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1、71 STERILITY TESTS Change to read: Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols () to specify this fact. 6 These Pharmacopeial procedure
2、s are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures.The test is applied to substances, preparations, or articles which, according to the
3、 Pharmacopeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test.6 Add the following: PRECAUTIONS AGAINST MICROBIAL CONTAMINATION The test for sterility is carried out u
4、nder 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 microorganisms that are to be revealed in the test. The working
5、conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.6 Change to read: CULTURE MEDIA AND INCUBATION TEMPERATURES6 Media for the test may be prepared as described below or equivalent commercial media m
6、ay be used provided that they comply with6 the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi. 6 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.
7、 However, it will also detect aerobic bacteria. SoybeanCasein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.Fluid Thioglycollate Medium l-Cystine 0.5 g Sodium Chloride 2.5 g Dextrose Monohydrate/Anhydrous6 5.5/5.0 g Agar 6 0.75 g Yeast Extract (water-soluble) 5.0 g Pan
8、creatic Digest of Casein 15.0 g Sodium Thioglycollate 0.5 g or Thioglycolic Acid 0.3 mL Resazurin Sodium Solution (1 in 1000),freshly prepared 1.0 mL Purified Water 1000 mL pH after sterilization: 7.10.2.Mix the l-cystine, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein wit
9、h the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 0.2. If filtration is necessary, heat the solution again wit
10、hout boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change indicative of oxyge
11、n uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature between 2 and 25 in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating t
12、he containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container. Do not use the medium for a longer storage period than has been validated.6 Fluid Thioglycollate Medium is to b
13、e incubated at 3035. For products containing a mercurial preservative that cannot be tested by the membrane filtration method, Fluid Thioglycollate Medium incubated at 2025 may be used instead of SoybeanCasein Digest Medium provided that it has been validated as described in Growth Promotion Test of
14、 Aerobes, Anaerobes, and Fungi. Where prescribed or justified and authorized, the following alternative thioglycollate medium might 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. Sterilize as
15、 directed above. The pH after sterilization is 7.1 0.2. Heat in a water bath prior to use and incubate at 3035 under anaerobic conditions.6 SoybeanCasein Digest Medium Pancreatic Digest of Casein 17.0 g Papaic Digest of Soybean Meal 3.0 g Sodium Chloride 5.0 g Dibasic Potassium Phosphate 2.5 g Dextr
16、ose Monohydrate/Anhydrous6 2.5/2.3 g Purified Water 1000 mL pH after sterilization: 7.30.2.Dissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 1 N sodium hydroxide so that, after sterilization, it will have a
17、 pH of 7.3 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 2 and 25 in a sterile well-closed container, unless it is intended for immediate use. Do not use the medium for a longer storage period than h
18、as been validated.6 SoybeanCasein Digest Medium is to be incubated at 22.5 2.5.Media for Penicillins or Cephalosporins Where sterility test media are to be used in the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Fl
19、uid Thioglycollate Medium and the SoybeanCasein Digest Medium as follows. To the containers of each medium, transfer aseptically a quantity of -lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Determine the quantity of -lactamase required to inactivate the anti
20、biotic by using a -lactamase preparation that has been assayed previously for its penicillin- or cephalosporin-inactivating power. noteSupplemented -lactamase media can also be used in the membrane filtration test. Alternatively (in an area completely separate from that used for sterility testing),
21、confirm that an appropriate amount of -lactamase is incorporated into the medium, following either method under Method Suitability6 Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be
22、 observed as a confirmation that the -lactamase concentration is appropriate. Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and the Method Suitability6 Test Aerobic bacteria Staphylococcus aureus 6 ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276 Ba
23、cillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134 Pseudomonas aeruginosa1 ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275 Anaerobic bacterium Clostridium sporogenes2 ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437, NBRC 14293 Fungi Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594 Asp
24、ergillus brasiliensis (Aspergillus Niger) ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455 1 An alternative microorganism is Kocuria rhizophila ( Micrococcus luteus) ATCC 9341.6 2 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is desired, is Bacteroides vulgatus (ATCC 8
25、482).6 6 The media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.Sterility Incubate portions of the media for 14 days. No growth of microorganisms occurs.Growth Promotion Test of Aerobes, Anaerobes, and Fungi Test each lot of re
26、ady-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. 6 Suitable strains of microorganisms are indicated in Table 1.Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following microorganisms, using
27、a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate portions of alternative thioglycollate medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portion
28、s of SoybeanCasein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus niger, Bacillus subtilis, and Candida albicans. Incubate for not more than 3 days in the ca
29、se of bacteria and not more than 5 days in the case of fungi. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed-lot.6 The media are suitable if a clearly vis
30、ible growth of the microorganisms occurs.6 DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION Fluid A preparation Dissolve 1 g of peptic digest of animal tissue in water to make 1 L, filter or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 0.2. Dispense into containers, and steriliz
31、e using a validated process.preparation for penicillins or cephalosporins Aseptically add to the above Preparation, if necessary, a quantity of sterile -lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered (see
32、 Media for Penicillins or Cephalosporins).Fluid D To each L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 0.2, dispense into containers, and sterilize using a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as “sterile pathway.”Fluid K
33、 Dissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 0.2. Dispense into containers, and sterilize using a validated process. Change to read: METHOD SUITABILITY6 TEST Carr
34、y 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.Membrane Filtration After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small num
35、ber of viable microorganisms (not more than 100 cfu) to the final portion of sterile diluent used to rinse the filter.Direct Inoculation After transferring the contents of the container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medi
36、um, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the medium.In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as a positive control. Incubate all th
37、e containers containing medium for not more than 5 days.If clearly visible growth of microorganisms 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 act
38、ivity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.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 antim
39、icrobial 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 Suitability6 Test.This method suitability6 is performed (a) when the test for sterility has to be carried out
40、on a new product; and (b) whenever there is a change in the experimental conditions of the test. The method suitability6 may be performed simultaneously with the Test for Sterility of the Product to be Examined.Change to read: TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED Number of Articles to Be
41、 Tested Unless otherwise specified elsewhere in this chapter or in the individual monograph, test the number of articles specified in Table 3. If the contents of each article are of sufficient quantity (see Table 2), they may be divided so that equal appropriate portions are added to each of the spe
42、cified media. notePerform sterility testing employing two or more of the specified media. If each article does not contain sufficient quantities for each medium, use twice the number of articles indicated in Table 3. Table 2. Minimum Quantity to be Used for Each Medium Quantity per Container Minimum
43、 Quantity to be Used (unless otherwise justified and authorized) Liquids 6 Less than 1 mL The whole contents of each container 140 mL Half the contents of each container, but not less than 1 mL Greater than 40 mL, and not greater than 100 mL 20 mL Greater than 100 mL 10% of the contents of the conta
44、iner, but not less than 20 mL Antibiotic liquids 1 mL 6 6 Insoluble preparations, creams, and ointments to be suspended or emulsified Use the contents of each container to provide not less than 200 mg Solids Less than 50 mg The whole contents of each container 50 mg or more, but less than 300 mg Hal
45、f the contents of each container, but not less than 50 mg 300 mg5 g 150 mg Greater than 5 g 500 mg 6 Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long) Surgical dressing/cotton/gauze (in packages) 100 mg per package Sutures and other individually packaged s
46、ingle-use material The whole device Other medical devices The whole device, cut into pieces or disassembled Table 3. Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch Number of Items in the Batch*6 Minimum Number of Items to be Tested for Each Medium (unless
47、otherwise justified and authorized)*6 Parenteral preparations Not more than 100 containers 10% or 4 containers, whichever is the greater More than 100 but not more than 500 containers 10 containers More than 500 containers 2% or 20 containers, whichever is less For large-volume parenterals 2% or 10
48、containers, whichever is less Antibiotic solids Pharmacy bulk packages (5 g) 20 containers Pharmacy bulk packages (5 g) 6 containers Bulks and blends See Bulk solid products Ophthalmic and other noninjectable preparations Not more than 200 containers 5% or 2 containers, whichever is the greater More than 200 containers 10 containers If the product is presented in the form of single-dose containers,apply the scheme shown above for preparations for parenteral use. 6 Catgut and other surgical sutures for veterinary use 2% or 5
