Establishing whether or not a substance is genotoxic is critical in risk assessment, as it determines whether the substance can be assessed as a threshold or non-threshold toxin. The OECD Test Guidelines (TGs) on genetic toxicology are considered the “gold standard” on how the favoured tests should be conducted, with industry sectors recommending specific tests, or combinations thereof, to fulfil regulatory requirements on this endpoint.

In recent years concerns have been raised over OECD TG 486, the in vivo unscheduled DNA synthesis assay (UDS assay). A major limitation is that it is only truly applicable to substances that induce DNA damage in the liver, and of the type that is repaired by nucleotide excision (i.e. mainly bulky adducts); as the assay does not detect DNA damage that is repaired by other mechanisms or is not repaired at all, it could potentially result in a false negative response. And, where a response is positive, there is no indication of the fidelity of the DNA repair, meaning that the assay provides little or no information on potential mutagenic activity resulting from mis-repair or mis-replication. Indeed, caveats have been placed on its use as a follow-up for in vitro positive results by the ICH (2014) and ECHA (2017), and the OECD considered that it meets the criteria for deletion from its guidelines (OECD, 2016). Unsurprising then, that the UDS assay has also come under scrutiny in a recent EFSA publication.

In considering the limitations of the UDS assay, EFSA made use of the newly developed EURL ECVAM database (which comprises genotoxicity and carcinogenicity data on over 700 substances giving positive results in the (Ames) bacterial reverse mutation test, OECD TG 471). The sensitivity of the UDS assay to predict (genotoxic) carcinogenicity was found to be around 60%, compared to 88% for the transgenic rodent gene mutation assay (TGR, OECD TG 488) and 91% for the in vivo mammalian comet assay (OECD TG 489). Furthermore, the UDS assay failed to detect around 40% of Ames-positive substances that had also given positive results in other in vivo genotoxicity tests.

In line with the ICH and ECHA, EFSA concluded that the UDS assay is no longer acceptable as a follow-up to positive in vitro tests for new substance dossiers. For substance re-evaluations that already have UDS assay data, only positive responses are considered adequate to assess genotoxic potential; for negative responses, the reliability and significance of the results require evaluation in a weight-of-evidence approach (taking into account the type of DNA damage, metabolism, toxicokinetics etc.). It is inevitable that, for some substances, additional tests such as the TGR or the in vivo comet assay will be needed to enable a final conclusion to be drawn.


ECHA (2017). European Chemicals Agency. Guidance on information requirements and chemical safety assessment, chapter R.7a: endpoint specific guidance. Version 6.0.

EFSA Scientific Committee, Hardy A, Benford D, Halldorsson T, Jeger M, Knutsen HK, More S, Naegeli H, Noteborn H, Ockleford C, Ricci A, Rychen G, Silano V, Solecki R, Turck D, Younes M, Aquilina G, Crebelli R, Gurtler R, Hirsch-Ernst KI, Mosesso P, Nielsen E, van Benthem J, Carfi M, Georgiadis N, Maurici D, Parra Morte J and Schlatter J, 2017. Scientific Opinion on the clarification of some aspects related to genotoxicity assessment. EFSA Journal 2017;15(12):5113, 25 pp.

EURL ECVAM (2017). European Union Reference Laboratory for Alternatives to Animal Testing. EURL ECVAM genotoxicity and carcinogenicity consolidated database of Ames positive chemicals.

ICH (2014). International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk (M7).


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