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.