J. Soc. Cosmet. Chem., 31, 1-10 (January/February 1980) GRAS antimicrobial agents for cosmetic products jONj. KABARA, Department of Biomechanics, Michigan State University, East Lansing, M148824. Received.June 7, •979. Presented at Annual Scientific Seminar, Society of Cosmetic Chemists, May 1979, Dallas, Texas. Synopis Data are presented for extending and enhancing the ANTIMICROBIAL activity of a new preservative, Lauricidin © (monolaurin). Chemicals well known in the food and COSMETIC industry to have antioxidant and chelating properties were shown to be enhancers. Butylated hydroxy toluene (BHT) and certain alkyl derivatives were examined for biocidal activity. Structure-function studies indicate that the butyl chain was the most active of the alky derivatives (C•-C•0) examined. The removal of the alkyl chain in the para position increased biological activity as did the change of the tert butyl groups from the 2,6- to the 3,5-position. Although the methoxy derivative of BHT was not very active, another anisole derivative, butylated hydroxy anisole (BHA), was very active. While ethylenediaminetetraacetic acid (EDTA) has little antimicrobial activity of its own, it was a strong enhancer of Lauricidin against gram (-) organisms. Combinations of Lauricidin, BHA, and EDTA were shown to have wide spectrum activity. Kill times for the combination in water which were normally slow became much faster when the combination was placed in alcoholic solutions. The addition of Lauricidin and BHA to commercial mouth-washes greatly enhanced their previously poor cidal action. Lauribic, a proprietory combination of Lauricidin and sorbic acid (GRAS MATERIALS), was shown under identical conditions to be often more active than parabens or sorbic acid. INTRODUCTION Antimicrobials are employed as antiseptics, as disinfectants and (in lower concentra- tions) as preservatives for foods, cosmetics and drugs. Despite the rather large number (over 60) of antimicrobials available, each has important physical, chemical or toxicological limitations, so that interest in the development of new and better antimicrobials remains high. Traditional preservatives such as ethanol at a concentration of not less than 20%, chloroform at 0.25%, sucrose at 67% and benzoic acid at 0.1% are still used and as such are a testimony to their effectiveness. Severe limitations in their applicability however restrict their use in modern products. Our laboratory became interested in the role of fatty acids and their derivatives as potential cosmetic and food preservatives (1-7). Lauricidin © (Monolaurin), product of this research effort, fulfills many of the needed criteria of the ideal cosmetic preservative. This surfactant is the first nonionic to possess bacteriostatic and

2 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS bactericidal activity. Lauricidin is recognized as a GRAS food emulsifier and thus has no toxicity in the usual sense of the word. As a nonionic it represents a chemical family which has a low order of irritability (7). The one limitation of Lauricidin is its lack of gram (-) activity. This paper represents our initial effort to achieve wide spectrum activity for Lauricidin by combining it with other food grade chemicals. In our studies tert-butyl toluene (BHT), tert-butyl anisole (BHA), and/or ethylenediaminetetraacetic acid (EDTA) were used in combination with Lauricidin (5). Both BHT and BHA were discovered to possess antibacterial activity earlier but because of patent filings, a public announcement of their antimicrobial potential was delayed until this announcement. During this hiatus a number of papers dealing with the antimicrobial properties of these and similar phenolic compounds have appeared (8-19). MATERIALS AND METHODS SOURCE OF PRESERVATIVES The nonionic germicide Lauricidin and Lauribic were obtained from Med-Chem Labs (Monroe, MI) tert-butyl-hydroxy-toluene (BHT) and - anisole (BHA) were obtained from Ashland Chemical Co. (Dublin, OH) and Eastman Organic Chemicals (Roches- ter, NY) respectively. Ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma Chemical (St. Louis, MO). SOURCE OF MICROBIOLOGICAL MEDIA AND OTHER CHEMICAL Except for the protease peptone, which was ordered from Difco Laboratories (Detroit, MI), the various broths and agar were purchased from BBL, Div. Becton, DicKinson and Co. (Cockeyville, MD). Tween 80 © (poloxyethylene sorbitan mono-oleate) was bought from Sigma Chemical Co. (St. Louis, MO). Inorganic chemicals used in the experiments were obtained from Mallinckrodts, Inc. (St. Louis, MO). PREPARATION OF PRESERVATIVE SOLUTION AND/OR SUSPENSIONS Standard solutions of Lauricidin, BHT/BHA, EDTA individually or in combination were prepared by first dissolving the weighed lipophilic compounds in 1 or 2 ml of isopropyl alcohol at 60 ø and immediately adding 199 or 98 ml of trypticase soy broth (60øC). Standard solutions or suspensions at 10 mg/ml were serially diluted two-fold with additional broth to a concentration of 125 3tg/ml. If a compound or mixture were found to have inhibitory values at concentrations lower than 125 3tg/ml, additional dilutions were carried out. MICROORGANISMS The microorganisms used for these studies were picked from those frequently or typically encountered in contaminated cosmetics and/or from those of clinical significance (1,2). Strain differences within a genus were not significant for the compounds tested by our technique.