ANTIBACTERIAL AGENTS IN HUMAN SKIN 371 tissue homogenizer equipped with a tight-fitting pestle until successive aliquots of the suspension gave counts reproducible to within 2% or less. Before use, the formulation was warmed to 30øC and agitated continu- ously with a magnetic stirrer. The composition of the test solution is given in Table I. The activity of the preparation was determined by scintillation counting of aliquots after homogenization. Table I Composition of Test Solution Ingredients g/100 ml Hexachlorophene Labeled 0.400 • 311.5 /•c Unlabeled 0. 350 TCC Labeled 0.092 • 311.5 /•c Unlabeled 0. 658 Ivory Soap a 2.0 Total gc/100 ml 623.0 Procter & Gamble Co., Cincinnati, Ohio. The formulation was applied 24 hours after depilation and after in- spection of the animals to insure absence of abrasions or irritation. Prior to application of test material, the rats were anesthetized and circles hav- ing a diameter of 3.5 cm were circumscribed on each prepared skin site with waterfast ink. A 0.1-ml sample of the test solution was spread evenly over the test site by rubbing briskly with the tip of the delivery pipet for $ min. At various intervals after application, ranging from 0 to 8 min in order to permit deposition of variable quantities of material on the skin, the test sites were thoroughly rinsed with tap water to remove excess test material. Immediately after rinsing, each animal was sacrificed and the skin at each application site was excised, spread on aluminum foil, and counted using a Nuclear Radiation Detector Model D-34' (mica window mass of 1.4 mg/cm 2 with an effective diameter of 2.778 cm) connected to a Nu- clear-Chicago Analyzer/Scaler 8725.* Triplicate skin samples, each having an area of 0.363 cm 2, were taken from each counting area using a cork borer. These samples were dried and burned using the Schoeniger combustion method. The radioactive * Nuclear Chicago, Des Plaines, Ill. 60018.

372 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS CO2formed was trapped in phenethylamine solution* and counted using standard procedures. The averages for each set of samples (in micro- curies) were plotted against the observed surface counts. The relation- ship between surface counts and total radioactivity in and on the skin is shown in Fig. 1. 48 • 40 eo • S2 0 24 0 5 10 15 20 25 Surface Counts x iO-3/Min. Figure 1. Relationship between surface count and radioactive content of skin * This solution consists of 27% v/v absolute methanol, 27% v/v 2-phenethylamine, 0.5% w/v PPO (2,5-diphenyloxazole), and 0.91% w/v POPOP [p-bis-2-(5-phenyloxazolyl)benzene] and toluene, q.s. to 100%. ,' i The average counting efficiency for the recovery of radioactivity ap- plied to skin using the Schoeniger combustion technique was 56.2%. This efficiency was determined by burning samples of skin to which known quantities of radioactivity had been applied. For the human study, five adult male and five adult female subjects were employed. The subjects were instructed to adhere to their usual habits in washing their hands using a control bar soap identical with that which was supplemented with bacteriostats when the antibacterial soap solutions were prepared. The use of hand creams, lotions, or other prep- arations was not permitted during the experimental periods.