The comet assay – how to recognise “good data”
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1
Huntingdon Life Sciences, Genetic Toxicology, United Kingdom
Testing of potentially genotoxic materials currently involves use of in vitro assays such as the Ames test (gene mutations), the chromosome aberration assay and in vitro micronucleus assay (predominantly using human peripheral lymphocytes to detect clastogenicity and/or aueuploidy) and the mouse lymphoma assay (gene mutations). In addition, one in vivo assay, predominately the in vivo micronucleus assay, is “normally” required to satisfy regulatory testing requirements and on occasion a second in vivo assay is required to assist in interpretation of results. Following release of the OECD 489 guideline “In vivo mammalian alkaline comet assay” in September 2014 the in vivo comet assay is now considered to be the second in vivo genetic toxicology assay of choice, effectively replacing the use of the UDS assay.
The comet assay can be used to detect single strand and double strand breaks by measuring the median %tail intensity under alkaline conditions (pH>13). Following the JaCVAM validation trial, subsequent publication of the OECD 489 guideline recommended using the liver (site of metabolism) and glandular stomach (site of contact) of rats. However, any tissue can be examined if experimental competency with the tissue of interest has been proven. A number of key areas need careful consideration when performing the assay and interpreting the data. Both duration and temperature of tissue preparation have been shown to be critical parameters for obtaining “good data” along with the correct electrophoresis conditions to detect ‘weak’ effects. Study design requires careful thought in terms of animals per group, slides analysed per animal and cells analysed per slide and to facilitate this a special interest group within industry has published recommendations on statistical analysis of the comet assay. The sensitivity of the comet assay within the laboratory can be determined with the use of ‘power curves’ and identifying a high sensitivity could consequently allow a reduction in the use of animals. Furthermore, the OECD 489 guideline stipulates the use of control charts (c-charts or X-bar charts) to monitor the temporal drift of historical control data enabling the laboratory to ensure that the experimental methodology employed is under control.
Keywords:
Comet,
JaCVAM,
Tail Intensity,
Control charts,
Power curves
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:
Barfield
W
(2015). The comet assay – how to recognise “good data”.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00054
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Received:
08 May 2015;
Published Online:
23 Jun 2015.
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Correspondence:
Mr. William Barfield, Huntingdon Life Sciences, Genetic Toxicology, Huntingdon, Cambridgeshire, PE28 4HS, United Kingdom, barfielw@ukorg.huntingdon.com