The EURL ECVAM Scientific Advisory Committee (ESAC) advises the EURL ECVAM on scientific matters. An ESAC subgroup conducted an independent peer review of validation studies of two test methods and assessed their scientific validity for the purpose of genotoxicity. Genotoxicity refers to the effects of chemical substances that cause changes in the DNA of cells.

RS Comet and RSMN are separate in vitro assays that utilize reconstructed human skin models and are considered to be better models for predicting human response to chemical exposure via the skin due to their enhanced biological similarity to human skin tissue compared to traditional monolayer models.

The 3D reconstructed skin (RS) Comet Assay is an in vitro genotoxicity test that is an adaptation of the well-known alkaline Comet Assay and was developed in a three-dimensional reconstructed human skin model (RHS). Specifically, the method utilizes the Phenion® full-thickness skin model, which consists of two tissues made from primary and p53-competent cells of human origin, primary keratinocytes and fibroblasts. The aim of the method is to predict the potential of substances that cause DNA damage when associated with skin exposure.

The 3D Reconstructed Human Skin Micronucleus assay (RSMN) assay is also an in vitro genotoxicity test that transfers the well-known micronucleus test into a three-dimensional reconstructed human skin model (3D RHS). The test investigates the potential of test items to cause genotoxicity in the form of chromosome damage (clastogenicity and aneugenicity). The method utilizes the EpiDerm™ skin model (MatTek, Ashland, USA), which is grown from normal human epidermal keratinocytes derived from newborn foreskin tissue on specially prepared tissue culture inserts. The method is aimed at predicting the potential of substances primarily associated with dermal exposure to cause DNA damage.

The ESAC considered both assays to be biologically relevant. Both assays offer advantages for expanding the chemical range compared to conventional in vitro assays. However, the ESAC opinion recommends a slightly different testing strategy than that proposed by the test submitters. Specifically, the RS assay should be started because it covers the mechanism of action that tested positive in the standard in vitro test procedure. If the result is negative, the second RS assay should be performed for confirmation. A positive result in either the RS Comet or RSMN assay would then be sufficient to conclude that the test item is genotoxic in skin. The transferability, reproducibility and, when the tests are used in combination as proposed by ESAC, the predictiveness of RS Comet and RSMN are sufficient.

The tests are valuable components of a tiered testing strategy for assessing genotoxicity in the skin following dermal application.

Publication:
European Commission: Joint Research Centre, CLEWELL, R., CORSINI, E., GRECO, D., KIENHUIS, A., LEHMANN, D., RODRÍGUEZ, B., SEGNER, H. and TRALAU, T., (2025). ESAC Opinion on the Scientific Validity of the RS Comet and RSMN Test Methods. MENNECOZZI, M., CORVI, R. and BARROSO, J. editor(s), Publications Office of the European Union, Luxembourg, 2025, https://data.europa.eu/doi/10.2760/2520536, JRC140306. https://publications.jrc.ec.europa.eu/repository/handle/JRC140306

Additional information:
https://tsar.jrc.ec.europa.eu/test-method/tm2020-01
https://tsar.jrc.ec.europa.eu/test-method/tm2020-04