Test No. 446A: ToxTracker gene reporter assay for the identification of genotoxic hazard and genotoxic/non‑genotoxic mechanism of action

This Test Guideline describes a stem cell-based reporter assay that identifies the genotoxic properties of chemicals by combining six fluorescent reporter genes that are selectively activated by different cellular signalling responses. Two of the signalling responses are associated with direct genotoxicity. The other four stress response pathways are associated with cytotoxic effects like oxidative stress or protein damage that indirectly may lead to genotoxicity. The six genetically engineered reporter genes, each expressing a different GFP-tagged reporter, are exposed to the test chemical both with and without an exogenous source of metabolic activation. Activation of the GFP reporter genes is measured by flow cytometry. The differential induction of each reporter compared to the solvent control cells is used to predict genotoxicity and inform on the genotoxic/non-genotoxic mechanism of action of the test chemical. Simultaneously, cytotoxicity of the tested chemicals is determined by relative cell count in chemical-exposed cultures compared to the solvent control cultures.