234 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Results from the series of tests shown in Table I indicate that the percent reductions could be duplicated between tests when 6 to 8 panelists were used. Reducing the contact time between the skin of the forearm and the streaked plate from 4 hours to 2 hours did not affect the residual antibacterial activity of the skin and did reduce discomfort to the panelist. In later experiments a 30-minute contact time of the streaked petri dishes with the skin was shown to be equally effective. During these first experiments, some of the skin-attached streaked plates became con- taminated with resident skin bacteria. To avoid such problems in subsequent experi- ments, a spontaneous streptomycin-resistant colony from Staphy/ococcus epidermidis ATCC 14990 was isolated. This bacteria was designated as S. epidermidis JEH-51. One mil- ligram of streptomycin sulfate was also incorporated per milliliter of trypticase soy agar to inhibit the growth of resident skin bacteria on the stre'aked plates. In an attempt to improve test sensitivity and to further decrease the coefficient of variation, the inoculum on the streaked agar plates was also raised from 100-300 CFU to 1000-2000 CFU. The next series of experiments was designed to determine if a buildup of triclocarban germicidal activity on the deodorant soap-washed forearms compared to placebo soap (no triclocarban)-washed forearms could be shown as a function of the number of washings. Seven or eight different panelists were used for each experiment. The panelists washed for an average of 5 to 13 washes over a period of 2 to 4 days, with at least a 2-hour drying out time between washes. The data for these runs are presented in Table II. The reduction in bacterial levels observed with the deodorant soap after 5 washings Table II A Comparison of the Residual Antibacterial Activity of a Deodorant Soap (1.553 Triclocarban) as a Function of the Number of Washings • Test No. of No. of Applications Number Panelists b Mean (range) c • + S.D. % Reduction p-Value 4 8 5.25 (4-7) 0. 1722 q- .5172 32.7 N.S. d 5 8 5.13 (5-6) 0.0821 q- .1548 17.2 N.S. 4 + 5 e 16 5.19(4-7) 0.1271 q- .3717 25.4 N.S. 6 8 7.25 (7-8) 0.1400 q- . 1574 27.6 .05 7 7 9.57 (8-10) 0.2429 q- .2520 42.8 .05 8 8 13.0 (13) 0.1439 q- .1170 28.2 .02 • The placebo soap used had the same base composition as the deodorant soap. The test bacterium was S. epidermidis JEH 51. The desired control inoculum was 1,000-2,000 CFU/plate using a 3/2,000 dilution of the overnight culture. Actual control mean log CFU/plate ranged from 3.0365 to 3.2764 with coef- ficients of variation of 1.21 to 3.38%. The contact time of the inoculated plates on the washed forearms was 30 minutes. Other abbreviations are given in Table I. b Different panelists used in each test. c All panelists participated in a minimum number of supervised washings (the minimum number given under "Range") over a 2-4 day period. In addition, panelists were issued color-coded test bars, and instructed to perform any ad. lib. washings of their forearms with the test bars and to record the number of such washings. The total of the supervised plus ad./lb. washings is the maximum number of applications for any panelist in the test (the maximum number under "Range"). The "mean" is the average number of applications per panelist. d N.S. = nonsignificant (p .05). e Data from test 4 and 5 combined.

SOAP BAR ANTIBACTERIAL ACTIVITY 235 was not statistically significant, even when the results from two independent tests were combined. This finding suggests that the magnitude of the reduction after 5 washings is less than that observed with 7 or more washings, which gave significant reductions. Since the magnitude of the reductions after 7, 9, and 13 washes are statistically indis- tinguishable, the extent of triclocarban residual antimicrobial activity deposited on deodorant soap (1.5% triclocarban)-washed skin in the agar patch test apparently reaches a maximum after 7 washes and remains at that level at least through 13 washes. A plausible interpretation of this result is that after 7 washes, the amount of antimi- crobial activity that is absorbed on the skin is in equilibrium with the amount of antimicrobial material that desorbs off the skin with additional washings. Increasing the number of CFU/plate in the experiments in Table II did reduce the coefficients of variation of the control plate counts. However, the magnitude of the reductions observed in Table II are smaller than those observed in Table I. Since there were a number of differences in experimental procedures between the two sets of tests, described in the table footnotes, the actual reason(s) for the smaller magnitudes observed in Table II remain(s) an area for further investigation. Nevertheless, statistically sig- nificant reductions were observed with 6-8 panelists in both sets of tests after 7-8 applications of the deodorant bar soap. CONCLUSION The agar patch test is a useful method for determining residual antimicrobial activity on soap-washed skin. This method eliminates the need for chemicals to migrate through agar to reach the deep-seeded organisms, since the organisms are streaked on the surface of the agar which is in direct contact with the soap-washed skin. Contamination of the contact plates by skin bacteria can be reduced by using a streptomycin-resistant strain and streptomycin-containing media. This method can be used for evaluating antimi- crobials or antimicrobial products which are intended to be washed off the skin after use. In principle, any relatively flat area of the human body could be used in this test, although the volar surfaces of the forearms are the most conveniently studied site. Using this method, it was shown that repeated washing with a deodorant soap con- taining 1.5% triclocarban deposits measurable germicidal activity on panelists' skin. REFERENCES (1) A. R. Cade, Antiseptic soaps, a simplified in vivo method for determining their degerming efficacy, Soap Sanit. Chem., 26, 35-38 (1950). (2) P. B. Price, The bacteriology of normal skin a new quantitative test applied to a study of the bacterial flora and the disinfectant action of mechanical cleansing, d r . Infect. Dis., 63, 301-308 (1938). (3) H. J. G. Quinn, G. Voss, and H. S. Whitehouse, A method for the in vivo evaluation of skin sanitizing soaps, Appl. Microbiol., 2, 202-204 (1954). (4) J. J. Leyden, R. Stewart, and A.M. Kligman, Updated in vivo methods for evaluating topical antimicrobial agents on human skin, dr. Invest. Dermatol., 72, 165-170 (1979). (5) D. Taber, J. C. Lazanas, O. E. Fancher, and J. C. Calandra, The accumulation and persistence of antibacterial agents in human skin. dr. Soc. Cosmet. Chem., 22, 369-377 (1971). (6) D. Taber, A. B. Ward, and F. Yackovich, Use of antibacterial soap in the treatment of erythrasma of the toe webs, Cutis, 5, 991-997 (1969).