Møller, PeterLadeira, CarinaZiemann, ChristinaKnasmueller, SiegfriedMišík, MiroslavLouro, HenriquetaSilva, Maria JoãoOlsen, Ann KarinAzqueta, AmayaLangie, Sabine A.Bonassi, StefanoDusinska, MariaGajski, GoranCollins, Andrew2026-03-232026-03-232026-03Møller P, Ladeira C, Ziemann C, Knasmueller S, Mišík M, Louro H, et al. Towards a validation of the standard and enzyme-linked comet assay: a retrospective variability analysis. Arch Toxicol. 2026 Mar 10. Epub ahead of print.0340-57611432-0738http://hdl.handle.net/10400.21/22730AKO and MD were supported by the Horizon Europe project PROPLANET (Grant agreement number 10109842). SK and MM were supported by the RadExIORSBoost EU project HORIZON-WIDERA 2023 (Grant No. 101158832). GG is supported by the European Regional Development Fund project Rec-IMI (Grant No. KK.01.1.1.02.0007) and the European Union—Next Generation EU BioMolTox (Grant No. 533-03-23-0006).The comet assay is one of the most popular tests for genotoxicity in cell cultures, non-animal species, animals, and humans. It has high sensitivity to detect low levels of DNA damage, can be applied to non-proliferating cells, requires relatively few cells, is technically simple, and is low-cost. The Organisation for Economic Co-operation and Development (OECD) adopted in 2016 the in vivo comet assay for measurement of DNA strand breaks in animal tissues. There is a desire to expand the comet assay to genotoxicity testing in cell cultures, including the detection of oxidatively damaged DNA by incubation of gel-embedded nucleoids with DNA repair enzymes, especially formamidopyrimidine DNA glycosylase (Fpg), which converts oxidised purines to DNA breaks. Based on available information in the literature, this review provides a retrospective evaluation of the validation status of this assay, focusing on accuracy and reliability in genotoxicity testing in vitro. Information on accuracy is scarce, although limited evidence suggests levels of Fpg-sensitive sites are similar to those obtained by Fpg-linked alkaline unwinding and alkaline elution assays. Several ring studies have shown that estimated background levels of DNA breaks vary within and between laboratories. However, ring studies indicate good intra- and inter-laboratory reproducibility of the standard assay on ionizing radiation-exposed and the Fpg-linked assay on potassium bromate-exposed cells. Further studies are needed to assess the reproducibility in multiple laboratories using coded samples of non-genotoxins and genotoxins. Nevertheless, the available results indicate the comet assay is a reliable in vitro genotoxicity test.engComet assayDNA damageFPG-sensitive sitesIn vitro genotoxicityValidationjournal article10.1007/s00204-026-04317-y