AUTOMATION AND VALIDATION OF MICRONUCLEUS DETECTION IN THE 3D EPIDERM™ HUMAN RECONSTRUCTED SKIN ASSAY AND CORRELATION WITH 2D DOSE RESPONSES.

Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructive skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay’s fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 µg/ml methyl methanesulfonate (MMS) at 1750 µg/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 µg/ml, which was very similar to the topical LOGEL, when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm™. Our preliminary validation here demonstrates that the RMSN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimizing unnecessary in vitro tests by reducing the in vitro misleading positives.

2D vs. 3D cultures, Automated scoring, Binucleation rate, DNA repair, EpiDerm, Genotoxicity, Lowest observed genotoxic effect level (LOGEL), Metafer Slide Scanning Platform, Micronucleus assay, Reconstructed Skin Micronucleus (RSMN) assay

Acetone, Etoposide, Hydrogen peroxide, Methyl carbamate, Methyl methanesulfonate, Mitomycin-C, N-ethyl-N-nitrosourea, N-methylpurine-DNA glycosylase