The JaCVAM / OECD activities on the comet assay
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring DNA strand breaks in eukaryotic cells. This assay was adopted in the Organisation for Economic Co-operation and Development (OECD) Test guideline (TG) 489 on September 26, 2014. This TG is part of a series of TGs on genetic toxicology.
A formal validation trial of the this assay was performed in 2006-2012, coordinated by the Japanese Center for the Validation of Alternative Methods (JaCVAM), in conjunction with the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM), the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) . The assay was reviewed by the OECD genotoxicity experts based on the JaCVAM trial (2014) and in Rothfuss et al. (2010). This TG includes the recommended use and limitations of the comet assay, and is based on the final protocol used in the validation trial, and on additional relevant published and unpublished (laboratories proprietary) data.
The outline of this TG describes below: each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate). A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified. Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size DNA fragments migrate away from the head to the tail, and the intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage.
Acknowledgements
On behalf of the comet assay international validation management team, I appreciate the all collaborators and supporters involved with adapting TG 489. This validation study was supported by the MHLW funding in Japan.
References
OECD (2014), In Vivo Mammalian Alkaline Comet Assay, OECD Guidelines for the Testing of Chemicals, No.489, OECD Publishing, Paris.
OECD (2014), Reports of the JaCVAM initiative international pre-validation and validation studies of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens, Series on Testing and Assessment, Nos. 195 and 196, OECD Publishing, Paris.
Rothfuss, A. et al. (2010), Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2-and 4-week repeat-dose studies, Mutation Research, Vol., 702/1, pp. 40-69.
Keywords:
Comet Assay,
in vivo,
Validation study,
OECD,
Test guideline
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Oral Presentation
Topic:
The comet assay in Regulatory Toxicology
Citation:
Kojima
H
(2015). The JaCVAM / OECD activities on the comet assay.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00049
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Received:
30 Apr 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
PhD. Hajime Kojima, NIHS, JaCVAM, Tokyo, 158-8501, Japan, h-kojima@nihs.go.jp