Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation

Abstract

Despite regulatory directives requiring the reduction of animal use in safety testing, recent modifications to genotoxicity testing guidelines now propose the use of two in vivo genotoxicity assays as a follow-up to an in vitro positive (International Conference on Harmonization Consensus Draft Guidance S2[R1] released March, 2008). To address both goals, the in vivo comet and micronucleus (MN) assays can be successfully combined into one informative study. Combining these two assays with such differences in sensitivity, endpoints measured and the type of data generated significantly improves upon the current standard capabilities for detecting genotoxicity without requiring additional animals. But to take full advantage of the benefits of incorporating the comet assay in safety testing, these same differences must be recognized and considered. Developed from over 15 years experience using the in vivo comet and MN assays in genotoxicity testing of chemicals and pharmaceuticals, this paper presents guidelines for the appropriate experimental design, dose selection and data interpretation for combined in vivo comet/MN assay studies. To illustrate the approach, data from combined assay studies are presented and discussed.

Fig. 1

The progression of cytotoxicity. Before cellular swelling and/or condensation are visible by gross or microscopic examination of a tissue, the earliest detectable measurements of cell death can include an increase in the percentage of individual cells with LMW DNA diffusion and/or evidence of depleted glycogen (e.g. decreased cytoplasmic pallor) in tissue sections. With the lysis of the necrotic cell membranes, more advanced LMW DNA diffusion, increased plasma enzyme levels and/or cytoplasmic eosinophilia (i.e. inflammation) in the tissue may be detected. However, the stage at which tissue necrosis/apoptosis and post-necrotic effects (e.g. compensatory hyperplasia/hypertrophy) can be detected by histopathology is too advanced to be detected by the LMW DNA diffusion assay as complete digestion and clearance of the individual dead cells has occurred.

Fig. 2

Dose-related decreases in DNA migration and LMW DNA diffusion in response to cytotoxicity of pharmaceutical tested up to the MTD. For viewing on same graph scale with LMW data, OTM values were multiplied by 3. Star indicates statistically significant increase at P < 0.05.

Fig. 3

Hormetic (∩ shaped) dose response curve of pharmaceutical tested in the comet assay. For viewing the same graph scale with LMW data, OTM values were multiplied by 3. Star indicates statistically significant increase at P < 0.05.

Fig. 4

Possible dose response curves in the comet assay. While the common assumption is that genotoxicity is most likely to be detected at doses immediately below the MTD (- - -), the genotoxic effect as well as the confounding effects of tissue cytotoxicity (―) and even very steep dose response curves (….) can be detected in the comet assay at doses significantly lower than the MTD.

Fig. 5

Histopathology of glycogen depletion in the liver. Haematoxylin- and eosin-stained normal liver from the vehicle control animals (a) compared to the liver with depleted glycogen from animals dosed with ≥50 mg/kg of EMS (b).