Recommendations for design of the rat comet assay

Abstract

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.

Review of genotoxicity studies of glyphosate and glyphosate-based formulations

“…The latter are thought to represent dead or dying cells severely damaged by cytotoxicity. While ''hedgehogs'' are usually not included in tabulation of comet effects, they may be used as an additional measure of toxic effects (Smith et al, 2008).…”

The influence of the number of cells scored on the sensitivity in the comet assay

“…For in vivo samples the optimal number of cells scored could be different and recommendations for designing the comet assay with samples from rats, including experimental design issues, have been reported [11]. The comet assay has become an important tool for assessing DNA damage in exposed populations and it is the method of choice for population-based studies of environmental and occupational exposure to pollutants [2].…”

“…Several studies have reported the use of automated scoring, both with in-house systems and with commercially available equipment [5][6][7][8][9]. However, the impact on the sensitivity of the assay by increasing the number of cells scored has not yet been fully investigated and only a few studies have addressed the issue [6,10,11]. The aim of this study was to investigate if the assay sensitivity could be improved by increasing the number of cells scored in independent samples, compared with the standard number of 100 cells per sample.…”

Genotoxicity evaluation of chlorpyrifos: a gender related approach in regular toxicity testing

“…However, the procedure was slightly modified according the recommendation of Smith et al (2008). The 100 μl of HMP agarose 0.9% was placed on fully frosted slides.…”