j. Soc. Cosmet. Chem., 34, 35-39 (January/February 1983) FDA methodology for the microbiological analysis of cosmetics and topical drugs MICHAEL J. PALMIERI, Food and Drug Administration, Department of Health and Human Services, New York Regional Laboratory, 850 Third Avenue, Brooklyn, NY 1 I232. Received June I, 1982. Presented at the SCC-SIM Microbiological Seminar, New York, NY, Dec. 9, I981. Synopsis The microbiological analysis of many cosmetics and topical drugs often presents problems in detecting microbial contaminants. This occurs principally because of poor miscibility of these products with water and because of the presence of preservatives in their formulations. Both improved miscibility of product with medium and neutralization of several preservatives have been achieved by the use of modified letheen medium. A rapid screening test is performed initially to determine if the product is free of contamination. If microbial growth is observed, then plate counts and enrichments are employed to test for Staphylococcus aureus, yeasts, fungi, aerobes, anaerobes, and gram negative bacilli. INTRODUCTION This report deals with the methodology currently employed at the New York Regional Laboratory of the Food and Drug Administration (FDA) for the microbiological analysis of both cosmetic and topical drug preparations. During the period 1979 ß through 1981 approximately two hundred cosmetic and topical drug samples of both domestic and foreign origin were analyzed at the New York Laboratory. The kinds of products tested included skin creams and lotions, shampoos, hair rinses, powders, various eye cosmetics, ophthalmic solutions, and several types of topical ointments and disinfectants. All these product types and especially those with emulsion formulations have been shown at one time or another to contain a wide variety of microorganisms. Sixteen samples (8%) were found to be contaminated with various gram negative bacteria, a group of organisms of special concern because many are potentially pathogenic. Generally, preparations containing 10% or more alcohol, such as after-shave lotions, colognes, and skin refreshners do not present serious microbiological problems. Products with low water activity (aw) such as anhydrous oils, ointments, or petrolatum do not support the growth of large numbers of microorganisms and therefore are also relatively problem-free. 35

36 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Many types of cosmetic and topical preparations contain microorganisms, but their presence is difficult to detect because the cells are entrapped in oil-water emulsions or ointment based matrices. Physical and chemical procedures are employed to free entrapped cells so that they can grow in culture media. Many products contain preservatives which kill or inhibit the growth of microorganisms. In testing such products, it is desirable to neutralize the preservative so that any viable cells present can grow in the test culture medium. It may not be obvious why we want to look for microorganisms that cannot grow in a preserved cosmetic or topical drug. The problem is that although initially the preservative may work well in the product, upon storage its effectivensss can be reduced by chemical changes. Furthermore, upon application of a product to the body, the effectiveness of a preservative can be reduced due to dilution with moisture and organic matter or even by evaporation of the preservative. Previously inhibited microorganisms are now capable of growth and can be potentially pathogenic if they gain entrance into the body. The present methods employed by FDA for the microbiological analysis of cosmetics and topical drugs are outlined in the 5th edition of the Bacteriological Analytical Manual (BAM) (1). The ultimate objective of the examination is to provide a microbiological profile of the product which is as complete and comprehensive as possible. A considerable improvement in our ability to detect contaminating microor- ganisms has been achieved through the use of modified letheen broth as both an enrichment medium and diluent. Modified letheen, a highly enriched medium containing lecithin, polysorbate $0, and sodium bisulfite, supports the growth of most microorganisms. Both the lecithin and polysorbate 80 serve as surface-active agents and emulsifiers that promote microbial contact with the aqueous phase of the broth medium. These two ingredients also serve to inactivate or neutralize several kinds of preservatives, including quarternary ammonium compounds, parabens, and phenolics. Viable cells that are present but inhibited by these preservatives in products are therefore able to grow in culture media. Sodium bisulfite, another ingredient in modified letheen, neutralizes some of the aidehyde-type preservatives, e.g., 2% glutaraldehyde (2). In practice, we have found that modified letheen broth is a very effective and versatile medium. However, it seems possible that future research will lead to development of media that are even more effective in neutralizing preservatives than modified letheen. For example, Bruch and Kohn reported that polysorbate 20 is more effective than polysorbate $0 in neutralizing parabens (3). As in all microbiological testing, the sample should be analyzed as soon as possible after collection. The containers are examined visually for any defects or irregularities, such as cracks or leaks. Once established that a container is sound, the external surface is disinfected with a suitable antimicrobial agent and air dried under a laminar flow hood. The analytical methodology can be divided into four distinct stages: screening, plating, enrichment, and if required, identification of isolates. An initial screening test is performed to determine if the product is contaminated. If growth is detected in the screening media, then a variety of methods are used to determine plate counts of specific groups of microorganisms including aerobes, anaerobes, Staphylococcus aureus, yeasts, and molds. Every sample containing gram negative bacilli is subjected to an enrichment procedure for the enumeration and further identification of these organisms.