The primary purpose of adding antimicrobial preservatives to dosage forms is to prevent adverse effects arising from contamination by micro-organisms that may be introduced inadvertently during or subsequent to the manufacturing process. However, antimicrobial agents should not be used solely to reduce the viable microbial count as a substitute for good manufacturing procedures. There may be situations where preservative systems may have to be used to minimize proliferation of micro-organisms in preparations that are not required to be sterile. It should be recognized that the presence of dead micro-organisms or the metabolic by-products may cause adverse reactions in sensitized persons.
Any antimicrobial agent may show the protective properties of a preservative. However for the protection of the consumer, the concentration of the preservative shown to be effective in the final packaged product should be considerably below the concentrations of the preservative that may be toxic to human beings.
The following tests are provided to demonstrate, in multiple dose injectable, nasal, and ophthalmic products made with aqueous bases or vehicles, the effectiveness of any added antimicrobial preservatives, the presence of which is declared on the label of the product concerned. The tests and samples apply only to the product in the original, unopened container in which it was supplied by the manufacturer.
The organisms specified for use in the tests are intended to be representative of those that might be expected to be found in the environment in which the preparation is manufactured, stored and used. However, they should be supplemented by other strains or species, especially those likely to be found in the conditions under which a particular product is made or used, or that might offer a particular challenge to the type of product being tested. Single-strain challenges (rather than mixed cultures) should be used throughout.
Precautions:- Challenge tests should be conducted under conditions that prevent accidental contamination of the product during the test but the precautions taken to prevent contamination should not affect the survival of organisms in the product being examined.
Test organisms:- The following test organisms are used in the test.
Candida albicans ATCC 10231 (NCPF 3179)
Aspergillus niger ATCC 16404
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027 (NCIB 8626)
Staphylococcus aureus ATCC 6538 9 (NCTC 10788)
Medium:- For the initial cultivation of the test organism, use Soyabean Casein Digest Agar Medium or any other medium not less nutritive than the said medium.
Preparation of inoculum:- From a recently grown stock culture of each of the test organisms, subculture on the surface of a suitable volume of the above stated medium. Incubate the bacterial cultures at 30°C to 35°C for 18 to 24 hours and incubate the cultures of C.albicans and A. niger at 20°C to 25°C for 48 hours and 7 days respectively.
Using sterile saline solution, harvest the bacterial and C. albicans cultures and dilute suitably with the sterile saline solution to bring the count to about 1 x 108 per ml. Similarly harvest A. niger culture with sterile saline solution containing 0.05% w/v of Polysorbate 80 and adjust the spore count to about 1 x 108 per ml with sterile saline solution.
Alternatively, the stock culture organisms may be grown in a suitable liquid medium, and the cells may be harvested by centrifugation, washed and resuspended in sterile saline solution to give the required microbial or spore count.
Determine the number of colony-forming units (CFU) per ml in each suspension. This value serves to determine the size of inoculum to be used in the test. If the standardized suspensions are not used promptly, periodically monitor the suspensions by the plate-count method to determine any loss of viability.
Procedure:- Inoculate each original product container or product tube (when original container is not suitable for inoculation with sterile syringe fitted with needle, transfer 20 ml per capped bacterial tube) with one of the standard microbial suspensions using a ratio equivalent to 0.1ml of inoculum suspension to 20ml of product and mix. The final concentration should be between 1 x 105 and 1 x 106 micro-organisms per ml of the product. Determine the number of viable micro-organisms by the plate count method in each inoculum suspension and from there calculate the initial concentration of micro-organisms per ml of product being examined.
Incubate the inoculated containers or tubes at 20°C to 25°C. Determine the viable count (by the plate count method) at 7, 14, 21 and 28 days subsequent to inoculation. Record also any change observed in the appearance.
Interpretation:- The preservative is effective in the product examined if
(a) The concentrations of viable bacteria are not more than 0.1% of the initial concentrations by the 14th day,
(b) The concentrations of viable yeasts and mould remain at or below the initial concentration during the first 14 days and
(c) The concentration of each test micro-organism remains at or below these designated levels during the remainder of the 28 days test period.
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