What is a USP <61> Test?
The USP <61> - Microbial Enumerations Test is a product safety test found in Chapter 61 of the United States Pharmacopeia ('USP'). It is suggested for use by the FDA for pharmaceuticals as well as cosmetics and personal care products, to ensure that a product's preparation complies with pre-set specifications for microbiological quality.
What is a USP <61> Test?
The USP <61> - Microbial Enumerations Test is a product safety test found in Chapter 61 of the United States Pharmacopeia ('USP'). It is suggested for use by the FDA for pharmaceuticals as well as cosmetics and personal care products, to ensure that a product's preparation complies with pre-set specifications for microbiological quality.
The USP <61> test is similar to an aerobic plate count assay as both tests determine if a product is currently contaminated with bacteria or fungi.
The USP <61> test is a full quantitative analysis of a product to determine the Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC) present in the sample. It also includes a neutralization and recovery validation using specific microorganisms. This ensures that the methods used to determine the presence of these microorganisms are appropriate for each individual product.
Acceptable Guidelines for Microbial Limits
Cosmetics and personal care products are not expected to be completely free of all microorganisms; however, they must be completely free of high-virulence microbial pathogens, especially in eye-area products, and the total number of microorganisms in the product must be low. Since there are no widely accepted standards for the number of microorganisms, there are some temporary guidelines to follow.
For eye-area and baby products, no more than 100 colony forming units (CFU)/ g or ml of product is considered acceptable. For non-eye-area products, no more than 1000 CFU/ g or ml of product is considered acceptable. If the product is determined to have aerobic microbial counts at or below these levels, further testing under USP <62> – Test for Specified Microorganisms – or an equivalent test may be necessary to determine if those microorganisms are high-virulence pathogens.
Pathogens are opportunistic pathogens whose presence in a cosmetic or personal care product would be of particular concern, especially in eye-area cosmetics, including S. aureus, P. aeruginosa, and C. albicans. These three microorganisms along with B. subtilis and A. brasiliensis are used as indicator organisms for the neutralization and recovery validation portion of the test.
MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: USP (61), (62)
Clients are advised to review the status of their non-sterile product testing in light of the new USP changes. If your substances or products have been validated and tested using USP methods other than those provided in USP 35-NF 30 it is essential to re-validate and begin testing using USP 35-NF 30 tests. Many of the changes are designed to harmonize with the European Pharmacopeia methods. The new USP methods now include more organisms. These are major changes for microbial limits testing. Clients are advised to factor these new USP methods into their testing schedules.
USP Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
USP tests allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions. The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality.
USP Microbiological Examination of Nonsterile Products: Tests for Specified Organisms
USP tests allow determination of the absence of, or limited occurrence of, specified microorganisms that may be detected under USP testing conditions.
USP microbial enumeration tests include changes in pass/fail criteria and includes longer incubation durations than in previous editions.
The Preparatory Test is replaced by the Method Suitability for product inhibition. The growth promotion organisms and the methodology have been significantly updated to include more types of organisms and different growth media. The performance of the Suitability Test Method ensures that any antimicrobial activity inherent in the sample to be tested does not adversely affect the reliability of the test and that the test procedure to be routinely utilized is otherwise suitable for use with the sample.
More organisms have been specified for USP testing. Organisms such as Candida albicans, Clostridia species and bile-tolerant gram-negative bacteria may be required to be tested depending on specifications regarding product formulation.
Clients must now specify which microorganisms are required to be absent from their substances or products. This requirement is based on the unique characteristics of the product’s formulation process, raw materials, etc.
Amount of Sample required for USP <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
10 Grams/mls/patches for USP 61 test while 30 to 40 grams/mls/patches for USP 61 Suitability Test
Amount of Sample Required for USP <62> Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms
10 Grams/mls/patches for USP 62 test while 30 to 40 grams/mls/patches for USP 62 Suitability Test
The USP 61/62 test utilizes the Compendial Method, as set forth by USP, for the testing of non-sterile pharmaceutical products and other non-sterile article for bacterial load and pathogenic organisms. USP 61 testing includes a plate count and suitability testing, which validates the plate count. USP 62 testing evaluates the presence or absence of objectionable organisms. This test method has defined procedures for the detection of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella species, fungal organisms and a quantitative MPN for Bile Tolerant Gram Negative bacteria. USP 61/62 testing is recommended for all pharmaceutical raw materials, non-sterile products, liquids and powders, transdermal patches and dietary supplements.
The USP tests are designed to validate and determine the quantitation of organisms present and screen for the presence or absence of objectionable organisms in non-sterile pharmaceutical products.
The validation and suitability testing are performed once for each product. The testing verifies the validity of the testing method by demonstrating any antimicrobial properties of the product do not inhibit possible recovery of growth. Both validation and suitability are performed with inoculation of the product which describes the validation of the plate count preparation tests and plating method by demonstrating recovery of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus brasiliensis on the appropriate media. USP suitability testing verifies the validity of the testing method by demonstrating the recovery of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, bile-tolerant gram-negative bacteria, Clostridia species, Salmonella species and Candida albicans.
USP routine analysis is performed to determine the total aerobic microbial and total yeasts and molds counts. The plating of the sample can be performed by membrane filtration, pour plating, or the spread plate method. The sample is diluted in an appropriate medium, generally Phosphate Buffer Solution, then it is plated on Soybean Casein Digest Agar (SCDA) for total aerobic microbial count (TAMC), and Sabaroud Dextrose Agar (SDEX) for total yeasts and molds count (TYMC). Incubation is for 3-5 days at 30-35°C for TAMC and 5-7 days at 20-25°C for TYMC.
Turn Around Time for USP <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
3 to 7 days for USP61 test and 10 to 14 days for USP 61 Suitability Test
Turn Around Time for USP <62> Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms
5 to 7 days for USP 62 test while 10 to 14 days for USP 62 Suitability Test
USP routine tests for the determination of the presence of specified microorganisms; Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, bile-tolerant gram-negative bacteria, Clostridia species, Salmonella species and Candida albicans. The test involves an initial enrichment of the sample in Soybean Casein Digest Broth (SCDA) or other appropriate neutralizing media, and then streaking the enriched sample onto selective agars for determination of the presence of the defined objectionable organisms.
USP 61 and USP 62 tests are carried out for Microbiological Examination of Non-Sterile Products. The USP 61 test is performed in routine to determine the “total aerobic microbial count” (TAMC) and “total yeasts and molds counts” (TYMC). Enumeration of microorganisms can be carried out by membrane filtration, pour plating, or the spread plate method.
USP 62 is performed in routine to test the presence of specified microorganisms:
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, bile-tolerant Gram-negative bacteria, Clostridia species, Salmonella species and/or Candida albicans.
In the case of USP 62 testing, the sample is first enriched by inoculation in Soybean Casien Digest Broth (SCDA), or other appropriate neutralizing media, and then streaked onto selective agars for determination of the presence of specified / objectionable microorganisms.
USP <61> and USP <62> tests provide harmonization to existing European Pharmacopeia methods for testing non-sterile pharmaceuticals. USP <61> describes the microbial enumeration tests. USP requires that prior to routine enumeration testing, a suitability of counting method / Method Validation must be performed.
USP <62> describes growth and recovery of specific organisms. USP requires that prior to routine specified microorganisms testing, a suitability of test method / Method Validation must be performed.
USP 61 Validation/ Suitability
100 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus brasillensus are inoculated to demonstrate the validation of the plate count method and media used.
USP 62 Validation / Suitability testing
Demonstrates the recovery of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, bile-tolerant Gram-negative bacteria, Clostridia species, Salmonella species and Candida albicans.
