Regulatory Acceptance ofthe Successfully Validated 3T3 NRU InVitro Phototoxicity Test

As no standard guidelines for testing photoirritation potential, either in vivo or in vitro, had been accepted for regulatory purposes at the international level by the OECD, in 1991 the European Commission (EC) and the European Cosmetics, Toiletry and Perfumery Association (COLIPA) established a joint program to develop and validate in-vitro photoirritation tests. In the first phase of the study, which was funded by DG XI of the EC and co-ordinated by ZEBET, in-vitro phototoxicity tests established in laboratories of the cosmetics industry, and a new assay - the 3T3 NRU PT test, which is a photocytotoxicity test using the mouse fibroblast cell line 3T3 with neutral red uptake (NRU) as the endpoint for cytotoxicity - were evaluated.

In the prevalidation study conducted with 20 test chemicals, the 3T3 NRU PT in-vitro phototoxicity test was unexpectedly the only such test in which all 20 chemicals were correctly identified as phototoxic or non-phototoxic [16, 17]. Quite independently, a laboratory in Japan subsequently obtained the same correct results in the 3T3 NRU PT, when testing the same set of 20 test chemicals.

In the second phase of the study, which was funded by ECVAM and co-ordinated by ZEBET, the 3T3 NRU PT test was validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. A representative set of test chemicals covering all major classes of phototoxins was selected according to results from standardized photopatch testing in humans [18]. The results obtained for this in-vitro test under blind conditions were reproducible, and the correlation between in vitro and in vivo data was almost perfect. Therefore, the ECVAM Scientific Advisory Committee [19] concluded in 1998 that the 3T3 NRU PT is a scientifically validated test which is ready to be considered for regulatory acceptance. However, the EU expert committee on the safety of cosmetics, the Scientific Committee on Cosmetology and Non-Food-Products (SCCNFP), criticized that an insufficient number of UV-filter chemicals (widely used as sunblockers) were tested in the formal validation study. In a subsequent blind trial on UV filter chemicals, which was again funded by ECVAM and co-ordinated by ZEBET, the phototoxic potential of all test chemicals was predicted correctly in the 3T3 NRU PT in-vitro phototoxicity test [20]. Therefore, in 1998, the EU - having accepted the 3T3 NRU PT test as the first experimentally validated in-vitro toxicity test for regulatory purposes - officially applied to the OECD for worldwide acceptance of this in-vitro toxicity test. In 2000, the European Commission officially accepted and published the 3T3 NRU PT phototoxicity test in Annex V of Directive 67/548 EEC on the Classification, Packaging and Labelling of Dangerous Substances [21]. Thus, this in-vitro test is the first formally validated in-vitro toxicity test to be accepted into Annex V, and is the only phototoxicity test accepted for regulatory purposes in Europe. Meanwhile, in 2004 the OECD accepted the 3T3 NRU PT phototoxicity test at the worldwide level as the first in-vitro toxicity test to be introduced into the OECD Guidelines for the testing of chemicals [15].

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