Evaluation of chemicals requiring metabolic activation in the EpiDerm? 3D human reconstructed skin micronucleus (RSMN) assay

摘要

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm? is a promising new assay for evaluating genotoxicity of dermally applied chemicals. A global pre-validation project sponsored by the European Cosmetics Association (Cosmetics Europe - formerly known as COLIPA), and the European Center for Validation of Alternative Methods (ECVAM), is underway. Results to date demonstrate international inter-laboratory and inter-experimental reproducibility of the assay for chemicals that do not require metabolism [Aardema et al., Mutat. Res. 701 (2010) 123-131]. We have expanded these studies to investigate chemicals that do require metabolic activation: 4-nitroquinoline-N-oxide (4NQO), cyclophosphamide (CP), dimethylbenzanthracene (DMBA), dimethylnitrosamine (DMN), dibenzanthracene (DBA) and benzo(a)pyrene (B. aP). In this study, the standard protocol of two applications over 48. h was compared with an extended protocol involving three applications over 72. h. Extending the treatment period to 72. h changed the result significantly only for 4NQO, which was negative in the standard 48. h dosing regimen, but positive with the 72. h treatment. DMBA and CP were positive in the standard 48. h assay (CP induced a more reproducible response with the 72. h treatment) and B. aP gave mixed results; DBA and DMN were negative in both the 48. h and the 72. h dosing regimens. While further work with chemicals that require metabolism is needed, it appears that the RMSN assay detects some chemicals that require metabolic activation (4 out of 6 chemicals were positive in one or both protocols). At this point in time, for general testing, the use of a longer treatment period in situations where the standard 48. h treatment is negative or questionable is recommended.