j. Soc. Cosmet. Chem., 29, 757-766 (December 1978) Imidazolidinyl urea activity against Pseudomonas PHILIP A. BERKE and WILLIAM E. ROSEN Sutton Laboratories, Inc., 459 East First Avenue, Roselle, NJ 07203. April I4, 1978. Presented at Annual Scientific Meeting, Society of Cosmetic Chemists, December I977, New York, New York. Synopsis Pseudomonas contamination of cosmetics is a major concern in the cosmetic industry because pseudomonads are so widely distributed in nature, so adaptable, and so resistant to most antimicrobials. Eleven ATCC-type pseudomonads, representing ones of concern for contaminating cosmetic products, and seventeen "wild" pseudomonads, isolated from a variety of contaminated cosmetic products, were screened and were found to differ in their vulnerability to IMIDAZOLIDINYL UREA • alone or in combination with parabens. Screening experiments were carried out to study variables such as incubation time, incubation temperature and pH with the purpose of learning how to design and interpret Pseudomonas screening experiments. Experimental data are presented contrasting inadequate or marginal preservation of cosmetic lotions and shampoos with adequate preservation of these products. It was found, for example, that the parabens alone provided inadequate protection AGAINST PSEUDOMONAS growth in several creams, lotions, and shampoos. Addition of Imidazolidinyl Urea to these formulations is shown to enhance the range of effectiveness and to give adequate protection against Pseudomonas contamination. INTRODUCTION Pseudomonas contamination of cosmetic products continues to be a serious problem in the cosmetic industry (1, 2). Essentially all types of water-containing products are vulnerable to this versatile, adaptable, sometimes pathogenic, gram-negative bacterium (3). Because its nutritional requirements are minimal (3), it often survives and multiplies under conditions where other microorganisms cannot. Even in distilled water, Pseudomonas has been reported to grow to counts of one million per ml or more (4, 5). Contamination with the single species Pseudomonas aeruginosa has alone accounted for numerous recalls of products from the market (6). Pseudomonads are difficult to avoid because they are widely distributed in water and soil (3) and are commonly found on the skin and on particles of dust in the air (6). The solution to the problem of Pseudomonas contamination of cosmetics is good manufacturing procedures plus the incorporation of an effective preservative system, but the choice of a preservative system is complicated by several factors: most commonly used preservatives are not effective against Pseudomonas preservatives are frequently inactivated by other •GERMALL 115, registered trademark of Sutton Laboratories, Inc., Roselie, NJ 07203. 757

758 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS components of a cosmetic formulation and there are hundreds of Pseudomonas species and an almost limitless number of strains, which may differ in their vulnerability to antimicrobials. It was therefore of interest to examine the behavior of a variety of pseudomonads in the presence of Imidazolidinyl Urea • (7), a commonly used (8) cosmetic preservative, in a laboratory screening test. Of course no screening test can substitute for the testing of a finished product. Only by challenge testing the final cosmetic formulation can one hope to establish whether or not a cosmetic is adequately preserved. Nevertheless, screening experiments can sometimes provide general information, which the authors felt might help cosmetic chemists and microbiologists to recognize the complexities and limitations of all testing procedures with Pseudomonas. EXPERIMENTAL METHODS Since different screening methods often give different results (9), a modification of an AOAC screening test (10) was chosen which was consistent with general practice, although somewhat more rigorous than usual. The choice was made to ignore bacterial counting and to require complete kill. This unusually rigorous approach meant that a 99.9% kill would be recorded in this study as "no kill," because growth would occur in a subculture even if only one organism survived. Nevertheless, the authors felt that this adaptation of the phenol coefficient method (10), spread out over several days, more closely represented the needed activity against Pseudomonas. An incubation temperature of 37øC for P. aeruginosa is recommended by AOAC (10) and others (11), and is considered by many (12) as the optimum temperature for growth of most bacteria. Since the USP XIX (13) recommends an incubation temperature for bacteria of 30-35øC, the choice was made in the study reported here to routinely incubate at 35øC. A third basic choice was to incubate in dilute nutrient media (see below), following the often-repeated suggestion by Goldman (14) that such dilute media best simulate conditions of growth in cosmetics. Initial screening was done with 11 pseudomonads purchased from ATCC (15), including those types most likely to be found in cosmetics. A 0.3% solution (16) of Imidazolidinyl Urea (Germall 115-Sutton Laboratories, Inc., Roselie, N.Y. 07203) was challenged with approximately 106 pseudomonads/mi. All pH's were adjusted to 7.0, because for most bacteria the optimum pH for growth generally lies between 6 and 8 (3, 12, 17). Table I shows the ATCC pseudomonads tested and includes strains of P. aeruginosa recom- mended by the USP (13) for testing antimicrobial effectiveness (i.e., P. aeruginosa ATCC 9027), by the AOAC (10) for disinfectant testing (i.e., P. aeruginosa ATCC 15442), by the CTFA Preservation Subcommittee (11) for testing of lotions (i.e., P. aeruginosa ATCC 13388), by the FDA (18) for testing of antiseptics (i.e., P. aeruginosa ATCC 14502) and the neotype strain (19) which is also used for antibiotic testing (i.e., P. aeruginosa ATCC 10145). The other pseudomonads tested (P. cepacia, P. putida, P. stutzeri, P. fluorescens and P. aureofaciens) are neotype strains of pseudomonads of interest to cosmetic microbiologists, and are species generally found in soil and water. Cultures were maintained on Trypic Soy Agar (TSA, Difco Laboratories, Detroit, Michigan) at 5øC. They were transferred approximately once a month. •GERMALL 115, registered trademark of Sutton Laboratories, Inc., Roselle, NJ 07203.