IN VITRO PHOTOTOXICITY ASSAY 431 Table IV Comparison of Human, Mouse, Guinea Pig and In Vitro Data Guinea 7-Hour Test Agent Human Mouse Pig Exposure In Vitro Activity* Citralva - (7) • - (7) a Isocyclomone - (12.5) a - (10) a Dimyrcetal - (12) a NR Dihydromyrcenal - (1) a NR Bergamot Oil (Expressed) + (20) b + (10) b Lime Oil (Expressed) + (30) b + ( 15)b 8-Methoxy Psoralen + (0.01) b + (0.0003) b Angelica Root Oil NR + (3) a Fig Leaf Oil NR + (0.1) b Rue Oil NR + (3) e NR -(5) NR -(5) NR -(5) NR -(5) +(50) • +(1) 0.4% +(10) c +(5) 0.01% + (0.006) • +(0.0001) 100% +(25) c +(5) 0.08% +(ly +(0.1) 1.0% +(25) c +(5) 0.01% * Phototoxic activity expressed as activity in % 8-MOP refer to Table I for standard deviations. + = positive - = negative phototoxic response. NR = not reported. • Data reported by Weinberg and Springer (7). b Data reported by Forbes et al. (8). c Data reported by Tenenbamn et al. (2). d Data reported by OpDyke (9). e Data reported by OpDyke (10). (#) = concentration tested. experience, particularly for weak agents, is unclear in the absence of comparative studies. Further research is needed to determine the actual phototoxic potential of these materials. In relating positive in vitro findings to clinical experience, Weinberg and Springer (7) reported positive in vitro responses on heliotropine and lyral which could not be repro- duced in humans. In our previous study (2), we also found positive results in vitro on these compounds and could not reproduce these findings in the guinea pig model. Muller and Mitchell (14), using a similar in vitro method (15), found that a rubber chemical (Tetramethylthiuram-monosulfate) elicited positive phototoxic responses in vitro which was not seen in the mouse or human model. The data from these experi- ments indicate that the yeast assay may be eliciting a false positive response however, the population size used in vivo, which demonstrated negative results, may have been too small to accurately reflect the phototoxic potential of weaker agents. With respect to "quenching" a phototoxic response, published studies indicated that 10% topical application of benzophenone-4 prevents 8-Methoxy psoralen (4) and tetra- cycline (5) induced phototoxic reactions in humans. Additional work by Akin et al. (6) resulted in the elimination of a phototoxic response of 8-MOP in guinea pigs and hairless mice after a topical administration of an experimental sunscreen agent (7% N,N-Dimethyl-2-Ethylhexyl-P-Aminobenzoate and 3 % Benzophenone-3). Data gener- ated from the present in vitro assay indicated a potential to reduce potent and eliminate weak phototoxic test agents, demonstrating the extreme sensitivity of the method. The effectiveness of the benzophenone/methanol vehicle in reducing the phototoxic reaction

432 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS may indicate that the wave lengths responsible for the elicitation of the responses are absorbed by the benzophenone agent. Alternatively, the addition of the sunscreen may simply impede the diffusion of the phototoxin through the agar. Although a complete reduction in the phototoxic response was not observed, there are other absorbers and sunscreen agents that were not investigated which may possess the ability to totally inhibit the effects of phototoxic materials. The comparison of results from existing human, animal, and in vitro tests indicates that the in vitro assay may be useful as a screening technique to determine phototoxic poten- tial in humans and can eliminate or at least reduce the amount of animal testing re- quired to evaluate product safety. Applicability of the in vitro assay to evaluate a broad spectrum of raw materials will depend upon additional work to determine the extent of false positives which may result. In an in vivo test system, it has been speculated that the primary subcellular level of phototoxic damage may involve the cellular membrane, cytoplasmic organelles, and cross-links with DNA in the nucleus leading to chromosomal breaks. The mechanisms of phototoxic agents' actions in the yeast cells are not clear. Thus, it would be valuable to investigate the possible penetration of the phototoxic agent or its precursors through the cell wall of yeast. Nevertheless, the in vitro results obtained on a number of known phototoxic agents show agreement with published animal and human data. In summary, the yeast assay developed may prove to be a valuable prescreening tech- nique for the evaluation of phototoxic materials and a practical method for reducing the number of animals required for pre-market safety testing. In addition, the method may be useful to evaluate the potential of substances which may be used in eliminating the phototoxic effects of ingredients contained in consumer products. REFERENCES (1) A. Reitz, Untersuchungen mit photodynamischen stoffen (Photobiologischen sensibilisatoren), Centr. Bakteriol. Parasitenk, 45, 270-285 (1908). (2) S. Tenenbaum, J. C. DiNardo, W. E. Morris, B. A. Wolf, and R. W. Schnetzinger, A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials, Ce//Bio. Tox- icol., 1, 1-6 (1984). (3) J. H. Draize, "Dermal Toxicity" in Association of Food and Drug Officials of the U.S., Austin, Texas. Appraisal of the safety of chemicals in foods, drugs and cosmetics, pp 46-59 (1959). (4) J. A. Parrish, M. A. Pathak, and T. B. Fitzpatrick, Prevention of unintentional overexposure in topical psoralen treatment of vitiligo, Arch. Dermatol., 104, 281-283 (1971). (5) R. F. Dahlen, S. I. Shapiro, C. Z. Berry, and M. M. Schreiber, A method for evaluating sunscreen protection from longwave ultraviolet, J. Invest. Dermatol., 55, 164-169 (1979). (6) F. J. Akin, A. P. Rose, T. W. Chamness, and E. Marlowe, Sunscreen protection against drug-in- duced phototoxicity in animal models, Toxicol. App/. Pharmacol., 49, 219-224 (1979). (7) E. H. Weinberg and S. T. Springer, The evaluation in vitro of fragrance materials for phototoxic activity, J. Soc. Cosmet. Chem., 32, 303-315 (1981). (8) P. D. Forbes, F. Urbach, and R. E. Davies, Phototoxicity testing of fragrance raw materials, Fd. Cosmet. ToMco/., 15, 55-60 (1977). (9) D. L. J. OpDyke, Fragrance raw materials monographs angelica root oil, Fd. Cosmet. Toxico/., 13, 713 (1975). (10) D. L. J. OpDyke, Fragrance raw materials monographs rue oil, Fd. Cosmet. Toxicol., 13, 455 (1975). (11) W. M. Sams, The experimental production of drug phototoxicity in guinea pigs, Arch. Dermato/., 94, 733-777 (1966).