Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals

A A Reus,S Pfuhler,G J Carr,T R Downs,A Zeller,R Corvi,C A M Krul,K Reisinger

doi:10.1093/mutage/get051

Abstract

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Highlights

The standard battery of genotoxicity testing for regulatory purposes normally includes at least two in vitro assays to predict the genotoxic potential of pharmaceuticals, industrial chemicals, food additives and ingredients of beauty care products
Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data
One of the recommendations made by genotoxicity experts from academia, government and industry during the 5th International Workshop on Genotoxicity Testing [8] and a workshop supported by the European Union Reference Laboratory on Alternatives to Animal Testing (EURL ECVAM) [3] was to investigate the use of new test systems, e.g. reconstructed 3D human epidermal skin models

Summary

Introduction

The standard battery of genotoxicity testing for regulatory purposes normally includes at least two in vitro assays to predict the genotoxic potential of pharmaceuticals, industrial chemicals, food additives and ingredients of beauty care products. Compounds in the cosmetics industry cannot be evaluated in vivo because animal testing for this purpose is prohibited, as defined in the Seventh Amendment to the Cosmetics Directive [6], which may result in unnecessary loss of new ingredients if genotoxicity assessment relies solely on the current in vitro assays [7]. One of the recommendations made by genotoxicity experts from academia, government and industry during the 5th International Workshop on Genotoxicity Testing [8] and a workshop supported by the European Union Reference Laboratory on Alternatives to Animal Testing (EURL ECVAM) [3] was to investigate the use of new test systems, e.g. reconstructed 3D human epidermal skin models These models have the advantage of allowing topical application of the compounds, evaluation of formulations and poorly soluble compounds, as well as measurement of local toxicological effects in target cells. Non-carcinogenic, positive in in vitro genotoxicity assays (reviewed in ref. 29)

Materials and methods

Evaluation criteria

Results and discussion

Conclusion