J. Soc. Cosmetic Chemists, 16, 499-506(1965) Skin Substantivity as a Criterion the Evaluation of Antimicrobials THOMAS F. McNAMARA, Ph.D., MARIANNE L. STEINBACH, B.S., and BENJAMIN ,q •4w•q'?, • q * Presented before the New York Chapter, September 9, 1•6•, New York City Synopsis--Skin substantivity is defined as the avidity of a compound for skin tissue. Com- pounds possessing substantivity were evaluated by two major criteria: (a) qualitatively by measuring the comparative avidity for skin tissue and (b) quantitatively by titrating the release of the compound from tissue. A method was developed to determine each of these properties for compounds having antimicrobial properties. The application of such data to the selection of a compound for a particular topical use was discussed. INTRODUCTION The trend toward the topical application of antimicrobial sub- stances in cosmetic preparations and proprietary formulations has been increasing for several years. This is evidenced by the fact that the number and types of medicated cosmetics are now approaching in num- ber those of the nonmedicated ones. A large number of chemical com- pounds with similar antimicrobial, physical and chemical properties and toxicity levels are available for use in topical formulations. If one is to choose the best compound for topical application, an additional criterion must be established. Such a criterion for evaluating these compounds is their avidity for tissue and their elution from it in an active form (tissue substantivity). This report is concerned with the methodology used in the in vitro evaluation of four antimicrobial agents with regard to their tissue substantivity. * Warner-Lambert Research Institute, Morris Plains, N.J. 07950. 499

500 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS MATERIALS AND METHODS Agents: For use in these studies, samples of 4-amino-l-laurylquin- aldinium acetate monohydrate (Laurodin acetate),* 1,1 '-hexamethylene- bis[5-(2-ethylhexyl)biguanide] dihydrochloride (Sterwin #904),• alkyl- dimethylbenzylammonium saccharinate (Holichem Hg• 3300), •: and the 1-hexadecylpyridinium chloride (C.P.C.)õ were obtained from com- mercial sources. For the microbiological studies, solutions were prepared in the follow- ing manner. Aqueous or aqueous-alcoholic stock solutions of the com- pounds were prepared at concentrations of 1000 .a/mi. and stored in the refrigerator until used. In Vitro Tests: The organisms tested were obtained either from the American Type Culture Collection (AATCC) or from our own stock cul- ture collection (WLRI). Three basic media were used in these studies, brain heart infusion broth (Difco) for the bacteria, Sabouraud's dextrose broth (Difco) for C. albicans, and AATCC broth (Difco) for the determination of bactericidal activity of the compounds. (Exception: F. polymorphum was cultured in a mixture (1: 1) of brain heart infusion broth (Difco) and fluid thio- glycollate (Difco)). The determination of the minimum inhibitory concentration for each of the organisms was carried out by the twofold broth dilution method. The inoculum used, except for F. polymorphum, was 0.1 ml. of a 1:1000 dilution of a 50% transmission (Lumetron Colorimeter Model 402E equipped with M 465 filter) of a twenty- four-hour broth culture. For F. polymorphum, the inoculum used was 0.1 ml. of a 50•o transmission of a twenty-four-hour broth culture. All cultures were incubated at 37øC for forty-eight hours. The minimum inhibitory concentration was recorded as the lowest concentration of the compound at which there was no visible growth of the organism. The bactericidal activity was determined for each compound according to the U.S.F.D.A. (1) method for testing antiseptics and disinfectants. In those cases where a neutralizer was available to obviate possible bacteriostasis of the test substances, the appropriate neutralizer was added to the subculture medium. These neutralizing substances were: 0.05•o sodium thioglycollate for mercurials and other * Allen & Hanburys Ltd. t Sterwin Chemicals, Inc. { Hollichem Corp. õ K & K Laboratories, Inc.