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Mutat Res Genet Toxicol Environ Mutagen. 2015 May 1;783:6-12. doi: 10.1016/j.mrgentox.2014.09.006. Epub 2014 Sep 22.

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).

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Ulm University, Institute of Human Genetics, Ulm, Germany. Electronic address:
Japanese Center for the Validation of Alternative Methods, National Institute of Health Sciences, Tokyo, Japan.
Huntingdon Life Sciences, Huntingdon, UK.
University of Oslo, Department of Nutrition, Oslo, Norway.
Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany.
Novartis Institutes for BioMedical Research, Preclinical Safety, Basel, Switzerland.
Mitsubishi Tanabe Pharma Co., Chiba, Japan.
Helix3 Inc., RTP, NC, USA.
Covance Laboratories Ltd, Harrogate, UK.
Janssen Research & Development, Beerse, Belgium.
Boehringer Ingelheim Pharmaceuticals Inc., Nonclinical Drug Safety, Ridgefield, USA.
Sumitomo Chemical Co. Ltd., Osaka, Japan.
BioReliance by SAFC, Rockville, MD, USA.
The Procter and Gamble Company, Global Product Stewardship, Mason, OH, USA.
Public Interest Incorporated Foundation Biosafety Research Center, Shizuoka, Japan.
Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD, USA.


As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.


Genotoxicity testing; Laboratory proficiency; Test protocol; Tissue toxicity

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