1、4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 1 页，共 8 页 51 ANTIMICROBIAL EFFECTIVENESS TESTING 抗菌的效力测试Antimicrobial preservatives are substances added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the
2、manufacturing process. In the case of sterile articles packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses.抗菌防腐剂是实际被添加到有菌的剂量配方里的物质，使他们不受微生物滋长或是被无意中引进到生产过程中的微生物侵害。在无菌包装多
3、剂量配方的容器里，抗菌防腐剂被添加用来抑制由重复添加单剂量所引进的微生物的滋长。Antimicrobial preservatives should not be used as a substitute for good manufacturing practices or solely to reduce the viable microbial population of a nonsterile product or control the presterilization bioburden of multidose formulations during manufacturing
4、. Antimicrobial preservatives in compendial dosage forms meet the requirements for Added Substancesunder Ingredients and Processesin the General Notices. 抗菌防腐剂不应被作为在良好生产操作中的替代方式使用，或仅是用来减少一个有菌产品的微生物群数量，或是在生产时控制多剂量的微生物量。在剂量表里抗菌防腐剂与添加的要求必需符合规定。All useful antimicrobial agents are toxic substances. For m
5、aximum protection of patients, the concentration of the preservative shown to be effective in the final packaged product should be below a level that may be toxic to human beings. The concentration of an added antimicrobial preservative can be kept at a minimum if the active ingredients of the formu
6、lation possess an intrinsic antimicrobial activity. Antimicrobial effectiveness, whether inherent in the product or whether produced because of the addition of an antimicrobial preservative, must be demonstrated for all injections packaged in multiple-dose containers or for other products containing
7、 antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for multiple-dose topical and oral dosage forms and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids (see Pharmaceutical Dosage Forms (1151).所有有用的抗菌药剂都是有毒物质。为了最大的保护使用者，防腐剂的含量应被有效地显示在
8、产品的最终包装上，并应会处在一个可能对人体有毒的水平线下。添加的抗菌防腐剂含量能够被保持在一个最小的浓度，如果配方的活性成分具有一个固定的抗菌力。抗菌的效力，不管是产品里固有的或者是生产中4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 2 页，共 8 页添加的抗菌防腐剂，都必须在所有注入包装的多剂量容器里或其它包含抗菌防腐剂的产品上显示。抗菌防腐剂必须被显示在多剂量和口头剂量型和给其它剂量型，如眼药，耳药，鼻药，冲洗的和透析液。(看 Pharmaceutical Dosage Forms(a1151).This chapter provides tests to
9、demonstrate the effectiveness of antimicrobial protection. Added antimicrobial preservatives must be declared on the label. The tests and criteria for effectiveness apply to a product in the original, unopened container in which it was distributed by the manufacturer.本章提供的测试用来显示抗菌保护的效力。添加的抗菌防腐剂必须显示在
10、标签上。测试的效力和标准适用于一个产品在原有的、未打开过的经由制造商销售的容器里。PRODUCT CATEGORIES 产品分类 For the purpose of testing, compendial articles have been divided into four categories (see Table 1).The criteria of antimicrobial effectiveness for these products are a function of the route of administration.为了测试的目的，配制产品分开为四个分类（看表 1）
11、。抗菌效力的标准对于这些产品是一个管理的作用。Table 1.Compendial Product Categories 表 1: 配制药品分类Category类 别Product Description产 品 概 述1 Injections, other parenterals including emulsions, otic products, sterile nasalproducts, and ophthalmic products made with aqueous bases or vehicles.针剂，其它非肠胃药剂，包括乳剂、无菌耳用剂、鼻用剂和眼用剂含水的或其它介质的。2
12、 Topically used products made with aqueous bases or vehicles, nonsterile nasal products, and emulsions, including those applied to mucous membranes.局部使用产品，含水的或其它介质，如未灭菌耳用剂产品、软膏，包括那些涂在黏膜上的。3 Oral products other than antacids, made with aqueous bases or vehicles.口服产品，除了以水为基底或其它的介质的解酸剂。4 Antacids made
13、with an aqueous base.含水解酸剂。4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 3 页，共 8 页TEST ORGANISMS 测试生物体Use cultures of the following microorganisms1:Candida albicans ( ATCC No.10231), Aspergillus niger(ATCC No.16404),Escherichia coli(ATCC No.8739),Pseudomonas aeruginosa ( ATCC No.9027),and Staphylococcus au
14、reus ( ATCC No.6538).The viable microorganisms used in the test must not be more than five passages removed from the original ATCC culture. For purposes of the test, one passage is defined as the transfer of organisms from an established culture to fresh medium. All transfers are counted. In the cas
15、e of organisms maintained by seed-lot techniques, each cycle of freezing, thawing, and revival in fresh medium is taken as one transfer. 使用以下微生物的培养基：白念珠菌（ATCC NO.10231）,黑曲霉菌（ATCC NO.16404）,大肠杆菌（ATCC NO.8739）,绿脓杆菌(ATCC no.9027),和金黄色葡萄球菌( ATCC NO.6538). 在测试中使用活的微生物从原始的培养基移转次数不得超过五次。为了测试的目的，移转的定义是从一个已确
16、定的培养基移到一个新鲜的媒介里。所有的移转均应被计算在内的。一旦有机体被以种子库(seed-lot) 技术保存下来时，每个在新鲜媒介里冰冻的，融化的和再生的循环周期被认为是一次转移。A seed-stock technique should be used for long-term storage of cultures. Cultures received from the ATCC should be resuscitated according to directions. If grown in broth, the cells are pelleted by centrifugat
17、ion. Resuspend in 1/20th the volume of fresh maintenance broth, and add an equal volume of 20%(v/v in water)sterile glycerol. Cells grown on agar may be scraped from the surface into the 10%glycerol broth. Dispense small aliquots of the suspension into sterile vials. 种子存储技术将会被使用到培养的长期存储中。从 ATCC 得到的培
18、养基将会依照操作方向苏醒。如果在液体培养基里生长，细胞会通过离心法的方式被聚集成块。悬浮在 1/20 容量的新鲜的液体培养基里，并添加一个同等容量的 20%（V/V 水里）的无菌的甘油。从表面到 10%的甘油液体培养基里可能得到在琼胶培养基上的细胞生长。分配小部分的悬浮液到无菌的小瓶里。Store the vials in liquid nitrogen or in a mechanical freezer at no more than -50 .When a fresh seed-stock vial is required, it may be removed and used to i
19、noculate a series of working cultures. These working cultures may then be used periodically (each day in the case of bacteria and yeast)to start the inoculum culture.4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 4 页，共 8 页把小瓶子放在液化氮里或一个不超过-50 摄氏度的机械冷藏室里。当要求一个新鲜的种子存储瓶时，它可能被移除和使用来给一系列的有效的培养菌做接种。这些有效的培养基到时可以被定期
20、地（每天，当测试细菌和酵母时）使用来启动接种培养基。MEDIA 媒介All media used in the test must be tested for growth promotion. Use the microorganisms indicated above under Test Organisms. 测试中所有媒介的使用，必须做促生长测试。在有机体测试下使用上面指定的测试微生物。PREPARATION OF INOCULUM 接种体的准备Preparatory to the test, inoculate the surface of a suitable volume of
21、solid agar medium from a recently revived stock culture of each of the specified microorganisms. The culture conditions for the inoculum culture are described in Table 2 in which the suitable media are SoybeanCasein Digest or Sabouraud Dextrose Agar Medium (see Microbial Enumeration Tests (61) and A
22、bsence of Specified Microorganisms 62) ).准备测试，由最近再生的培养基的表面适当的接种特定的微生物。接种培养基的描述请参考表 2，表 2 里的适合的培养基是黄豆-消化酪蛋白 (soybean casein digest)或沙保罗氏葡萄糖琼脂培养基。（ 参看Microbial Enumeration Tests (61)和 Absence of Specified Microorganism 62）To harvest the bacterial and C. albicans cultures, use sterile saline TS, washin
23、g the surface growth, collecting it in a suitable vessel, and adding sufficient sterile saline TS to obtain a microbial count of about 1108colony-forming units ( cfu ) per mL. To harvest the cells of A. niger, use sterile saline TS containing 0.05%of poly sorbate 80, and add sufficient sterile salin
24、e TS to obtain a count of about 1108cfu per mL.为了获得细菌和白色念珠菌(C.albicans) 培养基，使用干净的盐水溶液(Saline TS ，注: 9 克氯化钠溶于1000 mL.的水)，清洗表面的生成物，把它收集在一个合适的容器里，并添加足够的盐水溶液以获得一个每毫升大约 1108 cfu 的微生物计数。为了获得黑曲霉菌 (A. niger)细胞，使用干净的盐水溶液含 0.05%的聚山梨醇酯 80(polysorbate 80)，并添加足够的盐水溶液来获得一个大约 1108cfu 每毫升的计数。4a49a29f2aba3a3e0de76a7
25、62fbfdd47.pdf 第 5 页，共 8 页Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., SoybeanCasein Digest Broth or Sabouraud Dextrose Broth)and the cells harvested by centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about
26、1108cfu per mL. NOTEThe estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not used within 2hours. 或者，一个合适的液体的培养液（如，黄豆-消化酪蛋白液 soybean casein digest broth 或沙氏琼脂培养液 sabouraud dextrose broth）可能生长出培养菌微生物
27、并通过离心法得到细胞，然后以无菌的盐水溶液清洗和重新悬浮以获得一个大约 1108cfu 每毫升的微生物计数。（注意 预计的接种浓度可能通过浊度计测量来挑战微生物。如果悬浮液没有在两个小时内被使用，请冷冻它）Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 2 to confirm the initial cfu per mL estimate. This value
28、 serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7days. 测出每个悬浮液的每毫升的菌落数量，使用在 TABLE2 里的媒介和微生物的恢复培养期的情况来确认估计每毫升的原始 cfu。这个值可以用在测试中做接种尺寸的比对。细菌和酵
29、母菌的悬浮液在 24 小时收获期里应被使用，但真菌的准备可在冷冻情况下储存 7 天。PROCEDURE 程序The test can be conducted either in five original containers if sufficient volume of product is available in each container and the product container can be entered aseptically (i.e., needle and syringe through an elastomeric rubber stopper),or i
30、n five sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred. 测试可以在五个干净的容器里操作，如果在每个容器里有足够容量的产品且产品容器能被无菌地装入（ 如，使用针和注射器穿过一个人造的橡胶瓶塞），或者在五个无菌的，加盖的、尺寸合适的，能移转产品的充足容量的细菌容器里。4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 6 页，共 8 页Inoculate each co
31、ntainer with one of the prepared and standardized inoculum, and mix. The volume of the suspension inoculum used is between 0.5%and 1.0%of the volume of the product. The concentration of test microorganisms that is added to the product (Categories 1,2,and 3)are such that the final concentration of th
32、e test preparation after inoculation is between 1105and 1106cfu per mL of the product. For Category 4 products (antacids)the final concentration of the test preparation after inoculation is between 1103and 1104cfu per mL of the product. 给每一个准备好的标准的接种体的容器做接种注射，并混合。接种悬浮液的使用量在产品量的 0.5%到1.0%之间。被添加到产品的测试
33、细菌浓度（类型 1.2.3）到最终的测试准备浓度在接种之后是每毫升1105and 1106cfu 之间。对于类型 4 产品（解酸剂）测试准备的最终浓度在接种后是每毫升产品 1103到 1104cfu 之间。The initial concentration of viable microorganisms in each test preparation is estimated based on the concentration of microorganisms in each of the standardized inoculum as determined by the plate
34、-count method.在每次测试准备的活的微生物的原始浓度是预计是基于每个标准接种体的微生物浓度，通过皿测法来预计的。Incubate the inoculated containers at 22.52.5 .Sample each container at the appropriate intervals specified in Table 3.Record any changes observed in appearance at these intervals. Determine by the plate-count procedure the number of cfu
35、present in each test preparation for the applicable intervals (see Procedure under Microbial Limit Tests (61). Incorporate an inactivator (neutralizer) of the specific antimicrobial in the plate count or in the appropriate dilution prepared for plating. These conditions are determined in the validat
36、ion study for that sample based upon the conditions of media and microbial recovery incubation times listed in Table 2.Using the calculated concentrations of cfu per mL present at the start of the test, calculate the change in log10values of the concentration of cfu per mL for each microorganism at
37、the applicable test intervals, and express the changes in terms of log reductions.在 22.52.5 的温度下将容器接种。如表 3，在适当的时间间隔里把每个容器做样品标示。记录下在每个间隔时间里出现的任何变化（参看 Microbial Limit Tests (61)下的程序） 。以平皿计数方式(plate count)在适当的时间间隔里计算出现的 cfu 数。使用特定的抗菌剂做为钝化剂（中和剂）或是适当的稀释量来做平皿4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 7 页，共 8
38、页计数。在表 2 中规定出培养基与不同菌种的各种接种状况与验收时间。在开始测试时使用每毫升多少CFU 来计算浓度，在适当的间隔测试对每个微生物计算每毫升 CFU 浓度的 log10 值的变化，并表述以 log为单位的减少变化量。Table 2.Culture Conditions for Inoculum Preparation 接种体准备的培养基Organism微生物Suitable Medium 适合的培养基IncubationTemperature接种温度Inoculum Incubation Time接种时间Microbial Recovery Incubation Time验收时间E
39、scherichia coli 大肠杆菌(ATCC No.8739)SoybeanCasein Digest Broth;黄豆-消化酪蛋白液SoybeanCasein Digest Agar黄豆-消化酪蛋白基32.52.518 to 24hours18 至 24 小时3 to 5days3 至 5 天Pseudomonas aeruginosa 绿脓杆菌(ATCC No.9027)SoybeanCasein Digest Broth;黄豆-消化酪蛋白液SoybeanCasein Digest Agar黄豆-消化酪蛋白基32.52.518 to 24hours18 至 24 小时3 to 5da
40、ys3 至 5 天Staphylococcus aureus金黄色葡萄球菌 (ATCC No.6538)SoybeanCasein Digest Broth;黄豆-消化酪蛋白液SoybeanCasein Digest Agar黄豆-消化酪蛋白基32.52.518 to 24hours18 至 24 小时3 to 5days3 至 5 天Candida albicans 白念珠菌(ATCC No.10231)Sabouraud Dextrose Agar;沙氏琼脂培养基Sabouraud Dextrose Broth沙氏琼脂培养液22.52.544 to 52hours44 到 52 小时3 t
41、o 5days3 至 5 天Aspergillus niger 黑曲霉菌(ATCC No.16404)Sabouraud Dextrose Agar;沙氏琼脂培养基Sabouraud Dextrose Broth沙氏琼脂培养液22.52.56 to 10days6 至 10 天3 to 7days3 至 7 天CRITERIA FOR ANTIMICROBIAL EFFECTIVENESS 抗菌效力标准The requirements for antimicrobial effectiveness are met if the criteria specified under Table 3
42、are met (see Significant Figures and Tolerancesunder General Notices).No increase is defined as not more than 0.5 log10 unit higher than the previous value measured. 4a49a29f2aba3a3e0de76a762fbfdd47.pdf 第 8 页，共 8 页抗菌效力的要求如 Table 3 下规定的标准（参看 significant figures and tolerances under general notices）。增
43、加值不高于现行测量值的 0.5 log10 单位的可视为没有增加。Table 3.Criteria for Tested Microorganisms 测试微生物的标准For Category 1 Products 对第一类产品Bacteria: 细菌 Not less than 1.0 log reduction from the initial calculated count at 7days,not less than 3.0log reduction from the initial count at 14days,and no increase from the 14dayscou
44、nt at 28days. 7 天里的原始计数量不少于 1.0 log 的减少量，在 14 天里原始计数量不少于 3.0 log 的减少量，在 14 天-28 天里计数量不增加。Yeast and Molds:酵母和霉菌No increase from the initial calculated count at 7, 14, and 28days.原始计数量在 7 天，14 天，28 天里没有增加。For Category 2 Products 对第二类产品Bacteria:细菌Not less than 2.0log reduction from the initial count at
45、 14days,and no increase from the 14dayscount at 28days.Yeast and Molds:酵母和霉菌No increase from the initial calculated count at 14and 28days.For Category 3 Products 对第三类产品Bacteria:细菌Not less than 1.0log reduction from the initial count at 14days,and no increase from the 14dayscount at 28days.Yeast and Molds:酵母和霉菌No increase from the initial calculated count at 14and 28days.For Category 4 Products 对第四类产品Bacteria,Yeast,and Molds:细菌、酵母和霉菌No increase from the initial calculated count at 14and 28days. 在 14-28 天里最初计算数量不增加。
