title: 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).
abstract: 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.
authors.affiliation: Ulm University, Institute of Human Genetics, Ulm, Germany. Electronic address: guenter.speit@uni-ulm.de.
authors.forename: Günter
authors.initials: G
authors.lastname: Speit
authors.affiliation: Japanese Center for the Validation of Alternative Methods, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Kojima
authors.affiliation: Huntingdon Life Sciences, Huntingdon, UK.
authors.forename: Brian
authors.initials: B
authors.lastname: Burlinson
authors.affiliation: University of Oslo, Department of Nutrition, Oslo, Norway.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Collins
authors.affiliation: Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Kasper
authors.affiliation: Novartis Institutes for BioMedical Research, Preclinical Safety, Basel, Switzerland.
authors.forename: Ulla
authors.initials: U
authors.lastname: Plappert-Helbig
authors.affiliation: Mitsubishi Tanabe Pharma Co., Chiba, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Uno
authors.affiliation: Helix3 Inc., RTP, NC, USA.
authors.forename: Marie
authors.initials: M
authors.lastname: Vasquez
authors.affiliation: Covance Laboratories Ltd, Harrogate, UK.
authors.forename: Carol
authors.initials: C
authors.lastname: Beevers
authors.affiliation: Janssen Research & Development, Beerse, Belgium.
authors.forename: Marlies
authors.initials: M
authors.lastname: De Boeck
authors.affiliation: Boehringer Ingelheim Pharmaceuticals Inc., Nonclinical Drug Safety, Ridgefield, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Escobar
authors.affiliation: Sumitomo Chemical Co. Ltd., Osaka, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kitamoto
authors.affiliation: BioReliance by SAFC, Rockville, MD, USA.
authors.forename: Kamala
authors.initials: K
authors.lastname: Pant
authors.affiliation: The Procter and Gamble Company, Global Product Stewardship, Mason, OH, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pfuhler
authors.affiliation: Public Interest Incorporated Foundation Biosafety Research Center, Shizuoka, Japan.
authors.forename: Jin
authors.initials: J
authors.lastname: Tanaka
authors.affiliation: Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD, USA.
authors.forename: Dan D
authors.initials: DD
authors.lastname: Levy
mesh_terms: D000818:Animals; D020552:Comet Assay / Q000379:methods* / Q000592:standards*; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004249:DNA Damage; D004493:Education; D006801:Humans
chemical_list: D004247:DNA
keywords: Genotoxicity testing; Laboratory proficiency; Test protocol; Tissue toxicity