, using a separate portion of medium for each of the following species of micro-organism: Aspergillus brasiliensis, Bacillus subtilis, Candida albicans. Incubate for notmorethan3days in the case of bacteria and not more than 5 days in the case of fungi. 取部分硫乙醇酸盐流体培养基，接种少量（不超过100CFU）下述试验菌：生孢梭菌、铜绿假单胞菌以及金黄色葡萄球菌，每种试验菌均使用单独一份培养基。取部分大豆-酪蛋白消化物培养基，接种少量（不超过100CFU）下述试验菌：黑曲霉、枯草芽孢杆菌以及白色念珠菌，每种试验菌均使用单独一份培养基。细菌培养不超过3天，真菌培养不超过5天。Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than 5 passages removed from the original master seed-lot. 采用菌种保藏技术（种子-批系统），以确保用于接种的活试验菌从原始主种子批的传代数不超过5。The media are suitable if a clearly visible growth of the micro-organisms occurs. 如果出现清晰可见的微生物生长，则该培养基是适合的。METHOD SUITABILITY TEST 方法适用性检测Carry out a test as described below under Test for sterility of the product to be examined using exactly the same methods except for the following modifications. 除了以下一些变动，其余完全按照供试品的无菌检测项下描述的方法进行检测。（完全按照，以下变动除外：）Membrane filtration. After transferring the contents of the container or containers to be tested to the membrane add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the final portion of sterile diluent used to rinse the filter. 薄膜过滤法 : 将待测容器的内容物转至薄膜后，采用冲洗滤器的最后一部分无菌稀释剂接种少量（不超过100CFU）活试验菌。Direct inoculation. After transferring the content of the container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medium add an inoculum of a small number of viable micro -organisms (not more than 100 CFU) to the medium. 直接接种法: 将待测容器的内容物（对于肠线或其它兽用手术缝合线：若干股线）转至培养基，再向培养基中接种少量（不超过100CFU）活试验菌。In both cases use the same micro-organisms as those described above under Growth promotion test of aerobes, anaerobes and fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days. 在这两种方法，采用的都是 需氧菌、厌氧菌和真菌的生长促进试验 项下提到的试验菌里的同一种菌株。进行生长促进试验，作为阳性对照。所有含培养基容器的培养时间不超过5天。If clearly visible growth of micro-organisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification. 如果供试品在接种后可观察到清晰可见的微生物生长，目测与不含供试品的对照容器相当。说明供试品在测定试验条件下无抗微生物活性，或者该活性被有效消除了。不需要对方法进行改变就可以用于无菌检查不需要对方法进行改进就可以用于无菌检查。If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the method suitability test. 如果含有供试品的容器中未观察到清晰可见的微生物生长，目测与不含供试品的对照容器相当。则供试品具有抗微生物活性，在测定试验条件下未能有效去除。需要变更测定条件以消除其抗微生物活性，并重复方法适用性检测。This method suitability test is performed: 方法适用性检测在以下情况下应进行：a) when the test for sterility has to be carried out on a new product; 当无菌检查法用于新的产品检测时；b) whenever there is a change in the experimental conditions of the test. 每当检测试验条件发生改变的时候。The method suitability test may be performed simultaneously with the test for sterility of the product to be examined. 方法适用性检测可与供试品无菌检查同时进行。TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED 供品的无菌检查The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test. 供试品的无菌检查可以采用薄膜过滤法或培养基直接接种法进行测定，应当包含适当的阴性对照。如果供试品中的溶剂在测定条件下无抗微生物活性，当只要产品性质允许时的话，薄膜过滤法可广泛应用于可过滤的水性制剂、醇溶性或油溶性制剂以及可与水性或油性溶剂混合或溶解的制剂。Membrane filtration. Use membrane filters having a nominal pore size not greater than 0.45 m whose effectiveness to retain micro-organisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for example, for strongly alcoholic solutions. Specially adapted filters may be needed for certain products, e.g. for antibiotics. 薄膜过滤法 使用标示孔径不大于0.45m的、已被证实可有效截留微生物的膜过滤器经确定可有效截留微生物的膜过滤器。比如，硝酸纤维素过滤器可用于水性、油性以及弱醇性溶液，而醋酸纤维素可用于强醇性溶液。对于某些产品，比如抗生素，可能需要特定的过滤器。The technique described below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are used the volumes of the dilutions and the washings should be adjusted accordingly. The filtration apparatus and membrane are sterilised by appropriate means. The apparatus is designed so that the solution to be examined can be introduced and filtered under aseptic conditions; it permits the aseptic removal of the membrane for transfer to the medium or it is suitable for carrying out the incubation after adding the medium to the apparatus itself. 以下描述的方法中使用的是50mm的薄膜，如果使用不同尺寸的薄膜，稀释和冲洗的体积应当相应进行调节稀释和冲洗的体积应当进行相应调节。过滤的设备和薄膜应当采用适当的方式进行灭菌。应对设备进行设计，以便供试品溶液可以加入并在无菌条件下进行过滤；设备应支持无菌状态下去除薄膜来进行培养基转运，或者适合将培养基加入设备中后进行培养。Aqueous solutions. If appropriate, transfer a small quantity of a suitable, sterile diluent such as a 1 g/L neutral solution of meat or casein peptone pH 7.1 0.2 onto the membrane in the apparatus and filter. The diluent may contain suitable neutralising substances and/or appropriate inactivating substances for example in the case of antibiotics. 水性溶液 如合适，将少量适当的无菌稀释剂（比如1 g/L中性肉或酪蛋白胨，pH 7.1 0.2）转至设备的滤膜上并进行过滤。在产品为抗生素等情形中，稀释剂可以包含适当具有中和作用和/或具有灭活作用的物质。Transfer the contents of the container or containers to be tested to the membrane or membranes, if necessary after diluting to the volume used in the method suitability test with the chosen sterile diluent but in any case using not less than the quantities of the product to be examined prescribed in Table 2.6.1.-2. Filter immediately. If the product has antimicrobial properties, wash the membrane not less than 3 times by filtering through it each time the volume of the chosen sterile diluent used in the method suitability test. Do not exceed a washing cycle of 5 times 100 mL per filter, even if during the method suitability test it has been demonstrated that such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to the culture medium or cut it aseptically into 2 equal parts and transfer one half to each of 2 suitable media. Use the same volume of each medium as in the method suitability test. Alternatively, transfer the medium onto the membrane in the apparatus. Incubate the media for not less than 14 days. 将供试品容器内容物转至滤膜表面，必要时可采用选定的无菌稀释剂稀释至方法适用性检测中使用的体积，但任何时候所检测的检供试品的数量均不得低于表2.6.1.-2规定的量。立即过滤。如果供试品具有抗微生物活性，采用选用的稀释剂过滤冲洗滤膜至少3次，每次冲洗的体积按照方法法适用性检测中使用的体积。每个冲洗循环不要超过5次及100mL/次，即使方法适用性检测已证明这样的循环并不能完全消除供试品的抗微生物活性。转移整个滤膜到培养基中或者将其在无菌条件下剪切成大小相同的量块然后将每一部分分别转移到2种适合的培养基中。每种培养基使用的体积和方法适用性检测中的一致。也可以将培养基加入设备中的滤膜上。所得培养基培养时间不得少于14天。Soluble solids. Use for each medium not less than the quantity prescribed in Table 2.6.1.-2 of the product dissolved in a suitable solvent such as the solvent provided with the preparation, water for injections, saline or a 1 g/L neutral solution of meat or casein peptone and proceed with the test as described above for aqueous solutions using a membrane appropriate to the chosen solvent. 水溶性固体 对于每个培养基，需要将不少于表2.6.1.-2中规定数量的供试品采用合适的溶剂（比如，产品伴有的溶剂、注射用水、生理盐水以及1 g/L中性肉或酪蛋白胨）溶解，采用适用于选用溶剂的薄膜按照上述水性溶液部分描述的方法进行测定。Oils and oily solutions. Use for each medium not less than the quantity of the product prescribed in Table 2.6.1.-2. Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the membrane by its own weight then filter, applying the pressure or suction gradually. Wash the membrane at least 3 times by filtering through it each time about 100 mL of a suitable sterile solution such as 1 g/L neutral meat or casein peptone containing a suitable emulsifying agent at a concentration shown to be appropriate in the method suitability test, for example polysorbate 80 at a concentration of 10 g/L. Transfer the membrane or membranes to the culture medium or media or vice versa as described above for aqueous solutions, and incubate at the same temperature and for the same times.油及油性溶液 对于每个培养基，需要选用不少于表2.6.1.-2中规定数量的供试品进行检测。粘度十分低的油和油性溶液可不经稀释直接采用干燥薄膜过滤。粘性较大的油需要选用十四烷酸异丙酯等合适的、在试验测定条件无抗微生物活性的合适无菌稀释剂进行稀释。让油依靠自身重力作用渗透金薄膜，然后逐渐通过压力或吸力进行过滤。用100mL含有适当乳化剂的1 g/L中性肉或酪蛋白胨等合适无菌溶液过滤冲洗滤膜至少3次，该溶剂中包含适量的乳化剂，其浓度（适用于方法适用性检测）在方法验证试验中表面适用，例如聚山梨醇酯80的浓度选用10 g/L,按照以上水溶液项下的方法同样操作，转移薄膜至培养基中，并在相同的温度温孵培养相同的时间。Table 2.6.1.-2 Minimum quantity to be used for each medium表2.6.1.-2用于每个培养基的最小数量Quantity per container每个容器中的数量Minimum quantity to be used for each medium unless otherwise justified and authorized最小使用数量(除非另有依据并获得许可)Liquids液体 less than 1 mL 小于1 mL 1-40 mL 1-40 mL greater than 40 mL and not greater than 100 mL 大于40mL，不大于100mLgreater than 100 mL 大于100mLAntibiotic liquids 抗生素溶液The whole contents of each container 每个容器的总内容物Half the contents of each container but not less than 1 mL 每个容器中内容物的一半，但不得少于1mL20 mL 10 per cent of the contents of the container but not less than 20 mL 该容器内容物的10%，但不得少于20mL1mL Insoluble preparations, creams and ointments to be suspended or emulsified 待悬浮或乳化的不溶性配制品、乳膏、油膏The whole