Antimicrobial Effectiveness Testing

Antimicrobial preservatives are substances added to nonsterile dosage forms to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile articles packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses.

Antimicrobial effectiveness, whether inherent in the product or whether produced because of the addition of an antimicrobial preservative, must be demonstrated for all injections packaged in multiple-dose containers or for other products containing antimicrobial preservatives. Antimicrobial effectiveness must be demonstrated for multiple-dose topical and oral dosage forms and for other dosage forms such as ophthalmic, otic, nasal, irrigation, and dialysis fluids.

This chapter provides tests to demonstrate the effectiveness of antimicrobial protection. The tests and criteria for effectiveness apply to a product in the original, unopened container in which it was distributed by the manufacturer.

The following challenge organisms are used in USP testing: Candida albicans (ATCC No. 10231), Aspergillus niger (ATCC No. 16404), Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), and Staphylococcus aureus (ATCC No. 6538).

The test is conducted either in five original containers, if sufficient volume of product is available in each container and the product container can be entered aseptically (i.e., needle and syringe through an elastomeric rubber stopper), or in five sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred. Each container is inoculated with one of the prepared standardized inoculums.

Each container is incubated and sampled at specified intervals. The number of colony forming units present in each test preparation for each interval is determined by the plate-count method. An appropriate neutralizer is utilized in the plate count or in the appropriate dilution prepared for plating. These conditions are determined in the validation study for that sample based upon the condtions of media and the microbial recovery incubation times.

Using the calculated concentrations of cfu per mL present at the start of the test and the concentration of cfu per mL for each microorganism at each test interval, changes in log10 values are calculated and expressed in log reductions.

The requirements for antimicrobial effectiveness are met if the criteria specified in Chapter; Table 3 are met. No increase is defined as not more than 0.5 log10 unit higher than the previous value measured.

Criteria for Tested Microorganisms
For Category 1 Products
Bacteria:Not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 7, 14, and 28 days.
For Category 2 Products
Bacteria:Not less than 2.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 14 and 28 days.
For Category 3 Products
Bacteria:Not less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days.
Yeast and Molds:No increase from the initial calculated count at 14 and 28 days.
For Category 4 Products
Bacteria, Yeast, and Molds:

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