IN VITRO PHOTOTOXICITY ASSAY 429 Table I Effect of UV Exposure Dose on In Vitro Phototoxicity Test Results Test Test Agent Concentration 7 Hours Exposure Phototoxic Activity* After 18 Hours Exposure Heliotropine** 5% 0.02 + 0.004 0.02 + 0.001 Lyral** 5% 0.01 + 0.007 0.004 m 0 Lime Oil 5% 0.01 -+ 0.004 0.01 -+ 0 Rue Oil 5% 0.01 -+ 0.004 0.0l _+ 0.002 Angelica Root Oil 5% 0.08 m 0.04 0.04 m 0.002 Fragrance Oil I 5% 0.01 + 0.001 0.01 + 0.003 Fragrance Oil J 5% 0.01 + 0.006 0.01 + 0.003 Fragrance Oil K 5% 0.01 + 0.001 0.01 m 0.003 Fragrance Oil L 5% 0.03 -+ 0.007 0.03 + 0.02 Bergamot Oil (Expressed) 1% 0.4 + 0.07 0.5 + 0.08 Fig Leaf Oil O. 1% 1.0 -+ 0 0.6 -+ 0 8-MOP 0.01% 100 -+ 0 100 _+ 0 * Phototoxic activity expressed as activity in % 8-MOP. ** Data previously published in reference (2). + = Standard deviations expressed as activity in % 8-MOP. no disturbance in the growth pattern was noted in non-irradiated plates, this may suggest that incorporation of benzophenones and/or sunscreen agents into the test sample may simply reduce the ability of the mixture to disperse evenly through the agar. Table II Comparison of Results From the 18-Hour Exposure In Vitro and the Modified In Vivo Assays* 18-Hour In Vitro Assay Modified In Vivo Assay Test Test Activity in Test # of Animals Positive/ Agent** Concentration % 8-MOP Concentration # of Animals Tested Fragrance A Oil 5% 0.004 _+ 0.001 50% 4/5 Fragrance B Oil 5% 0.002 -+ 0.001 50% 3/5 Fragrance C Oil 5% 0.0001 _+ 0 50% 3/5 Fragrance D Oil 5% 0 50% 0/5 Fragrance E Oil 5 % 0 50% 0/5 Fragrance F Oil 5% 0 50% 0/5 Fragrance G Oil 5% 0 50% 0/5 Fragrance H Oil 5% 0 50% 0/5 Fragrance I Oil 5% 0.01 _+ 0.003 50% 3/5 Fragrance J Oil 5% 0.01 --- 0.003 50% 2/5 Fragrance K Oil 5% 0.01 -+ 0.003 50% 1/5 Fragrance L Oil 5% 0.01 -+ 0.003 50% 2/5 Fragrance M Oil 5% 0 50% 0/5 Fragrance N Oil 5% 0 50% 0/5 Fragrance O Oil 5% 0 50% 0/5 Fragrance P Oil 5% 0 50% 0/5 * Non-irradiated controls for both assays were negative. ** Fragrance oils A through P are complex mixtures containing a number of raw materials. + = Standard deviation expressed as activity in % 8-MOP. Fragrance oils A-H previously reported in reference (2).

430 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table III Quenching Effects of Benzophenones and Sunscreen Agents for the 7-Hour In Vitro Assay Test Vehicle/Activity in % 8-MOP Test Agent Test 10% Benzo- Concen- . phenone-2 tration Methanol* 90% Methanol 10% Benzo- phenone-4 90% Methanol 10% Benzo- 10% Benzo- phenone-4 phenone-4 10% Octyl 10% Octyl Methoxy Dimethyl Cinnamate PABA 80% Methanol 80% Methanol Heliotropine 5% 0.02 Lyral 5 % 0.01 Lime Oil 5% 0.01 Rue Oil 5% 0.01 Angelica Root Oil 5% 0.08 Bergamot Oil (Expressed) 1% 0.4 Fig Leaf Oil 0.1% 1.0 8-MOP 0.01% 100 0.008 b 0 0 0 0.008 b 0 0 • 0 - 0 0 • 0 - 0 • 0 a 0 0.01 4- 0.007 03 0 0.4 4- 0.03 0.1 • 0.2 • 0.5 4- o o • o • 40-80 + 28.0 50 • 50 • a Non-uniform growth around the perimeter of the treated disc. b Results represented by one data point. 4- = Standard deviation expressed as activity in % 8-MOP. * See Table I for standard deviations. Benzophenones supplied by BASF--Wyandotte Corp., Parsippany, NJ. Octyl Methoxy Cinnamate supplied by Givauden Corp., Clifton, NJ. Octyl Dimethyl Paba supplied by VanDyke & Company, Inc., Belleville, NJ. Table IV depicts published data on 10 fragrance raw materials, comparing the 7-hour in vitro assay to human and animal models (2,7,8-10). For the materials identified, agreement is shown between the methods. In general, the yeast assay is the most sensi- tive, followed by the mouse, guinea pig, and human models regarding the concentra- tion required to elicit a positive response. DISCUSSION The 18-hour in vitro assay modified to reduce the exposure time to 7 hours yielded a comparable 8-MOP concentration curve and relative 8-MOP activity for the materials tested. This modification makes the assay more practical by allowing the investigator to initiate and complete the treatment and exposure phases of the assay within a working day, leaving only the incubation and observation of the responses to be performed two days later. The use of the standard 8-MOP curve, when run concurrently with the assay, allows the data to be relative and a quantitative assay will result. Discrepancies between in vitro and in vivo results have been investigated whereby posi- tive in vitro responses could not be duplicated in the guinea pig model which has been used successfully by a number of investigators (11-13) to demonstrate phototoxic po- tential. When a modification of the animal assay was performed, encompassing mul- tiple applications and exposures to UVA at 10 times the in vitro test concentration, agreement between the assays was achieved. The relevance of these findings to human