TEST METHODS FOR SCREENING FRAGRANCES 61 bles I and VI. Of these 32 compounds, 25 were found to be allergenic for the guinea pig in 1 or more of the 4 tests used. This includes 4 compounds found to be allergenic only in the OET and 3 compounds found to be allergenic only in 1 or more of the other 3 tests. The remaining 7 compounds were found to be nonallergenic in all 4 tests on guinea pigs. These 7 compounds are described in the literature (22) as sensitizing for humans, but without confirmatory data such as a positive history, a positive patch test or re-exposition. In view of our negative findings on the guinea pig, we consider that the reported allergenicity of these compounds for humans might perhaps reflect a possible cross-sensitizing capacity. It can be seen from Table I that all the compounds with a well-established sensitizing capacity for man were found to be allergenic for the guinea pig in one or more of the 4 tests used. This could be expected for compounds 6f the "incrimination" Types B, C, D, and E. By contrast, the test results for compounds of Type A were divergent. These compounds induced hypersensitivity in guinea pigs when the incriminating data were accurate, and failed to do so when their reported allergenicity was based on an assump- tion or on an unconfirmed observation. The nonallergenicity of the first 6 compounds of Type A listed in Table I, as well as that of thymol, all of which failed to induce hypersensitivity in the guinea pig in any of the 4 tests used, may be considered fairly well-established, because they were negative in the OET even when tested undiluted (benzophenone and thymol could not be tested undiluted because of systemic toxicity, see Table I) and were also negative in the intradermal tests. On the other hand, 4 other compounds of Type A were shown to be weak sensitizers because they were positive in the OET at a concentration of 30 per cent. IV. DISCUSSION The OET is a procedure proposed for testing on guinea pigs the skin irritating and allergenic capacities of chemical compounds intended for use in perfumes, cosmetics, and dermatics. In the OET, the compounds to be tested are applied undiluted and in a descending series of concentrations. By establishing a dose-response curve, the minimal irritating and minimal sensitizing concentrations of a compound can be de- termined quantitatively. The end-point reactions are read on an "all or none basis," thus largely excluding subjective bias in the evaluation of the results. A total of 32 compounds described in the literature as allergenic for man were tested by the OET. For purposes of comparison they were also tested by the DT, the test with "FCAT, and the MT described by Magnusson and Kligman (4, 5). The highest number ß of allergenic compounds were detected by the OET, somewhat fewer by the FCAT and the MT, and a few by the DT. However, certain compounds were detected exclusively by the OET and others exclusively by one or more of the intradermal tests (DT, MT and/or FCAT). The reliability of the OET, i.e., its predictive value for man, was investi- gated by us, as had been done by Magnusson and Kligman, testing on animals the sensi- tizing properties of fragrances known from clinical experience to be allergenic for man and some known to be innocuous. Of the compounds tested, all those with well-es- tablished allergenic'ity for man were detected by the OET (see Tables I and VI). On the other hand, compounds with an unconfirmed clinical allergenicity yielded divergent results in the animal tests, as expected.

62 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table VII Allergenicity of Compounds Tested in Humans by the Maximization Test (47) and in Guinea Pigs by 4 Different Procedures Humans Guinea Pigs MT a OET DT b MT b FCAT • Test cone, Test cone. Compounds % Results % Results Results Results Results Amyl-cinnam•c aidehyde 3 - 100 .... Diethyl phthalate 5 - 100 .... Methyl-ionone 5 - 100 .... Ionone 4 - 100 .... Hydroxycitronella[ 6 - 100 .... Vanillin 1 - 100 + + + + Cinnamic alcohol 2 - 100 - - + + Coumarin 4 - 10 + - - - 3 - Euge nol 4 - 10 + + + + 3 - Geraniol 3 - 10 + - + + 3 - Heliotropin 3 - 30 + - + + 10 - d-Limonene 4 - 100 + - + + 30 - aThe occlusive ehciting concentration application was only at the user concentration x 2. •DT, MT, FCAT: concentrations used see Section II of this paper. Table VII shows a comparison of the results of our animal tests with those obtained by Greif (47) who used the MT on human subjects for testing several fragrance com- pounds "which had successfully weathered the test of time," i.e., the innocuoushess of which for humans had been demonstrated by many years of practical use. This com- parison confirms the high predictive reliability of the guinea pig OET for humans. Ta- ble VII shows that some compounds, all negative in the human MT, were not aller- genic for the guinea pig in the OET even when tested in the undiluted form. Others did not sensitize guinea pigs at concentrations 2 to 100 times as high as the conventional concentrations used on humans. This shows that the guinea pig OET can be used to de- termine a quantitatively precise risk of sensitization for humans. Table VII also shows that the results of the intradermal tests on the guinea pig seem to be less predictive for man (see comments on Table VII) and that a better correlation was found between the human MT and the guinea pig OET. In a later paper, the correlation between the results of Kligman's Human MT and the OET, especially, will be discussed. V. SUMMARY An OET is proposed for the detection of skin irritation and contact hypersensitivity in- duced in guinea pigs by various compounds intended for use in perfumes and cos-