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10.6 EFFICACY OF ANTIMICROBIAL PRESERVATION​

10.6 EFFICACY OF ANTIMICROBIAL PRESERVATION​

      Antimicrobial preservatives are substances added to pharmaceutical preparations to prevent microbial proliferation or to limit microbial contamination that may occur during normal conditions of storage and use. They are used primarily in multiple-dose parenteral, oral, nasal, topical, ear, and eye preparations made with aqueous bases or vehicles. Antimicrobial preservatives should not be used as a substitute for good manufacturing practices or solely to reduce the viable microbial population of a nonsterile product or control the presterilization bioburden of multidose formulations during manufacturing.

      The efficacy of an antimicrobial preservative may be enhanced or diminished by many factors. Those include the active ingredient, the formulation, and the container or closure used for that product. The test for efficacy of antimicrobial preservation should therefore be carried out on the product as presented, wherever possible in its original, unopened container in which it was distributed by the manufacturer.

      During development of a pharmaceutical preparation, it should be demonstrated that the antimicrobial activity of the preparation provides adequate protection from microbial contamination during storage and use. The test described below is therefore designed to determine the efficacy of antimicrobial activity of the product. The test is not intended to be performed on a routine control basis.

      The test for efficacy of antimicrobial preservation in the preparation consists of challenging the preparation with a prescribed inoculum of suitable micro-organisms, storing the inoculated preparation at a prescribed condition, and withdrawing samples from the container at prescribed time intervals to count the remaining viable organisms.  

      The preservative properties of the preparation are adequate if, in the conditions of the test, there is a significant decrease or no increase in the number of micro-organisms in the inoculated preparation after the times and at the temperatures prescribed. The criteria of acceptance vary for different types of preparation according to the degree of protection intended.

Product Categories

      For the purpose of testing, products have been divided into four categories (see Table 1). The criteria of antimicrobial effectiveness for these products are a function of the route of administration.

Table 1 Product Categories

Test Organisms​

Aspergillus niger                               ATCC16404, DMST2 15538

Candida albicans                               ATCC 10231, DMST 5815

Escherichia coli                                 ATCC 8739, DMST 15537

Pseudomonas aeruginosa                  ATCC 9027, DMST 15501

Staphylococcus aureus                      ATCC 6538, DMST 8013

      Single-strain challenges, either ATCC or DMST, should be used throughout the test.

Media

      For the initial cultivation of the test organisms, select an agar medium that promotes vigorous growth of the respective stock culture, such as Soybean-casein digest agar medium for bacteria and Sabouraud dextrose agar medium for fungi (Appendix 10.2).

Preparation of Inoculum

      Begin the test by inoculating the surface of a suitable solid agar medium from the recently grown stock culture of each of the specified micro-organisms. Incubate the bacterial cultures at 30º to 35º for 18 to 24 hours, the culture of Candida albicans at 20º to 25º for 48 hours, and the culture of Aspergillus niger at 20º to 25º for 1 week or until good sporulation is obtained.

      To harvest the bacterial and Candida albicans cultures, use a sterile 0.9 per cent w/v solution of sodium chloride for dispersal and transfer of the surface growth into a suitable vessel. Add sufficient suspending fluid to reduce the microbial count to about 1 × 108 colonyforming units (CFU) per ml. To harvest the Aspergillus niger culture, use a sterile 0.9 per cent w/v solution of sodium chloride containing 0.05 per cent w/v of polysorbate 80 and adjust the spore count to about 1 × 108 CFU per ml by adding the same solution.

      Alternatively, the stock culture organisms may be grown in a suitable liquid medium, and the microorganisms may be harvested by centrifugation, washed, and dispersed in a sterile 0.9 per cent w/v solution of sodium chloride to give the required microbial or spore count.

      Determine immediately the number of colonyforming units per ml in each suspension by means of Plate Method (Appendix 10.2). This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to seven days.


1ATCC = American Type Culture Collection

2DMST = Department of Medical Sciences, Thailand

 

Procedure 

      To count the viable micro-organisms in the inoculated preparations, use the agar medium corresponding to that used for the initial cultivation of the respective micro-organisms. Ensure that any residual antimicrobial activity of the products is eliminated either by dilution, by filtration or by the use of a specific inactivator.

      If the product container can be entered aseptically, with needle and syringe through stopper, conduct the test in five original containers. If not, transfer a 20-ml portion of preparation from five containers to each of five sterile capped tubes. Inoculate each container with one of the prepared and standardized inoculum, and mix. The volume of the suspension inoculum used is between 0.5 per cent and 1.0 per cent of the volume of the product. The concentration of test micro-organisms that is added to the product (Categories 1, 2, and 3) are such that the final concentration of the test preparation after inoculation is between 1 × 105 and 1 × 106 CFU per ml of the product. For Category 4 products (antacids) the final concentration of the test preparation after inoculation is between 1 × 103 and 1 × 104 CFU per ml of the product. Determine the number of viable microorganisms in each inoculated suspension and calculate the initial concentration of micro-organisms per ml of preparation.

      Incubate the inoculated containers or tubes at 20º to 25º. Sample each container at the appropriate intervals specified in Table 2. Record any changes observed in appearance at these intervals. Determine by the Plate Method the number of CFU present in each test preparation for the applicable intervals. Using the calculated concentrations of CFU per ml present at the start of the test, calculate the change in log10 values of the concentration of CFU per ml for each micro-organism at the applicable test internals, and express the changes in terms of log reductions.

Evaluation

      The criteria for evaluation of antimicrobial activity are given in Table 2 in terms of the log reduction in the number of viable micro-organisms. No increase (NI) is defined as not more than 0.5 log10 unit higher than the previous value measured.

Table 2 Criteria for the Evaluation of Preservative Efficacy

 

APPENDICES • 10.6 EFFICACY OF ANTIMICROBIAL PRESERVATION​
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หมายเหตุ / Note : TP II 2011 PAGE 640-641