312 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IV Validation Experiments: Fragrance Chemicals--No Prior Knowledge Of Phototoxic Status Test Concentration In Percent (Response: +, -) In Vitro Human Skin Yeast Screen Ocmea 0.1 (-) 0.01 (-) Jasmea H 0.1 (-) 0.01 (-) Heliotropine 0.1 (--) 0.01 (-) Dihydromyrcenal 1.0 (-) 0.1 (-) Wetrahydromugol 5.5 (-) 0.1 (-) Lyral 13.75 (-) 1 (-) Dimyrcetal 12 (-) 1 (-) Indisan 2 (-) 10 (-) Disubstituted Carbo- polycylic Derivative 5 ( - ) 1 ( - ) Unsaturated Branched Chain Ketone 15 (--) 1 (--) o. 1 (+) 0.1 (+) o. 1(+) i (+) I (+) lO (+) lO (+) 10 NT • lO (+) lO (+) •Not tested. dermal system. This method of identifying phototoxic molecules has most recently been used to study oil of Verbena and Fig Leaf Extract which may have significant phototoxic activity (personal communication with Dr. D. Opdyke). Information from studies like these may be used to suggest concentration limits below which clinically significant phototoxic irritation is unlikely to occur alternatively, such information may be used to suggest a ban on use in exceptional cases. There were several fundamental reasons for our interest in Daniels' procedure. First, like many of our colleagues, we were charged with the photo-safety testing of large numbers of fragrance chemicals and formulations which, in contrast to pharmaceuti- cals, have a small individual profit margin. These realities emphasized the need to develop low cost screening procedures in order to maintain our responsibility to protect the human volunteers as well as the consumers. The generation of congeners Table V Example of Structural Modifications Affecting Photoreactivity • Inhibition Of Yeast Test Chemical & Phototoxic Concentration Structure UV-Exposed No UV (1%) 6-Methylcoumarin •/•o Yes No Yes Isomer Mix of 5 or 7- No No No Methyloctahydrocoumarin Transoctahydrocoumarin No No No 6-Methyloctahydrocoumarin •o No No No •8-Methoxypsoralen at 10 4% in the methanol vehicle was the concurrent control in every test plate.

PHOTOTOXIC ACTIVITY OF FRAGRANCES 313 within a fragrance class requires a rapid, inexpensive device to aid in the selection of the best candidate for commercial development, including scale-up production. The yeast assay accomplishes this objective. Similarly, the screen should provide a tool for molecular modification efforts in order to reduce the hazard of phototoxic activity from commercially desirable, but phototoxic series of analogues. The potential to accomplish this was illustrated in the coumarin experiment (Table V) where it was demonstrated that saturation of the molecule eliminated the phototoxic inhibition of yeast. Of course, our results were easily predicted on the basis of what is known regarding UV absorption in saturated and highly unsaturated molecules of this type. However, as novel molecules are encountered and these are modified by methods directed towards improving the commercial performance, our ability to correctly predict phototoxic properties may not be as accurate. Biological data are needed, but need not necessarily be generated in animals. The failure of physical methods (UVA absorption) to predict photobiologic activity was emphasized with Phantolid which was phototoxic in vivo yet failed to give a clear indication of its phototoxic potential during analysis of UV absorption data. Further, the UV absorption by a chemical in a flask may not reflect performance in vivo because of effects of excipients or metabolism modifying the chemical structure and consequently UV absorption characteristics. In terms of hazard assessment, the yeast system has not failed to detect a known phototoxic substance. To fail in such an identification (a false negative) would greatly weaken its value. False negatives would cause an unfounded sense of security in those that determine the concentration of test material to be put on human skin, whether it be on test volunteers or consumers. On the other hand, the finding of a phototoxic event in vitro which does not correlate with epidemiological data at the same concentration is not so serious a barrier to the safety assessment process. In these cases, one merely is given a quantal basis for caution. The test data in Tables III and IV suggest that the screen is more sensitive than the human, perhaps ten-fold or more. This information is used to set initial, safe doses for human volunteers. Initial concentrations tested in humans may be ten-fold higher than the highest no-effect dose for yeast without undue hazard. Phototoxic reactions in humans at this level, if any, should be at the lower limit of sensitivity and thus be mild and rapidly reversible. The failure of humans to react at any concentration following positive data in the screen does not necessarily constitute a failure of the assay rather, it reflects the need for understanding of the reasons why humans were protected from an intrinsically active molecule. This conservative line of reasoning is valid for virtually every screen in vitro used to predict human response. It applies for example, to such important tests as mutagenicity assays in bacteria, teratogenicity in cultured embryos, and carcinogen- icity screening in cell culture. When an adverse finding is obtained in vitro, the tiered battery approach appears to be a rational alternative to impulse condemnation. Thus a positive phototoxic response in vitro indicates, simply, that the potential to produce phototoxic reaction exists. Whether this potential is realized in vivo depends on a more complex series of factors, some of which are discussed below. One way in which the response of intact skin may be modified is through alteration of the test material to an intrinsically more or intrinsically less toxic metabolite. Metabolism plays an important role in systemic toxicology but the influence of metabolizing enzymes in skin on phototoxic responses to fragrance materials are likely