The EpiDerm 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48 h and 72 h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMNassay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals.

reconstructed skin micronucleus assay, RSMN, genotoxicity, aneugens, clastogens, CREST analysis, micronuclei, binucleated cells, cytokinesis block proliferation index, Il-1b, IL-2, IL-6, IL-8, IL-10, IL-12 p70, TNF-a, IFN-g, GM-CSF

acetone, ethanol, saline, mitomycin C, vinblastine sulfate, methyl methanesulfonate, E-ethyl-n-nitrosourea, dimethylbenzanthracene, cyclophosphamide, 4-nitrophenol, cyclohexanone, DMSO, ethy-1,3-hexanediol