USE OF THE COMET ASSAY TO DETECT GENOTOXIC EFFECTS IN RECONSTITUTED HUMAN SKIN.
This study by scientists at Procter & Gamble and Wella-Cosmital SA demonstrated that MatTek’s EpiDerm in vitro human tissue equivalent may provide an excellent platform for performing comet (genotoxicity) assays of dermally applied cosmetics and their constituents because EpiDerm is produced from primary (non-transformed) human keratinocytes and it possesses a human skin-like barrier function. Current in vitro methods in genotoxicity testing demonstrate excellent sensitivity but are suffering from low specificity. Those tests are therefore prone to “false positive” results (non-carcinogens with positive genetox results). The reasons for the lack of specificity may be diverse: inadequate metabolic capacity and p53 status of the commonly used cell lines (resulting in DNA repair deficiency) as well as stringent requirements of current testing guidelines with regard to cytotoxicity and dose. For all dermally applied compounds the disregard of the barrier function of the skin may be another important factor. Cosmetics are mostly applied to the skin and their safety assessment will be substantially affected by the EU cosmetics directive, which will prohibit the use of animal experiments starting from March 2009. Reconstructed three-dimensional (3D) human skin models like the EpiDerm human skin equivalent might offer a solution for the above mentioned problems. They are commercially available and resemble closely the human skin in terms of permeability, metabolic capacity and structure. The Comet assay is a valuable tool in genotoxicity testing as it detects a broad range of (primary) DNA damage in virtually any cell type, even in non-proliferating cells. The only prerequisite for this test is the creation of a single cell suspension without introducing artificial DNA damage, which is not easy to achieve for the skin. The Comet assay in human 3D skin models is a promising tool for investigating compounds with dermal exposure and may help replace animal experiments. Researchers from Procter & Gamble and Wella-Cosmital SA were able to demonstrate dose-response relationship both with the model mutagen (MMS) and UV irradiation. However, at this early stage of development researchers still see issues with reproducibility and are not yet able to simultaneously assess genotoxicity and cytotoxicity, as it is common practice in Comet assay protocols with monolayer cell cultures.
Comet assay, DNA damage, EPI-200, EpiDerm, Genotoxicity, UV light, UV-A/B radiometer
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