Abstract
The TK6 human lymphoblastoid cell line, characterized by a wild-type p53 status, is widely recognized as a robust in vitromodel for genotoxicity testing. It is routinely employed in assays compliant with OECD test guidelines, specifically the in vitromicronucleus assay（TG 487） and the gene mutation assay（TG 490）. The presence of functional p53 contributes to the cell line’s reduced false-positive rate compared to rodent-derived cells, enhancing its relevance for human risk assessment. This biological fidelity makes TK6 cells particularly valuable in regulatory toxicology. Recent technological advances have further expanded the utility of TK6 cells. For example, integration with error-corrected next-generation sequencing（ecNGS） enables ultra-sensitive detection of mutational events at low frequencies, thereby improving the mechanistic understanding of genotoxic agents. Additionally, the MultiFlow assay—an advanced flow cytometry-based multiplex assay—facilitates the simultaneous detection of multiple genotoxicity endpoints, increasing throughput and data richness. These innovations have enabled more detailed mechanistic analyses and the identification of novel biomarkers for DNA damage and repair processes. Moreover, genome editing technologies have produced TK6-derived mutants deficient in specific DNA repair pathways, thereby broadening the application of this model to fundamental research on DNA damage responses and cellular repair mechanisms. Such modified cell lines provide powerful tools to dissect genotoxic mechanisms and to evaluate cellular effects of chemical compounds in a controlled genetic background. The reliability and versatility of TK6 cells have led to their growing acceptance in regulatory submissions across multiple jurisdictions. As animal welfare concerns intensify and regulatory frameworks increasingly endorse the 3Rs principles（Replacement, Reduction, Refinement）, TK6 cells represent a scientifically validated alternative to in vivogenotoxicity testing. This review comprehensively summarizes the current state of TK6 cell applications in genotoxicity assessment, highlights recent technological advancements, and discusses future perspectives for their expanded use as an alternative to animal experiments.
