STANDARD OPERATING PROCEDURE FOR EFFECTIVENESS OF ANTIMICROBIAL PRESERVATIVES

1. OBJECTIVE :

1.1 The objective of preparation of this SOP is to define a procedure how to perform Effectiveness of Antimicrobial Preservatives in any dosage form

1.2 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.

1.3 There may be situations where a preservative system may have to be used to minimize the 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.

2. PRECAUTIONS:

2.1 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.

3. TEST ORGANISMS:

The following test organisms are used in the test.

Caandida albicans ATCC 10231 (NCPF 3179)*

Aspergillus niger ATCC 16404

Escherichia coli ATCC 8739

Pseudomonas aeruginose ATCC 9027 (NCIB 8626)

Staphylococcus aureus ATCC 6538 (NCTC 10788)

4. MEDIUM:

4.1 For the initial cultivation of the test organisms, use soybean casein digest agar medium or any other medium not less nutritive than the said medium.

5. PREPARATION OF INOCULUM:

5.1 From a recently grown stock culture of each of the test organisms, use Soyabean Casein Digest Agar medium or any other medium not less nutritive than the said medium.

5.2 The subculture on the surface of a suitable volume of the above-stated medium. Incubate the bacterial cultures at 30° to 35° for 18 to 24 hours and incubate the cultures of C.albicans and A.niger at 20° to 25°C for 48 hours and 7 days respectively.

5.3 Using the sterile saline solution, harvest the bacteria and C. Albicans cultures and dilute suitably with sterile saline solution to bring the count to about 1 x 108 per ml. Similarly, harvest A.niger culture with a 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.

5.4 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.

5.5 Determine the number of colony-forming units (CFU) per ml in each suspension. This value serves to determine the size of the 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.

6. PROCEDURE :

6.1 Inoculate each original product container or product tube ( when original container is not suitable for inoculation with a sterile syringe fitted with a needle, transfer 20 ml per capped bacteriological tube) with one of the standardized 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 x106 micro-organism per ml of product. 

6.2 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-count per ml of the product being examined.

6.3 Incubate the inoculated containers or tubes at 20° to 25°. Determine the viable count ( by the plate count method) at 7, 14, 21, and 28 days are subsequent to inoculation. Record also any change observed in appearance.

7. INTERPRETATION:

7.1 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 molds 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.

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