701 .JOURNAL OF THE SOCIETY OF GOS\IETIC CHEX, IlS-lb Purified samples of the two couxnarins, limettin and 7-methoxy-5- gcranoxycoumarin, were tested for phototoxic effects in tabhits at l and 0.5%, respectively. No adverse skin effects were observed, and conse- quently, these two coumarins are not considered to be phototoxic com- ponents of bergamot. Because the tluorescence intensity of bergapten soh•tions varic• with the ratio of alcohol to water, one experiment was conducted in which 70 and 95% alcoholic solutions containing bergapten at 0.008 aud 0.0008% were applied to four sites in close proximity on the clipped back of each of 12 rabbits. The more concentrated alcoholi, solution spread over ahout twice as much skin area as the 705/0 alcohol. Results (Table VII) show that at 24 hours, 9 of 12 animals gave a positive re- action to 0.0008% hergapten in 70% alcohol whereas only 5 of 12 reacted to the salne concentration of bergapten in 95% alcohol. In addition, the reactions were stronger with 70 than 95% alcohol. The fact that the agent spread over considerably more skin surface when applied in 95% alcohol suggests that there is less active agent per trait area, thereby accounting for these results (Fig. •). Figure 4. Area and intensit• of phototoxic reactions to alcoholic bergapten in rabbits. Riglit side, 95% alcohol left side, 70% alcohol upper, 0.0008% bergapten lower, 0.008% bergapten

PERFUME PHOTOTOXICITY 705 Table VII Phototoxic Effects of Bergapten on Rabbits at 24 Hours Bergapten Positive Response Concentration (%) Fraction In 70%Mcohol O.OO8 12/12 0.0008 9/12 In 95%alcohol 0.008 12/12 0.0008 5/12 Table VIII Comparison of Phototoxicity (Response Fraction) of Psoralens in 95% Alcohol on Clipped Rabbits Concentration (%) Compound 0. 1000 0. 0040 0. 0008 0. 00008 Bergapten 4/4 6/6 3/5 0/5 Psoralen 4/4 6/6 5/5 0/5 8-Methoxypsoralen 2/2 2/2 2/2 0/5 8-Hydroxypsoralen 0/6 The phototoxic activities of four related furocoumarins--bergapten, psoralen, 8-methoxypsoralen (8-MOP), and 8-hydroxypsoralen (8-HOP) (Fig. 1)--were compared using rabbits as test animals. The results, given in Table VIII, show that psoralen, 8-MOP, and bergapten are quite com- parable in phototoxic activity. 8-Hydroxypsoralen does not appear to be phototoxic. Relation between Fluorescence Characteristics and Phototoxicity A study of fluorescence intensity, emission, and excitation peaks was conducted to find out if these might be useful measurements for predict- ing phototoxicity. The Aminco-Bowman spectrophotofiuorometer was used. Data (Table IX) for various concentrations of bergapten in 70 and 95% alcohol show that the excitation peaks for bergapten range from 320 to 360 nm depending on concentration. The emission peak was consistent at 480 nm. The fluorescence intensity of bergapten is greater in 95% than in 70% alcohol. However, the relative intensities of fluorescence (the product of the emission peak height in arbitrary units and the meter multiplier setting) show a relatively straight line re- lation when plotted against the log of the bergapten concentrations.