Effectiveness of Antimicrobial Preservatives in Pharma industry
The efficacy of antimicrobial preservation of a pharmaceutical preparation on its own or, if necessary, with the addition of a suitable preservative has to be ascertained during the development of the product.
The primary purpose of adding antimicrobial preservatives in Pharma industry 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 in Pharma industry should not be used solely to reduce the viable microbial count as a substitute for good manufacturing procedures.
There may be situations where a preservative system may have to be used to minimize proliferation of micro-organisms in preparations that are not required to be sterile.
It should be recognised that the presence of dead micro-organisms or the metabolic by-products may cause adverse reactions in sensitised 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 parenteral, otic, nasal, ophthalmic, oral, and topical products made with aqueous bases or vehicles, the effectiveness of any added preservatives, during the shelf
lives of the preparations to ensure that the antimicrobial activity has not been impaired by storage.
The tests apply only to the product in the original, unopened container in which it was supplied by the manufacturer.
The test consists of challenging the preparation in its final container with a prescribed inoculum of suitable microorganisms, storing the inoculated product at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples removed. The preservative properties of the product are considered adequate if, in the conditions of the test, there is a significant fall or no increase in the number of microorganisms in the inoculated preparation after storage for the times and at the temperatures prescribed.
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 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.
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
Aspergillus niger ATCC 16404
Escherichia coli ATCC 8739
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
For the initial cultivation of the test organism, use Soyabean Casein Digest Agar Medium for bacterial cultures and Sabouraud-dextrose agar for C albicans and A. niger, or any other media not less nutritive than the said media.
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 media. 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 percent 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 standardised suspensions are not used within 2 hours, it should be stored in a refrigerator. Periodically monitor the stored suspensions by the plate-count method to determine any loss of viability.
Inoculate each original product container or product tube (when original container is not suitable for inoculation with a sterile syringe fitted with needle, transfer 20 ml per capped bacterial tube) with one of the standard
microbial suspensions using a ratio equivalent to 0.1 ml of inoculum suspension to 20 ml of product and mix.
The final concentration should be between 1 x 105 and 1 x 106 microorganisms 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.
The preservative is effective in the product examined if
(a) the concentration of viable bacteria are not more than 0.1 percent of the initial concentrations by the 14th day,
(b) the concentrations of viable yeasts and moulds 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-day test period.
NOTE — The test for effectiveness of antimicrobial preservatives in Pharma industry is non-mandatory and is not intended for use
for routine control purposes.