Investigating the application of 3D skin models in micronucleus assay and comet assay

Conventional two-dimensional (2D) cell culture models for genotoxicity testing often yield false-positive results due to their limited metabolic capacity and lack of tissue architecture. Three-dimensional (3D) reconstructed human skin models offer a more physiologically relevant alternative for evaluating genotoxicity. In this study, the reproducibility, dose-response, and predictive performance of the micronucleus test (MNT) and comet assay were systematically assessed using 3D skin models EpiSkin-MNT and T-Skin. A panel of reference chemicals, including both genotoxic and non-genotoxic agents, was tested. Cytotoxicity was measured using ATP and adenylate kinase (AK) assays; genotoxicity endpoints included micronucleus frequency and comet assay parameters such as tail intensity and tail moment. Both assays exhibited clear dose-dependent increases in genotoxic markers for positive controls, while negative controls showed no significant response. Use of the DNA repair inhibitor aphidicolin in the comet assay enhanced the sensitivity of DNA damage detection. The integration of 3D skin models with MNT and comet assays provides a robust, reproducible, and physiologically relevant platform for genotoxicity assessment, supporting the transition from animal-based methods and aligning with regulatory trends.