Sterility test

By Asim Ahmad

1. STERILITY TEST FOR PARENTRAL PRODUCTS

2. DEFINITION: Sterility Testing: It is a testing procedure applied to products intended to be sterile before marketing, to check that these products are free from all living microorganisms.

3. Sterility tests are performed on random samples from the batch and must be carried out under aseptic conditions in order to avoid accidental contamination of the product during the test using, for example, a laminar air flow cabinet.

4. Growth of microorganisms, if present in samples under test, is usually checked by incubating over specified culture Media at specified temperature for specified time.

5. Methods of Sterility Test

6. Direct Inoculation of Culture Media This test is performed by direct transferring of the test sample into the culture media. However, in case of large volumes such as intravenous fluids, a concentrated medium may be added directly to the preparation in its container.

7. Membrane Filtration Method The membrane filtration method requires the test sample to first pass aseptically through a membrane filter capable of retaining microorganisms. The filter is rinsed and then the membrane is transferred into the appropriate test medium.

8. CULTURE MEDIA USED FOR STERILITY TESTING

9. Fluid Thioglycollate Medium Primarily intended for culturing anaerobic bacteria. However, it also detects aerobic bacteria.

10. Soybean–Casein Digest Medium : Suitable for culturing both fungi and aerobic bacteria.

11. Tryptic Soya Broth A nutritious medium that supports the growth of a wide variety of microorganisms, especially common aerobic and facultatively anaerobic bacteria.

12. Media for Penicillins or Cephalosporins If sterility testing of samples having Penicillins or Cephalosporins is to be performed, then the above mentioned media are treated with β-lactamase sufficient to inactivate the amount of antibiotic in the sample under test.

13. Suitability Tests for Media

14. Sterility The sterility behavior of each culture medium is confirmed by incubating a portion of the media at the specified incubation temperature for 14 days. No growth of microorganisms should occur.

15. Growth promotion test of Aerobes, Anaerobes and Fungi Each lot of the medium is checked for growth promotion of micro-organisms. Strains to be tested Aerobic micro-organisms: Staphylococcus aureus, Pseudomonas aeruginosa. Anaerobic micro-organisms: Clostridium sporogenes Fungi : Candida albicans , Aspergillus niger .

16. Inoculate portions of Fluid Thioglycollate Medium with a small number of the stated aerobic and anaerobic micro-organisms using a separate portion of medium for each of the species of microorganism. Incubate for not more than 3 days.

17. Inoculate portions of Soybean–Casein Digest Medium with a small number of the stated Fungi using a separate portion of medium for each of the following species of microorganism. Incubate for not more than 5 days. The Medias are suitable if a clearly visible growth of the microorganisms occurs.

18. Storage If prepared media are stored in unsealed containers, they can be used for one month and if stored in tight containers, the media can be used for one year.

19. Minimum quantity and Number of Articles to Be Tested Unless otherwise specified in the individual monograph, test the minimum quantity and number of articles specified.

20. Quantity per Container Minimum Quantity to be Used Liquids (other than antibiotics) Less than 1 mL The whole contents of each container 1–40 mL Half the contents of each container, but not less than 1 mL 40-100 mL 20 mL >100 mL 10% of the contents of the container, but not less than 20 mL Antibiotic liquids 1 mL Other preparations soluble in water or in isopropyl myristate The whole contents of each container to provide not less than 200 mg 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 - 300 mg Half the contents of each container, but not less than 50 mg 300 mg–5 g 150 mg > 5 g 500 mg

21. Number of Items in the Batch Minimum Number of Items to be Tested for Each Medium Parenteral preparations < 100 containers 10% or 4 containers, whichever is the greater 100 - 500 containers 10 containers > 500 containers 2% or 20 containers, whichever is less For large-volume parenterals 2% or 10 containers, whichever is less Antibiotic solids Pharmacy bulk packages (<5 g) 20 containers Pharmacy bulk packages (> 5 g) 6 containers Ophthalmic and other noninjectable preparations Not more than 200 containers 5% or 2 containers, whichever is the greater More than 200 containers 10 containers

22. Membrane Filtration Method Use membrane filters having a nominal pore size not greater than 0.45 µm whose effectiveness to retain microorganisms has been established. Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions. Cellulose acetate filters are used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products (e.g., for antibiotics). The filtration apparatus and membrane are sterilized by appropriate means.

23. Procedure Aqueous solutions Transfer the contents of the containers to be tested to the membrane, if necessary, after diluting with the chosen sterile diluent. 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 diluents. Transfer the whole membrane to the culture medium or cut it aseptically into two equal parts, and transfer one half to each of two suitable media. Incubate the media for not less than 14 days.

24. For soluble solids (other than antibiotics). Use quantity of the product prescribed in Tables 1 and 2 for each medium, dissolved in a suitable solvent and proceed as described above for Aqueous Solutions.

25. For oils and oily solutions Low viscosity solutions 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 . Filter the oily solutions by applying the pressure or suction gradually. Wash the membrane at least 3 times by filtering about 100 mL of a suitable sterile solution. Transfer the membrane to the culture medium as described above for Aqueous Solutions and incubate at the same temperatures and for the same times.

26. For ointments and creams Use quantity of the product prescribed in Tables 1 and 2 for each medium. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1% in isopropyl myristate as described above, by heating, if necessary, to not more than 40 ºC. Filter as rapidly as possible, and proceed as described above for Oils and Oily Solutions

27. For solids for injection other than antibiotics Constitute the test articles as directed on the label, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.

28. Direct Inoculation of the Culture Medium Transfer the quantity of the preparation to be examined prescribed in Tables 1 and 2 directly into the culture medium so that the volume of the product is not more than 10% of the volume of the medium, unless otherwise prescribed.

29. If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance. For large volume products, it may be preferable to use a concentrated culture medium that undergoes subsequent dilution with the product. Where appropriate, the concentrated medium may be added directly to the product in its container.

30. Procedure For oily liquids Use media to which have been added a suitable emulsifying agent e.g., polysorbate 80 at a concentration of 10 g per L.

31. For ointments and creams Prepare by diluting to about 1 : 10 with the chosen emulsifying agent in a suitable sterile diluent. Transfer the diluted product to a culture medium not containing an emulsifying agent and incubate for not less than 14 days.

32. For solids Use quantity of the product prescribed in Tables 1 and 2 for each medium. Transfer the material so obtained to 200 mL of Fluid Thioglycollate Medium, and mix. Similarly, transfer the same quantity to 200 mL of Soybean–Casein Digest Medium, and mix. Proceed as directed previously.

33. OBSERVATION AND INTERPRETATION OF RESULTS The culture media is examined throughout the incubation period at intervals for macroscopic evidence of microbial growth. If the medium becomes turbid then after 14 days transfer portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 days.

34. If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless there has been a fault in the test conditions.

35. If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.