PERFUME PHOTOTOXICITY 697 radiation sources was •neasured* using a Perkin-Elmer monochromator with strip chart and tape recorders (8). Over 90t•o of the ultraviolet (UV) radiation output (between 300 and 400 nm) of the Inspectolite was found to be at 365 ----q- 5 nm. The total UV output was about 3000 /•watts/cm 2 at a distance of 10 cm from the source and 1900/•watts/cm 2 at 15 cm.* The first step was to isolate coumarins and [urocoumarins from bcrgamot and test them for phototoxicity on human subjects (Appendix I). As these first preparations were not entirely pure, this was con- sidered a screening step. Phototoxicity tests were then performed on nine subjects using oil of bergamot, the coumarin-psoralcn nonvolatile fraction, coumarin-psoralen components separated by thin-layer chro- matography (TLC), and an ethyl ether-precipitated portion of the cou- marin-psoralcns. Thus, in addition to oil of bergamot, which contains about 2% coumarin-psoralens, tests were performed on TLC band 1 con- sisting mainly of bcrgamottin, band 2 which was mainly 7-mcthoxy-5- geranoxycoumarin, band 3 which was about 96:4 limettin-bergapten, band 4 which contained elements of band 3 plus bergaptol, and an ethyl ether-precipitated portion which was recrystallized three times with methanol and was mainly bergapten. The chemical formulas are shown in Fig. 1. The human studies were performed according to the method of Burdick (10). The skin of the forearm was tape-stripped to glistening. The agent (0.05 ml) was applied to the stripped area (5 X 12 cm), allowed to remain undisturbed for 5 minutes, and then irradiated with the Inspectolite for 40 minutes at a distance of 8-10 cm. The arm was examined at 24 and 48 hours. RESULTS Results (Table I) show that phototoxicity, consisting of erythema, edema, and sometimes vesiculation, was easily obtained on stripped • Measuremcnts by Dr. M.P. Thekaehara, Goddard Space Flight Center, Greenbelt, Md. t Inverse square law is applicable if the source is assumed to be 8.2 cm behind the front of filter. The Inspectolite was calibrated with a G.E. Spectral Irradiance Standard lamp, a 1000-watt quartz-iodine lamp standardized by Eppley Laboratories, Newport, R.I. After the Inspectolite has been calibrated, it can be checked from time to time with a Jagger UV dose-rate meter fitted with a diaphragm over the photocell to reduce the UV output intensity (9). An important factor to note, especially in biologic experiments involving different exposure times, is that the output increases as the instrument warms up.

698 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS CI H• CH• (•H• CH• OCH CH=CCH•CH•CH:C •' OC2CH:C-CH2CH•CH= C'•CH• '•'C H• BEROAMOTTIN 7-Methoxy t •eranoxy coumar•n geranylether (0.05% ,n bergareal) 5% OCH• 5•7-Dimethaxy caumorin (0.3ø/8 ,n bergoreo,) PSORALEN BERGAPTEN OCH• 5 - Melhoxypsoralen X ANTHOTOX IN (0 33 % •n bergarno 0 8-Me•haxypsaralen OH O[••O O••"O BERGAPTOL OH 5-Hydroxypsoralen XANTHOTOXOL (hydrolysis product of bergapten) 8-Hydroxypsaralen Figure 1. Structural formulas of test psoralens and coumarins skin with oil of bergamot, the complete coumarin-psoralen fraction, and those portions which contained bergapten. Figure 2 shows the skin reactions obtained on one of the subjects tested with these ma- terials. Figure 3 shows hyperpigrnentation at the application site 23 days after a positive reaction to 10% bergamot. In this subject, the pigmentation effect lasted about 1 month' in some individuals it is re- ported to last much longer (11). Limettin To evaluate limettin more precisely as a possible phototoxic agent, and to define further the limits of bergapten phototoxicity, tests were conducted on pure samples of both bergapten and limettin, in addition