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

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.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…Two in vitro genotoxicity approaches using three-dimensional skin models have been proposed: i) the human reconstructed skin micronucleus (RSMN) assay using the EpiDerm™ model, and ii) the Comet assay, which detects complementary DNA damage, including damage indicative of the occurrence of gene mutations (EpiDerm™, Phenion™). Both models are currently evaluated in a validation study coordinated by Cosmetics Europe (Aardema et al, 2010;Reus et al, 2013). Once validated, the RSMN and the three-dimensional skin Comet assay may be used as follow-up tests for positive results from the current in vitro genotoxicity test battery in a weight-of-evidence approach.…”

Rebooting the generally recognized as safe (GRAS) approach for food additive safety in the US

“…Ongoing work by ECVAM and Cosmetics Europe aims to establish and validate new methods for genotoxicity testing in reconstructed human 3D skin models (micronucleus test and comet assay) (Maurici et al, 2005;Aardema et al, 2010;Reus et al, 2013), again with the key goal of reducing false positives. The Comet assay is now being validated in a joint effort between Cosmetics Europe and BfR, Germany.…”

Alternative Methods to Animal Testing for the Safety Evaluation of Cosmetic Ingredients: An Overview

“…If the Ames test is negative and the in vitro micronucleus test is positive, then the substance may be considered an in vitro mutagen and further testing may be essential to clarify the clastogenic potential of the substance. In this case, either the Comet assay in mammalian cells [44] or in the 3D-reconstructed human skin model [45], or the micronucleus test in the 3D-reconstructed human skin model should be considered [46].…”