j. Soc. Cosmet. Chem., 31,133-143 (May/June 1980) Sunscreen testing methods: In vitro predictions of effectiveness ROBERT M. SAYRE, PATRICIA POH AGIN, DEBORAH L. DESROCHERS, EDWARD MARLOWE, Department of Photobiology Research, Schering-Plough, Inc., P.O. Box 377, Memphis, TN 38151. Received November 1979. Presented at Society of Cosmetic Chemists Annual Scientific Meeting, December 6-7, 1979, New York, New York. Synopsis Six sunscreen formulas were tested for water resistance by in vivo and in vitro methods. Comparison of results from the in vitro method, t•sing hairless mouse epidermis as a matrix for forward scattering analysis, with in vivo data revealed that protection levels determined by each method were quite similar. Products which were easily removed from htm•an skin were also removed from hairless mouse epidermis by water exposure. The results indicate that not only is choice of a sunscreen important, but also choice of the vehicle containing the sunscreen. Contrary to previous reports, products containing p-aminobenzoic acid were found not to be substantive. However, with the proper choice of sunscreen and vehicles, products from minimal to maximal SPF categories can be designed to resist removal by water. This study presents a practical advance in IN VITRO METHODS for rapid, accurate EVALUATION of SUNSCREEN St•bstantivity. INTRODUCTION Several types of in vitro tests have been utilized to commend the virtues of different sunscreening ingredients, to indicate the attributes of sunscreening products, to aid formulatots in designing products for specific needs, and to aid consumers in purchasing them. In the final analysis, it is human experience which must ultimately be the guide determining the usefulness and reliability of these methods. Several years ago this laboratory became concerned about the use and abuse of the results of in vitro methods in presentations of the resulting information to scientists, regulators, and consumers. Due to this concern, we have systematically designed studies to compare the results of different in vitro techniques to the results of equivalent human tests. This paper presents the first practical advance in in vitro methods for evaluation of sunscreen substantivity, a culmination of our previous endeavors. Basically all in vitro tests of sunscreen effectiveness depend upon either analyzing the solution absorption spectrum of the active ingredients of products or determining the forward scattering or transmission spectrum of thin films of products. Forward 133

134 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS scattering is particularly useful when dealing with either tissue samples or with physical sunscreens, because it collects all the light transmitted regardless of how it may have been scattered. During the past year, this laboratory has published several papers comparing the predicted effectiveness of sunscreening products with comparable human Sun Protec- tion Factor (SPF) tests on identical amounts of product (1,2,3). The results of these studies showed that solution tests of sunscreening effectiveness always indicated that sunscreening products should be many times more effective than they were in actual human use. These studies also showed that the solution tests could not correctly predict even the relative ordering of the effectiveness of a set of products. Based on these studies, extreme caution should be used in making any prediction of sunscreen effectiveness based on data from solution spectral analysis of sunscreening products. The other major type of in vitro test used has been examination of the transmission or absorption spectral characteristics of thin films of sunscreening products. Groves and Robertson have long been advocates of this type of testing (4-6). Recently in collaboration with Dr. Gordon A. Groves (University of Queensland, Australia), this laboratory examined a set of products to determine how well the thin film spectral technique compared with human tests. The results indicated that in vitro thin film tests in which skin is not included failed similarly to solution tests. At this time, only one in vitro test of sunscreen eflScacy remains which appears to produce results consistent with human tests. That test utilizes separated epidermis as a matrix on which sunscreen products can be spread and their forward scattering spectra analyzed. Published results for over twenty products indicate good agreement with actual human tests for a variety of different sunscreening ingredients and product vehicle types (1,2). We have also applied the forward scattering technique to other in vitro analyses, including examining the transmission properties of melanins of skin. The photoprotec- tive and photoreactive behavior of phaeomelanin impregnated skin has been reported from this laboratory in collaboration with Chedekel et al. (7). In that study phaeome- lanin-impregnated skin was shown to have a forward scattering spectrum similar to skin containing naturally occuring melanin. Our procedure was also used in a comparative UV transmission study of black and Caucasian epidermis and stratum corneum with Kaidbey and Kligman (8). The predictive techniques used for sunscreens were utilized in that study to demonstrate that the in vitro estimates of the transmission of skin were reliable in both the UVB and UVA portions of the spectrum. A third application of this method tested the possibility that butylated hydroxytoluene (BHT) could act as an oral sunscreen. This was investigated in collaboration with Black and colleagues (9,10). The epidermis of BHT-fed mice transmits less radiation than those of normal-diet mice. Subsequent chemical investiga- tions showed that only minute levels of BHT could be found in the epidermis of these animals. Our modified predictive test for sunscreening effectiveness provided an estimate that a greater amount of UV radiation would be required to produce erythema in BHT-fed animals. The DRF (dose reduction factors) calculated were consistent with those previously reported by DeRios and colleagues (11). Apparently a slight increase in skin thickness caused by the BHT results in less radiation being transmitted through the epidermis more energy is therefore required to produce erythema. This protective effect was easily detected using our in vitro technique.