Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT)

Gamma‐H2AX immunofluorescence for the detection of tissue‐specific genotoxicity in vivo

“…Recommendations from different expert groups (Burlinson et al, ; Uno et al, ; Speit et al, ) and OECD guidance (OECD , TG489) were followed, along with an earlier published experimental protocol, including tissue choice and sampling, preparation of single‐cell suspension and slides, followed by DNA unwinding, electrophoresis, neutralization of slides (Struwe et al, ; Guérard et al, ; Plappert‐Helbig and Guérard, ). Parts of liver, kidney and jejunum were sampled and processed for comet assay evaluation in accordance with the OECD guidance.…”

Outcome of the public consultation on the draft opinion providing clarification of some aspects related to genotoxicity assessment

“…Further, the impact of cytotoxicity with regards to positive comet assay results was not taken into account nor the fact that much of the comet assay results cited were principally from a single laboratory. As we noted in our comments to the mandate, many of the studies used in this earlier analysis were conducted well before the establishment of OECD TGs for either the comet or TGR assays, and in a recent publication (Speit et al, 2015)*, an expert group concluded that cytotoxicity could be an important confounder of comet results. Therefore, any evaluation regarding the performance of the comet assay drawn from these databases should re-evaluated in this context.…”

Main issues addressed in the 2014–2015 revisions to the OECD Genetic Toxicology Test Guidelines

“…The project was included on the TG program Work Plan, and Canada, France, The Netherlands, and the United States of America were identified as lead countries. A core group of experts from the lead countries (who are the authors of this article) was nominated to assist the WNT and the OECD secretariat to coordinate the revision of TGs and the drafting of an "Overview of the set of OECD Genetic Toxicology TGs and updates performed in -2015" [OECD, 2016a to provide information on the revision process and a summary of the key changes. This group submitted project proposals for the revision of the TGs for in vivo and in vitro assays.…”

“…It is not the goal of the current article to provide extensive details about the individual TGs. Further details are provided in the "Overview of the set of OECD Genetic Toxicology TGs and updates performed in -2015" [OECD, 2016a, as well as within the guidelines themselves, all of which are published [OECD, 2016b].…”

“…Although studies have been published reporting the use of the comet assay to investigate the induction of DNA damage in sperm, extensive discussion during the drafting of the TG for the comet assay led to the exclusion of germ cells from the tissues that could be used for testing with TG 489. There were two main reasons for this outcome: (1) much longer exposures and sampling times than those recommended in TG 489 would be necessary for a biologically significant analysis of DNA damage in mature sperm; and (2) the high and variable background levels of DNA damage that have been reported in the literature [Speit et al, ]. Protocol modifications together with improved standardization and validation studies were deemed necessary before the comet assay could be applied to sperm to generate data for regulatory submissions.…”