A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay

Abstract

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, β-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.

Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standardin vitrogenotoxicity assays

“…When the RSMN assay is used in combination with the RS Comet assay as intended, which is as a follow-up to positive results from the standard in vitro testing battery with a choice of RSMN or RS Comet, or both, depending on the genotoxicity endpoint(s) affected ( 17 , 52 , 53 ), the sensitivity increases to 89% while the specificity remains high. A manuscript with a detailed analysis of the predictive capacity of these two assays in a ‘battery’ approach is in preparation.…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…When comparing complex organs with cells cultured in standard 2D monolayers, 2D cultures do not have the ability to represent the functionality of an organ, whereas cells cultured in 3D resemble the organ structure better, due to their more “in vivo-like” behavior for key parameters such as cell viability, proliferation, differentiation, morphology, gene and protein expression and function [ 195 , 196 ]. For genotoxicity assessment, robust protocols for 3D models have been established for skin, airways and liver tissue equivalents [ 10 , 197 ].…”

Systems for Genetic Assessment of the Impact of Environmental Factors

“…The process of establishing metabolic pathways and assessing xenobiotic toxicity is a technically complex and time-consuming process, it requires the use of both in vitro cell systems and in vivo animal models before conducting human clinical trials [142]. While animal tests are still the primary standard method for assessing the genetic safety of xenobiotics, scientists are increasingly focusing on the concept of '3R', guided by principles designed to reduce, improve, and replace animal models when testing for genotoxicity [167,168]. In such a situation, the in vivo genotoxic potential is assessed further only after a positive result has been obtained with the in vitro basic test systems [112].…”