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.

706 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IX Fluorescence Intensity and Excitation and Emission Peaks of Bergapten in Alcoholic Solutions Relative Bergapten Excitation Emission Intensity Concentration (%) Peak (nm) Peak (nm) (units) • In 95%alcohol 0.0100 360 480 50 0.0080 360 480 41 0.0010 330 480 26 0.0008 330 480 25 0.0001 320 480 6 In 70%alcohol 0.0080 360 480 26 0.0007 332 484 16 • Product of the emission peak height in arbitrary unim and the spectrophotofluorometer meter multiplier setting. Table X Comparison of Photochemical Properties and Phototoxic Capacity of Four Psoralens and One Coumarin Alcohol Relative Concen- Concen- Fluorescence Photo- Test tration tration Excitation Emission Intensity toxic Substance (%) (%) Peak (nm) Peak (nm) (units) Effects Bergapten 0. 001 95 330 480 26 Yes 8-MOP 0. 001 95 320 480 I Yes 8-MOP 0. 001 70 320 480 2 Yes Psoralen 0. 010 95 395 460 65 Yes Psoralen 0. 001 95 389 460 5 Yes Psoralen 0. 0001 95 389 460 1 Yes 8-HOP 0.100 95 385 480 0.1 No Limettin 0. 00001 95 335 420 55 No The data of Table X show that there is no apparent connection be- tween phototoxic activity and fluorescence intensity, excitation or emis- sion peaks. In further pursuit of the mechanism of the photobiologic activity of psoralens, exploratory electron spin resonance studies were conducted. Absorption curves were obtained* on 0.02% solutions of 8-MOP and 8-HOP, each in 95% ethanol. The test materials were first frozen with liquid nitrogen in a quartz tube and then irradiated with the Inspectolite for $0 minutes at a distance of 5 cm. The frozen solutions were scanned * Work performed by Dr. H. Kon, National Institutes of Health, Bethesda, Md.