J. Soc. Cosmet. Chem., 36, 425-433 (November/December 1985) A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials. II. Modification and assessment of low level activity* JOSEPH C. D•NARDO, BARBARA A. WOLF, WILLIAM E. MORRIS, SAUL TENENBAUM, and RICHARD W. SCHNETZINGER, Revlon Research Center, Inc., 2121 Route 27, Edison, NJ 08818. Received November 7, 1984. Synopsis There are numerous reports in the scientific literature which describe photobiological effects of materials in cell and animal models. In an effort to develop a quick and inexpensive phototoxicity screen as well as to reduce the number of animals required to predict product safety, a quantitative in vitro assay using Saccha- romyces cerevisiae was developed and reported previously by the present authors. Activity of test materials in the assay was measured by zones of growth inhibition around the perimeter of a treated disc and compared to a concentration curve based on activity of 8-methoxy psoralen, a known phototoxic agent. The present study reports on a modification of the original method undertaken to reduce the time necessary to complete the assay without affecting the quantitative ability to assess a response. Additionally, "low level" in vitro phototoxic activity for a number of complex fragrance oil materials was compared to test results from an animal model, and the ability of benzophenones and sunscreen agents to inhibit a phototoxic response is also reported. In vitro results from the modified assay compared to published animal and human data for a number of fragrance raw materials showed good agreement between the methods. The in vitro assay devel- oped has shown to be useful as a prescreening tool for evaluating phototoxic potential of fragrance ingre- dients as well as an effective means of reducing the number of animals required for experimentation prior to assessing consumer safety. INTRODUCTION The use of a microorganism to observe a photobiologic effect has been reported as early as 1908 by Reitz (1). Since that time, a number of studies have been published refining various techniques and procedures which evaluate these effects for a variety of agents. Currently, there has been a heightened interest by the scientific community in utilizing such in vitro systems in an attempt to reduce the number of animals required for sub- stantiating product safety. As part of a continuing research effort to comply with such interest, a number of studies were initiated to develop and validate a quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials. The second in a series of research papers on alternatives to animal toxicity studies. 425

426 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS This paper presents data on a modification of the exposure time for the quantitative i, vitro assay reported by Tenenbaum eta/. (2) and the assessment of low level activity for a number of fragrance oil materials previously reported. In addition, an investigation of "quenching," the potential to inhibit a phototoxic response, was conducted by incor- porating benzophenone and sunscreen agents into the standard methanol vehicle. The results from these procedures were compared to published animal and human data. METHODS LIGHT SOURCE/ULTRAVIOLET EMISSION READINGS A bank of four black light tubes (Westinghouse F40BL, 320-400 nm, peak 350 nm) was utilized for all assays. Ultraviolet intensity was measured using a long-wave J221 Black-Ray meter (Ultraviolet Products, Inc.). Energy readings for animal assays ranged from 1.2 to 1.8 mW/cm 2. Flux tbr the in vitro assay ranged from 1.0 to 1.2 mW/cm 2 for the 18-hour assay and 2.0 to 2.5 mW/cm 2 for the 7-hour assay. UV emissions for the in vitro assays were taken through the top of a petri dish (Falcon, No. 1029) which simulated the amount of UV energy reaching the test sites. TEST MATERIALS A 5% test material concentration was used for in vitro studies based on preliminary work which demonstrated that higher concentrations of the fragrance materials elicited antimicrobial activity, limiting the ability of the assay to evaluate the phototoxic po- tential of these materials. A 10-fold exaggeration of the in vitro concentration was used for in'vivo testing based on the greater degree of sensitivity exhibited by the yeast assay compared to the animal model. 18-HOUR IN VITRO ASSAY Saccharomyces cerevisiae (isolated from Fleishman's Yeast) was used in an agar overlay technique wherein 3 ml of a 1% suspension of yeast (approximately 1.7 X 105 or- ganisms/ml) in molten plate count agar (Difco Laboratories No. 0479-01) was added to a 20-ml agar plate. Quarter-inch blank paper discs (Baltimore Biological Laboratory, No. 31039) were impregnated with 25 ptl of the test compound dissolved in methanol and were allowed to dry for 15 minutes at room temperature prior to being placed on the surface of the agar plate. Two plates containing test materials were exposed to UVA (Westinghouse F40BL tubes) for 18 hours at a distance of 31 cm and then incubated at 31-35øC for 48 hours. An additional plate, not irradiated but incubated for 48 hours, served as a control. Phototoxic materials were identified by a clear zone of growth inhibition in the irra- diated plates and none in the non-irradiated plate. A negative phototoxic response is indicated by absence of growth inhibition on both plates. The diameter of the zone of inhibition was measured by an antibiotic zone reader (Fisher-Lilly, Inc.). To quantify the phototoxic activity of the test materials, a curve of 8-Methoxy psoralen concentrations (ICN Pharmaceuticals, Inc. Lot #103296), ranging from 0.01% to 0.0001% (with the latter being the lower limit of detection for the assay) versus zone of