LABORATORY TESTING OF THE EFFECTS UPON THE SKIN OF TOPICAL AGENTS* By PETEI• I•'LESCH, M.D. University of Pennsylvania School of Medicine, Philadelphia, Pa. CLINICAL PROCEDURES for testing the effects of topical agents upon the human skin have numerous shortcomings. Such studies are time-con- suming, require large numbers of suitable human subjects, and the results cannot be objectively evaluated. Final proof of the efficacy of topical agents rests on clinical testing. Nevertheless, objective chemical studies of the interaction of topical agents with the human skin would be essential for the further development of a rational topical therapy, for predicting thera- peutic, irritating or sensitizing effects, and for screening purposes. The best tissue substrate for such studies is the epidermis, the most ac- tively metabolizing and regenerating part of the skin. Recent techniques, facilitating the separation and fractionation of human epidermis, have ad- vanced our knowledge of keratinization, the most important metabolic function of epidermal cells. In sharp contrast to previous theories, it has been shown, both by histochemical and direct chemical methods that the cystinc linkages in epidermal keratin, which greatly contribute to the chemi- cal stability and resistance of the skin surface, are already present in the Malpighian layer, the living portion of the epidermis. It has also been found that in keratins of the hair, nails and epidermis, small but significant amounts of sulfhydryl (--SH) groups are retained. Only a few of these --SH groups are chemically reactive in water. After treatment with detergents, the number of chemically demonstrable SH groups increases. It has been generally assumed that detergents produce this effect by denatur- ing the protein molecule and thereby unmasking previously hidden --SH groups. The extent of this process varies with different detergents and it is believed that denaturation of the proteins of the skin surface is one of the mechanisms whereby detergents exert their irritating action. Powdered keratin is a suitable tissue substrate for the study of the effects of detergents. For testing the chemical action of substances incor- porated into ointment bases, in our experiments, whole epidermis served as a tissue substrate. Two types of methods were used. In the simpler * Presented at the September 7, 1954, Meeting, Chicago, Ill. 161

162 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS method, the epidermis was first pulverized or homogenized and then incubated with the topical agent. This method has the disadvantage that in a finely ground state the epidermis is much more vulnerable to chemical attacks than when its cellular organization is not disrupted. Therefore, the results obtained with this method must be checked with the more cumber- some second method. In the latter procedure, pieces of skin are treated under standard conditions with the ointments to be investigated and then incubated for several hours at 35 to 37øC. At the end of this period the skin is wiped free of ointments, the epidermis is separated from the corium, dried, defatted, and pulverized or homogenized. Chemical determinations are then carried out in the epidermal powders or homogenates. This method rather closely simulates in vivo conditions of exposure. With both types of methods we have studied the concentration of--SH groups in the epidermis. Sulfhydryl groups react with a variety of chemi- cal substances, are influenced by a number of physical agents, and can com- bine with topical allergens. Preliminary results, showing the effects of sul- fur, ammoniated mercury, salicylic acid, etc., agreed with those previously obtained with conventional methods. A new method for the in vitro study of penetration of topical agents through human skin also utilizes strips of skin from surgical or autopsy ma- terial. The substances to be tested are incorporated into ointment bases and these ointments, as well as the ointment bases alone, are rubbed into the skin under standard conditions. The pieces of skin are then incubated at 35øC. for several hours. The surface is thoroughly wiped free of oint- ments and the epidermis is removed. On the surface of the denuded corium, suitable spot tests are carried out to check whether the substance, whose absorption is being studied, has penetrated through the epidermal barrier. (This procedure is discussed in detail in a later publication.) This new method of ours has several advantages. It utilizes human skin and thus the results have a more direct bearing on clinical practice than have observations made on animals. The procedure is simple and well suited for serial studies. Moreover, it is the only experimental method for the study of percutaneous penetration of toxic substances in man. It is also the only method which permits correlation between the rate or extent of percutaneous penetration and chemical changes brought about in the epidermis. The main disadvantage of the method is the use of dead skin. Cellular death may affect the epidermal barrier which prevents the absorption of many substances. Against this valid objection the following facts may be brought up: (1) In our experiments, absorption of salicylic acid followed the same pattern in dead skin as has previously been described in living skin. (2) Dead skin is impermeable to vitamin A in the author's opinion, this substance does not penetrate through living skin either. (3) While the