230 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ACKNOWLEDGEMENTS This work was supported by the Cosmetic, Toiletry, and Fragrance Association, Inc. We wish to thank Norman Estrin, Mel Goodman, Gary Henderson, Lester Isbrandt, Aaron Cooper, John McNally, Ira Rosenberg, C. T. Desmond, Bernard L. Kabocoff, M. Graber, Jack Winstead, and other members of the CTFA Nitrosamine Task Force for their advice and guidance. Specific methods are available from CTFA. REFERENCES (1) R. Montesano and H. Bartsch, Mutagenic and carcinogenic N-nitroso compounds: Possible environ- mental hazards, Murat. Res., 32, 179-228 (1976). (2) L. Griciute, "Carcinogenicity of N-nitroso Compounds and Their Possible Role in the Development of Human Cancer," in Environmental Carcinogens, Selected Methods of Analysis, H. Egan, Ed., IARC Scientific Publication No. 18 (International Agency for Research on Cancer, Lyon, France, 1978), Vol. 1, pp 3-9. (3) D. Schmiihl, Problems of dose-response studies in chemical carcinogenesis with special reference to N-nitroso compounds, CRC Critical Rev. Toxicol., 6, 257-281 (1979). (4) L. R. Ember, Nitrosamines: Assessing the relative risk, Chem. Eng. News, 20-26 (March 31, 1980). (5) W. Lijinsky and S.S. Epstein, Nitrosamines as environmental carcinogens, Nature, 225 (5227), 21- 23 (1970). (6.) E. A. Walker, P. Bogovski, and L. Griciute, Eds., Environmental N-Nitroso Compounds Analysis and Formation, IARC Scientific Publication No. 14 (International Agency for Research on Cancer, Lyon, France, 1976). (7) P. N. Magee, R. Montesano, and R. Preussman, "N-Nitroso Compounds and Related Carcinogens," in Chemical Carcinogens, C. E. Searle, Ed., ACS Monograph 173 (American Chemical Society, Wash- ington, D.C., 1976), pp 491-625. (8) P. Bogovski and E. A. Walker, Eds., N-Nitroso Compounds in the Environment, IARC Publication No. 9 (International Agency for Research on Cancer, Lyon, France, 1975). (9) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans.' Some N-Nitroso Compounds (International Agency for Research on Cancer, Lyon, France, 1978), Vol. 17. (10) E. A. Walker, L. Griciute, M. Castegnaro, and R. E. Lyle, Eds., Environmental Aspects of N-Nitroso Compounds, IARC Scientific Publication No. 19 (International Agency for Research on Cancer, Lyon, France, 1978). (11) E. A. Walker, L. Griciute, M. Castegnaro, M. Borzsonyi, and W. Davis, Eds., N-Nitroso Compounds.' Analysis, Formation, and Occurrence, IARC Scientific Publication No. 31 (International Agency for Research on Cancer, Lyon, France, 1980). (12) T. Y. Fan, J. Morrison, D. P. Rounbehler, R. Ross, and D. H. Fine, N-Nitroso-diethanolamine in synthetic cutting fluids: A part-per-hundred impurity, Science, 196, 70-71 (1977). (13) I. Schmeltz, S. Abidi, and D. Hoffman, Tumorigenic agents in unburned processed tobacco: N- nitrosodiethanolamine and 1,1-dimethylhydrazine, Cancer Lett., 2, 125-132 (1977). (14) T. Y. Fan, U. Goff, L. Song, D. H. Fine, G. P. Arsenault, and K. Bieman, N-Nitrosodiethanol- amine in cosmetics, lotions, and shampoos, Food Cosmet. Toxicol., 15, 423-430 (1977). (15) W. Lijinsky, M.D. Reuber, and W. B. Manning, Potent carcinogenicity of nitrosodiethanolamine in rats, Nature, 288, 589-590 (1980). (16) I. Rosenberg, The CTFA program on nitrosamines: An overview, CTFA Cosmet. J., 30-37 (July/ August/September 1981). (17) J. T. H. Ong, and B. S. Rutherford, Some factors affecting the rate of N-nitrosodiethanolamine formation from 2-bromo-2-nitropropane- 1,3-diol and ethanolamines, J. Soc. Cosmet. Chem., 31, 153- 159 (May/June 1980). (18) J.'L. Ho, H. H. Wisneski, and R. L. Yates, High pressure liquid chromatographic-thermal energy determination of N-nitrosodiethanolamine in cosmetics, J. Assoc. Off. Anal. Chem., 64 (4), 800-804 (1981). (19) M.D. Erickson, D. B. Lakings, A.D. Drinkwine, and J. L. Spigarelli, Quantitative analysis of N- nitrosodiethanolamine (NDELA) by high performance liquid'chromatography-thermal energy analyzer detection, J. Soc. Cosmet. Chem:, 36, 217-225 (1985).

j. Soc. Cosmet. Chem., 36, 231-236 (May/June 1985) Evaluation of substantivity and antibacterial activity of soap bars on human skin by an in vivo agar patch method FRANK YACKOVICH and JOHN E. HEINZE, Armour-Dial, Inc., Armour Research Center, 15101 N. Scottsdale Road, Scottsdale, AZ 85260. Received February 28, 1984. Synopsis An in vivo method, the "agar patch test," has been developed for determining the efficacy of antibacterial agents deposited on human skin from soap bars. This method has the advantage over previously published methods of eliminating the need for germicides to migrate through agar to reach the bacteria since the test bacteria are streaked on the surface of the agar medium and placed in direct contact with the soap- washed skin. This method was used to demonstrate the buildup of germicide activity as a function of the number of washings on forearms washed with a deodorant soap containing 1.5 % 3,4,4'-trichlorocarbanilide (triclocarban) compared to forearms washed with a placebo soap (no active ingredient). The results indicate that the extent of triclocarban residual activity deposited on skin washed with the deodorant bar (1.5% triclocarban) in the agar patch test reaches a statistically significant level after 7 washes and remains at that level at least through 13 washes. INTRODUCTION There are a number of in vivo methods for measuring the number of bacteria on skin, the substantivity of germicides on skin, or the degerming activity of soaps and soap germicides (1-9). Eigen et al. (9) stated that their in vivo seeded contact plate method could demonstrate differences in residual antibacterial activity remaining on skin after washing with a liquid antibacterial cleanser or antibacterial soap and thoroughly rinsing with water. The advantages of this method over previously published methods were that it allowed free movement of the•a-nelists, controlled the size and type of bacterial population, measured efficacy and not quantity of antibacterial agents found on the skin, and was easy to perform. The disadvantage is that only soap germicides that readily desorb off the skin and migrate through the agar to reach deeply seeded bacteria can be tested by this method. Thus, this method does not work well with the triclocarban (3,4,4'-trichlorocarbanilide) because it has very low water solubility and does not rap- idly diffuse in agar media. The "agar patch test" was developed as a modification of the method of Eigen et al. (9). The newly developed method eliminates the need for an antibacterial agent to migrate through agar to reach the bacteria since the bacteria are surface-streaked on the agar which is in contact with soap-washed skin. EXPERIMENTAL AGAR PLATES Falcon 1008 petri dishes (35 X 10 ram) without lids containing 11 ml of nutrient agar (Difco), mannitol salt agar (Difco), or trypticase soy agar (BBL) with 1 mg/ml 231