A crosslinking positive control for any in vivo comet assay tissue

OECD TG 489 recognizes that some modifications of the in vivo comet assay (e.g., longer electrophoresis times) can detect DNA crosslink inductions expressed as DNA migration decreases. But the application of these methods in regulated safety testing has been limited because identifying an in vivo crosslinking positive control for every tissue that can be evaluated with the comet assay can be very challenging. Most crosslinking agents induce crosslinks in just one tissue (e.g., site of contact) with the agent's toxicity limiting exposure and effects induced in other tissues. This complication can require different positive control compounds with additional animals for each compound to adequately evaluate crosslinks in the multiple tissues typically evaluated with the comet assay. Our objective was to develop a method for generating concurrent in vivo crosslinking positive control comet data for any tissue without using additional animals. We accomplished this by preparing for each tissue evaluated extra comet slides from the conventional positive control (EMS) dosed animals and ex vivo crosslinking the heavily damaged cells on these slides with 10% neutral buffered formalin (NBF) immediately before lysis. These ex vivo crosslinked slides were then electrophoresed with the rest of the study slides with the same standard conditions and electrophoresis time. The conventional comet study slides prepared from the liver, duodenum, glandular stomach, and lung tissues collected from the EMS dosed animals and lysed without ex vivo crosslinking expressed a statistically significant DNA migration increase when compared to the concurrent vehicle control dose group. The ex vivo crosslinked slides from the EMS dose group tissue samples expressed a statistically significant decrease in DNA migration when compared to the concurrent vehicle control group. Although no statistical analysis was conducted, it is clear by the data that the ex vivo crosslinked slides from the EMS dose group tissue samples also expressed a significant decrease compared to the same samples from the EMS dose group without the ex vivo NBF treatment. This data demonstrate that the method can be easily incorporated into any existing comet study design to evaluate crosslink induction in any comet assay tissue with minimal effort or cost and without the use of any additional animals.