Frontiers in Genetics (Jun 2015)
Validation of the 3D Skin Comet assay using full thickness skin models: transferability and reproducibility
Abstract
The 3D Skin Comet assay was developed to improve the in vitro prediction of the genotoxic potential of dermally applied chemicals. For this purpose, a classical read-out for genotoxicity (i.e. comet formation) was combined with reconstructed 3D skin models as well-established test systems. Five laboratories (BASF, BfR (Federal Institute for Risk Assessment), Henkel, Procter & Gamble and TNO Triskilion) started to validate this assay using the Phenion® Full- Thickness (FT) Skin Model and 8 coded chemicals with financial support by Cosmetics Europe and the German Ministry of Education & Research. There was an excellent overall predictivity of the expected genotoxicity (>90%). Four labs correctly identified all chemicals and the fifth correctly identified 80% of the chemicals. Background DNA damage was low and values for solvent (acetone) and positive (methyl methanesulfonate (MMS)) controls were comparable among labs. Inclusion of the DNA-polymerase inhibitor, aphidicolin (APC), in the protocol improved the predictivity of the assay since it enabled robust detection of pro-mutagens e.g., 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene. Therefore, all negative findings are now confirmed by additional APC experiments to come to a final conclusion. Furthermore, MMC, which intercalates between DNA strands causing covalent binding, was detected with the standard protocol, in which it gave weak but statistically significant responses. Stronger responses, however, were obtained using a cross-linker specific protocol in which MMC reduced the migration of MMS-induced DNA damage. These data support the use of the Phenion® FT in the Comet assay: no false-positives and only one false-negative finding in a single lab. Testing will continue to obtain data for 30 chemicals. Once validated, the 3D Skin Comet assay is foreseen to be used as a follow-up test for positive results from the current in vitro genotoxicity test battery.
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