FIRST PHASE INTER-LABORATORY VALIDATION OF THE IN VITRO EYE IRRITATION TESTS FOR COSMETIC INGREDIENTS: (2) EVALUATION OF CHORIOALLANTOIC MEMBRANE (CAM) TESTS

SHIGENOBU HAGINO1,2, HIROSHI ITAGAKI1,2, SHIGEMI KINOSHITA1,3, NAOKO TANI1,3, TSUNEAKI NAKAMURA1,4, NAOKO ONO1,4, KIMIYO KONISHI1,5, HAJIME KOJIMA1,6, YASUO OHNO1 AND AKIRA TAKANAKA7
1Japan Cosmetic Industry Assoc. (JCIA), 4th floor Hatsumei Bldg., 9-14, Tranomon, 2-chome, Minato-ku, Tokyo 105, Japan; 2Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 223, Japan; 3P0LA Corporation, 560 Kashio-cho, Totuka-ku, Yokohama 244, Japan; 4Lion Corporation, 100 Tajima, Odawara-shi, Kanagawa, 256, Japan; 5SUNSTAR Inc., 3-1 Asahi-machi, Takatsuki-shi, Osaka 569, Japan; 6Nippon Menard Cosmetic Co., Ltd., 4-66 Asakusa. Ogaki-si, Gifu 503, Japan; 7Div. Pharmacol. Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan

Regular Article :AATEX 3(4):137-145

Abstract
The chorioallantoic membrane (CAM) test using fertile hen's egg was evaluated in five laboratories as an alternative method to predict eye irritation of cosmetic ingredients. Nine surfactants and physiological saline were used as coded samples. Test procedures were controlled under the same standard operating procedure (SOP) among participants. CAM responses to the chemicals was measured using two systems; the macroscopic observation method (HET-CAM method) and trypan blue staining method.
The rank correlation coefficients between the lead laboratory and the other four laboratories were 0.77-0.99 for HET-CAM method and 0.88-0.93 for trypan blue staining method, respectively. The inter-laboratory variations of both methods were relatively small except for cases of non irritating samples. The correlation coefficient and the rank correlation coefficient between the HET-CAM scores and the maximum total scores of Draize eye irritation tests were 0.75 and 0.91, respectively. Those between the amount of trypan blue staining and the maximum total scores of Draize eye irritation tests were 0.95 and 0.91, respectively. When we compared these in vitro results with individual scores in the Draize eye irritation test. HET-CAM results showed a good correlation to the changes in the conjunctive. On the other hand, trypan blue staining method showed a good correlation to those in cornea.
From these results, we conclude that the MET-CAM method and trypan blue staining method using CAM are promising alternative methods to the Draize eye irritation test. Further evaluation of these methods using a wider range of cosmetic ingredients is under way.

Key words: CAM, validation study, Draize eye irritation test, alternatives, surfactant, MET-CAM, trypan blue staining, in vitro.


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