THE RECONSTRUCTED SKIN COMET ASSAY SHOWS GOOD INTRA- AND INTER-LABORATORY REPRODUCIBILITY: RESULTS FROM THREE LABORATORIES.

EU policy marks the need for a broad replacement of animal tests in relation to the 7th Amendment to the Cosmetics Directive. For cosmetics ingredients as well as many chemicals, the skin is the main route of exposure. The currently used standard in vitro genotoxicity assays, if used in a test battery approach, show a high percentage of irrelevant positive results. Moreover, there is no follow-up assay available in the target tissue skin and the use of in vivo assays is not allowed to further evaluate a positive response obtained from the in vitro assays. Genotoxicity assays using reconstructed human skin could however serve this purpose. EpiDerm™ skin models were topically exposed to vehicle or test compounds for 3h, followed by cell isolation and measurement of DNA damage using the Comet assay methodology. Inter-laboratory reproducibility of the 3D skin Comet assay was demonstrated for MMS and 4NQO and results showed good concordance with in vivo data. In the next phase of the project, the assay was performed with 3 coded compounds shipped to 3 different laboratories. The results of the collaborative study showed good reproducibility within and between laboratories. However, considerable intra- and inter-experimental variability was often observed in the background level of DNA damage in 3D skin tissues, with solvent control values sometimes greater than 40%. To resolve these problems we worked on several aspects of the experimental procedure and promising results were seen with the use of “underdeveloped” tissues, which are shipped at an earlier stage (5 days before regular shipment) and are then matured in our laboratories. Results thus far indicate that the comet assay in reconstructed 3D skin models is a relevant model for the safety assessment of chemicals with dermal exposure. This work was sponsored by the European Cosmetics industry (COLIPA) and the European Center on Validation of Alternative Methods (ECVAM).

Comet assay, EPI-200, EPI-201, EpiDermTM , Genotoxicity, Interlaboratory reproducibility, Reproducibility, Transferability

4NQO, Acetone, MMS