SALICYLANILIDE COMPOSITION FOR SOAPS AND COSMETICS 353 Figure 1.--Comparison of antibacterial activity of soap containing 0.3% diaphene (No. 1) with three commercial antibacterial soaps. For this purpose shaved fresh calfskin was used in the experiment. A surface area of 100 square centimeters was marked out with a marking pencil and one gram of product under investigation was applied to the area, distributed as evenly as possible, and rubbed in lightly. The skin por- tions marked out were carefully separated and one square centimeter areas were cut out and immediately sliced with a freezing microtome in a thick- ness of 35 microns parallel to the surface. Six slices with one square centi- meter surface area were then collected and boiled briefly with 1 ml. of 1 per cent sodium hydroxide in a test tube. The volume was made up to 10 ml. with standard nutrient broth and the solution adjusted to pH 7 to 8 with a few drops of dilute acetic acid solution, after it had been determined previously that Micrococcus pyrogenes var. aureus grows unhindered at this pH. Further control experiments with untreated skin pieces showed that these do not weaken the effectiveness of the substance under investigation toward Micrococcus pyrogenes var. aureus and may be therefore neglected. The broth tubes prepared as above were then further diluted in geometric series and inoculated with 0.15 ml. of a twenty-four-hour Micrococcus pyro- genes var. aureus culture. The growth of this micro/3rganism was read after twenty-four and forty-eight hours at 37øC.

354 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Conversion of the inhibition coefficients into gamma of active agent showed that the upper layers of the skin down to 210 microns contained more than 100•, of active agent. Since it must be assumed that a large part of the substance remained on the skin surface an accurate determina- tion of the quantity in these layers was not undertaken. In the following six slices down to a depth of 420 microns we found 10 gamma and down to 630 microns we still found 5 gamma. Under the conditions of our experi- ments there was no dependence on the time nor on the nature of the vehicle. We must assume that these experiments demonstrate the penetrability of the active agent. By penetration we refer to material imbedded in the philosebaceous apparatus and sudoriferous ducts. Diaphene was demon- strated to be present in these deeper layers in concentrations of fifty to one hundred times more than the quantity necessary for inhibition in the in vitro tests. The use of fresh calfskin provides a simplified and efficient method of evaluating soap or detergent bars containing bacteriostats. Skin disc substantivity tests may be conducted by applying aqueous solutions of the lather of an antiseptic treated bar to fresh calfskin and subjecting the treated skin to clear water leaching for five minutes. In performing this test a section of fresh calfskin (free of wrinkles) about 3 X 4 in. is subjected to a washing procedure in the same manner that the back of the hand might be washed using the treated soap either in liquid form or as a lather. The treated skin is then rinsed thoroughly with moderate rubbing in a manner similar to washing the hands. When this treatment is completed discs are cut out of the center portion of the calfskin piece with an instrument such as a cork borer. The resulting discs are placed on nutrient agar with the epidermis side down. The agar is previously seeded with Micrococcus pyogenes var. aureus. After incuba- tion at 37 ø for twenty-four hours zones of inhibition are read and compared with the controls. Using this fresh calfskin method, one can demonstrate the synergistic relationship between 5,4'-dibromosalicylanilide and 3,5,4'-tribromosalicyl- anilide. Skin substantivity tests were conducted by applying 5 per cent aqueous solutions of soap containing 0.5 per cent (based on the soap) of 5,4'- dibromosalicylanilide (5,4'-di BrSA) or 3,5,4'-tribromosalicylanilide (3,5,4'- tri BrSA), or mixtures thereof, to fresh calfskin and subjecting the treated skin to clear water leaching for five minutes. Then, the skin discs were placed on nutrient agar, seeded with Micrococcus pyogenes var. aureus, and incubated at 37øC. for twenty-four hours. The results of these tests, shown in Table 6, demonstrate that mixtures containing 65 per cent to 98 per cent of the tribromo derivative are unexpectedly and unusually high in antibacterial action. This discovery is the subject of a patent referred to previously (4).