Assessment of the genotoxicity of tert-butyl alcohol in an in vivo thyroid comet assay.

Chronic exposure to high (20,000 ppm) concentrations of tert-butyl alcohol (TBA) in drinking water, equivalent to ~2100 mg/kg bodyweight per day, is associated with slight increases in the incidence of thyroid follicular cell adenomas and carcinomas in mice, with no other indications of carcinogenicity. In a recent toxicological review of TBA, the U.S. EPA determined that the genotoxic potential of TBA was inconclusive, largely based on non-standard studies such as in vitro comet assays. As such, the potential role of genotoxicity in the mode of action of thyroid tumors and therefore human relevance was considered uncertain. To address the potential role of genotoxicity in TBA-associated thyroid tumor formation, CD-1 mice were exposed up to a maximum tolerated dose of 1500 mg/kg-day via oral gavage for two consecutive days and DNA damage was assessed with the comet assay in the thyroid. Blood TBA levels were analyzed by headspace GC-MS to confirm systemic tissue exposure. At study termination, no significant increases (DNA breakage) or decreases (DNA crosslinks) in %DNA tail were observed in TBA exposed mice. In contrast, oral gavage of the positive control ethyl methanesulfonate significantly increased %DNA tail in the thyroid. These findings are consistent with most genotoxicity studies on TBA and provide mechanistic support for non-linear, threshold toxicity criteria for TBA. While the mode of action for the thyroid tumors remains unclear, linear low dose extrapolation methods for TBA appear more a matter of policy than science.

Reducing risk of false positives in the in vivo comet assay and improving result reliability.

The risk of generating false positive in vivo comet assay results can be increased when procedural bias and/or technical variability is poorly controlled. This has been an ongoing concern since comet was first introduced into regulatory safety testing. But the proprietary nature of regulated studies and the 3Rs have limited the ability to conduct and publish the comparative in vivo studies necessary to determine the effect these factors can have on comet assay results when substances other than well characterized positive control compounds are evaluated in multiple tissues. That changed when Helix3 was asked to repeat for regulatory submission three independent in vivo comet studies with positive results generated by three other laboratories evaluating the effects of three different test substances on the liver, duodenum, and stomach. We repeated each study using the same test substance and experimental design as the original labs but with our standard quality control methods implemented to reduce procedural bias and variability. In every case, we generated negative results that regulatory authorities accepted over the initial positive results due to evidence of high technical variability and procedural bias in the original labs and studies. Meanwhile, the International Workshop on Genotoxicity (IWGT) compared >14 years of Helix3 comet historical control data (HCD) to HCD from 6 other experienced comet laboratories and concluded that our data exhibited the highest overall background % tail DNA levels with the lowest inter-study variability resulting in the highest quality HCD of all the labs evaluated. These case studies and the IWGT report suggest that our enhanced quality control methods and higher (>2 % mean of slide median tail DNA) background levels can effectively mitigate the nuisance factors that can generate false positive in vivo comet assay results. To facilitate a better understanding of the technical parameters that can significantly influence the comet results, we describe our enhanced procedures with justifications and examples.

Recommendations for safety testing with the in vivo comet assay.

While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data.

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

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.