304 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS potent phototoxic effects or if questions arise on bioavailability of test material in the microbiological system. Assays in yeast have been used to measure specific phototoxic substances such as bergapten (4). The original method has recently been adapated by us for the screening of fragrance chemicals in order to gain control over the rising cost of development, yet continue to address the ethical considerations incumbent on those who expose humans to new products. Our modified Daniels method is a sensitive, practical way for assessing the potential phototoxicity of chemicals in topically applied products. This test may not be satisfactory for the detection of systematically administered compounds like demeclocycline and sulfonamide (5), possibly because hepatic metabolic alteration is necessary for the production of a phototoxic molecule. Additional studies using metabolic activation systems, perhaps in the form of cell culture, or additional indicator organisms such as staphlococci (6) may be necessary to expand the sensitivity spectrum of the test to include the assessment of materials administered systematically or altered metabolically in the skin. It is unlikely however, that these refinements will be needed to test fragrance materials in the vast majority of cases. MATERIALS AND METHODS MATERIALS Discs--Schleicher and Schuell, Inc., Kean, New Hampshire 740-E 9.52 mm-diameter absorbent paper discs. Micropipette--Oxford Laboratories, 1702501 adjustable 10-50/,tl. UV Bulbs--F15T8 BLB Sylvania (or equiv.) 320-400 nm, Peak 370 nm. Plates--Falcon tissue culture cluster dish #3004, 1950 Williams Drive, Oxnard, California 93030. These plates contain four 35-mm wells arranged in a square. Irradiation with UVA is made through the plastic lid which reduces the amount of transmitted light by only a small degree. UV Meter--Sunburn/UVA meter, Solar Light Co., Philadelphia, Pa. Microorganism--Fleischman's Active Dry Yeast, commercial food grade. The inoculum was prepared by dissolving 0.02 gm of lyophilized yeast in 15 ml of warm sterile distilled water. After preparing a uniform suspension by mixing, an 8-fold dilution was made using warm sterile distilled water. This final dilution was used to innoculate the agar surface by streaking with thoroughly moistened sterile cotton-tip swabs. The surface of the agar should contain no excess liquid at the time of application of test agent. Agar--Potato dextrose agar from Difco, 39 grams per 1000 ml of water, boil, autoclave, dispense into each well to nearly full. Test Diluent--Methanol absolute, Baker Chemical Company or equivalent. Positive Control--8-Methoxypsoralen (8-MOP) diluted in methanol to 10-4%. METHODS With the exception of substituting Saccharomyces for Candida albicans, the method- ology is similar to that described by Daniels. All dilutions of test substances are done

PHOTOTOXIC ACTIVITY OF FRAGRANCES 305 with pure methanol. Each assay is done in duplicate. Each four-well plate contains a complete test unit. Three wells are used to test three successive ten-fold dilutions of test substance, usually 10, ! and 0.1%. The fourth well is used to test the 8-MOP positive control at 10-4%. This provides a control for each unit and chemical to assure that no reduction in assay sensitivity occurs. Precisely 40/al of each dilution is spotted uniformly on a paper disc and allowed to air-dry for 15 min. During this time the inoculum of yeast is prepared by adding 0.02 gm of yeast granules to 15 ml of water. Further dilute 1:8. After innoculation of the agar surfaces, the treated discs are applied to the center of the agar surface and pressed against it lightly with forceps. The discs are applied in sequence to be sure that no contamination of test material occurs: the positive controls are applied last and the lowest concentration of test compound applied to all plates before the next higher concentration is handled. Duplicate plates are exposed to UV immediately at room temperature with a UV surface flux at the plate of 1.5 mw/cm 2. Radiation is measured through the plastic well tops. A second set of duplicate plates is incubated in the dark. Irradiation is discontinued after 18 hours and zones of inhibition are measured at 48 hours after innoculation or when contrast is adequate. Contrast continues to increase with continued growth of yeast so that maximum contrast exists in about 4-5 days. Measurements of zones of inhibition are made using a caliper and metric rule and are expressed in terms of millimeters of inhibition from the disc edge. In deciding whether the data are meaningful, we use the following. A. Criteria for concluding that a phototoxic effect did occur.' 1. A readily perceptible zone of inhibition in a dose-related manner only around the test disc exposed to UV light. Only one concentration need be positive (as long as it is the highest concentration tested). 2. Either no inhibition or appreciably less inhibition of yeast occurs around similarly treated discs protected from UV light. This consideration allows for the evaluation of fragrance materials which are both phototoxic to yeast and also directly fungistatic or fungicidal in the manner of a chemotherapeutic agent or disinfectant. B. Criteria for concluding that a phototoxic effect did not occur.' 1. No zone of inhibition was observed in the highest concentration tested. 2. Zones of inhibition in UV exposed plates are no greater than those in plates protected from UV light. C. Criteria for concluding results are unacceptable or equivocal, possibly requiring a repeat study.' 1. Atypical effects from the 8-MOP control. 2. Greater inhibition in the dark than in the UV light. 3. Poor contrast, making measurements of inhibition unreliable and likely to vary between observers. 4. Presence of substantial bacterial contamination. 5. Grossly disparate zones of inhibition between duplicate plates (making suspect the identity of the material on one or the other spot).