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Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correl

Chapman, Katherine; Thomas, Adam D; Wills, John; Pfuhler, Stefan; Doak, Shareen H; Jenkins, Gareth JS


Katherine Chapman

Adam Thomas
Senior Lecturer in Human Genetics and Genomics

John Wills

Stefan Pfuhler

Shareen H Doak

Gareth JS Jenkins


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 reconstructed 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 and 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 (H2O 2), 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 RSMN assay may be a valuable followup to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives. © The Author 2014.


Chapman, K., Thomas, A. D., Wills, J., Pfuhler, S., Doak, S. H., & Jenkins, G. J. (2014). Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correl. Mutagenesis, 29(3), 165-175.

Journal Article Type Article
Acceptance Date Mar 27, 2014
Online Publication Date Mar 27, 2014
Publication Date Jan 1, 2014
Journal Mutagenesis
Print ISSN 0267-8357
Electronic ISSN 1464-3804
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
Volume 29
Issue 3
Pages 165-175
Public URL
Publisher URL


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