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).

306 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 6. Inverse dose relationships, making suspect the test agent identity or proper dilution used to prepare the materials. REPORTING OF THE RESULTS AND CAUTIONS In reporting results, the character as well as diameter of any zone of inhibition is reported. Comments on the relative phototoxic potential of two compounds should be made with caution since a possible influence on zone diameter is very likely to be the rate of chemical diffusion in the agar system. Thus a very phototoxic material could conceivably appear weak, simply because of poor diffusion in the aqueous system. On the other hand, comparisons between compounds are possible when it can be presumed on a structural basis or from other data that the compounds have similar diffusion characteristics. When questions on successful diffusion arise, a similar study can be constructed using shaker cultures and measuring inhibition by determining per cent transmittance of light compared to untreated cultures as a measure of growth of yeast. In routine assays of large numbers of samples, we have constructed a one page report format (results page) which also serves as the laboratory notebook entry. This page contains the information needed to identify the study number and compound plus the laboratory notebook references. It also contains a small section in which the reagent weights and calculations are shown which were used to make the reagent dilutions. There is also a result section in which the millimeters of inhibition of yeast growth are recorded for each plate at each of the three concentrations and a comparable section for recording inhibition in the plates kept in the dark. Finally, this section of the page contains the results of the 8-MOP control at 10-4%. Near the bottom of the page is a small section for conclusion. This section has blanks indicating whether positive control validation was achieved and whether a phototoxic response (in % of test agent) was achieved. Also at the bottom is a place for signatures of the investigators and date. RESULTS The process of adapting Daniels' method began with reproducing his results. Figure 1 depicts schematically how this was accomplished. The Petri plate was filled with nutrient agar and inoculated with Candida albicans. Immediately after inoculation, the air-dried paper disc containing the test materials was placed on the seeded agar and exposed to ultraviolet light. The exposure we found appropriate was 1.5 to 2.0 milliwatts per centimeter per second which is approximately the intensity that we measured during a sunny day this past summer. The l$-hour exposure period accumulates about 30 joules. This exposure produces only slight retardation of yeast growth. A dark control is used to identify antifungal effects that do not require ultraviolet light as an essential component. The lightly shaded areas are intended to show the freshly seeded agar and the dark shading indicates developed contrast after 48 hours of incubation. For illustrative purposes, an idealized test agent (the "unknown") is shown. Unknowns are routinely diluted to 10, 1 and 0.1% concentra- tions and should produce zones of inhibition which are dependent on concentration. In those cases where phototoxic activity is present as shown, the UVAoexposed