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Mutat Res. 2013 Jan 20;750(1-2):40-9. doi: 10.1016/j.mrgentox.2012.08.009. Epub 2012 Sep 27.

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

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Marilyn Aardema Consulting LLC, Fairfield, OH 45014, USA.


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 (BaP). In this study, the standard protocol of two applications over 48h was compared with an extended protocol involving three applications over 72h. Extending the treatment period to 72h changed the result significantly only for 4NQO, which was negative in the standard 48h dosing regimen, but positive with the 72h treatment. DMBA and CP were positive in the standard 48h assay (CP induced a more reproducible response with the 72h treatment) and BaP gave mixed results; DBA and DMN were negative in both the 48h and the 72h 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 48h treatment is negative or questionable is recommended.

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