Antimicrobial effectiveness test

The test for antimicrobial effectiveness is used to demonstrate the effectiveness of any added antimicrobial preservative(s). Compendial referenees include USP 24 Chapter (51) Antimicrobial Effectiveness Test JP XIII General Information 3, Pre-servatives-Effeetiveness Tests and the Ph. Eur. 3rd ed., Biologieal Tests, 5.1.3. Effi-eacy of Antimierobial Preservation. 

The selection of the preservative system for multiuse new products is the responsibility of the R D formulation group. Typical shelf specifications are 80 to 120% of label specifications. The appropriate preservative system for the particular formulation should be demonstrated to be effective by microbial challenge to at least 75% and preferably 50% of the target concentration. It is recommended that during development the product be formulated with preservative concentrations of 100, 75, and 50% of the labeled amount and be subjected to antimicrobial effectiveness testing to determine the lowest effective preservative concentration. 

The release and shelf-life specifications are established based on both the premarketed stability data for the preservative system concentration and the antimicrobial effectiveness test results. 

Abbreviations TAMC = total aerobic count TCYMC = total combined yeasts and molds AET = antimicrobial effectiveness test CCl = container-closure integrity A = water activity. 

A stability-indicating presrvative assay can be justified only at all time intervals as a substitute for the USP Antimicrobial Effectiveness Test by confirming preservative efficacy at 50, 75, and 100% of the lower shelf-life specification. 

If the study outlined above in paragraph 1 was not conducted during product development, it is recommended that QA and/or the technical services groups imder-take a study to justify the cmrent specifications and the elimination of routine antimicrobial effectiveness testing within the stability program. 

All preservative systems for both parenteral and nonsterile dosage forms should meet the 3 log reduction at 14 days for bacteria, that is, USP category I requirements. Both EP and BP Antimicrobial Effectiveness Testing would be run only if specially requested by the marketing group. 

USP 24 Testing Chapters (51) Antimicrobial Effectiveness Testing, (61) Microbial Limit Tests and (71) Sterility Tests, United States Pharmacopeial Convention, Inc., Rockville, MD, 2000. USP 24 Informational Chapters (1116) Microbiological Evaluation of Clean Rooms and other Controlled Environments, (1111) Microbiological Attributes of Pharmaceutical Articles, (1151) Pharmaceutical Dosage Forms, (1225) Validation of Compendial Methods, and (1231) Water for Pharmaceutical Purposes, United States Pharmacopeial Convention, Inc., Rockville, MD, 2000. 

This study should be independently repeated a sufficient number of times to establish an upper limit of cfu for the particular plating conditions. There is a lower limit at which the ability of the antimicrobial effectiveness test to recover microorganisms becomes unreasonable. If the first plating is performed with 1 ml of 10 dilution, cfu in the range of 1 to 10 per ml would not be seen. On this dilution plating, only the lower number of cfu may be reduced to three, allowing as few as 30 cfu/ml survivors to be reported. 

U.S. Pharmacopeia (USP) (2007), Antimicrobial effectiveness testing, general chapter <51>, U.S. Pharmacopeial Convention, Rockville, MD. 

USP (51) antimicrobial effectiveness testing. United States Pharmacopeia, 24 US Pharmacopeial Convention, Inc. Rockville, 2000 1809. 

Testing Chapters Antimicrobial Effectiveness Testing, Ch. 51 Microbial Limit Tests, Ch. 61 Sterility Tests, Ch. 71. In U.S.P. 24. 

If a multidose formulation is required, an antimicrobial agent, also called a preservative, must be added to the formulation for regulatory compliance. The preservative must meet USP requirements and the Antimicrobial Effectiveness Test as stated in the USP. The most often used preservatives for proteins or recombinant products are phenol at 0.3 to 0.5% chlorobutanol, also at 0.3 to 0.5%, and benzyl alcohol at 1.0 to 3.0%. 

The multidose formulation will be tested to determine its efficacy according to the Antimicrobial Effectiveness Test required by the USP.39 If the results support the USP requirements, international requirements must then be met. International markets require different preservatives, different concentrations, and different excipients of the formulation. In addition, the period required for the inhibition and/or killing of the challenge microorganisms may be different. International regulatory requirements for compliance should be well researched and understood by the scientific and the management staff. 

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