Developing an in vitro repeat-dose test as a 3Rs approach to detect non-genotoxic carcinogens

Currently there are no validated in vitro tests to detect non-DNA reative (non-genotoxic) carcinogens. Consequently, the 2-year rodent carcinogenicity test is the only validated approach to detect non-genotoxic carcinogens (NGC's). Indeed the continued use of the 2-year rodent bioassay, in an era when in vitro tests for genotoxicity are showing much promise, is often justified due to the need to detect these NGC's, which account for about 10-20% of carcinogens tested. About 12,000 such tests (severe by definition) are carried out in Europe annually.

The mechanisms behind NGC's carcinogenicity are varied, but often involve inducing long-term genome instability, indeed no cancers form without mass mutation induction. For example NGC's are said to promote proliferation, drive mitogenic signalling, cause DNA methylation abnormalities, abrogate DNA repair and induce oxidative stress, leading "indirectly" to long-term mutational events, not detectable in standard short-term tests. Therefore, given their ability to modify aspects of genome stability, we hypothesised that they should be detectable with modified in vitro approaches aimed at identifying DNA damage. We have now shown exactly that, i.e. using a modified "repeat dose" in vitro system for DNA damage detection with TK6 cells, we can detect DNA damage induced by several classic NGC's. Specifically, Nickel Chloride and Diethyl(Hexyl)phthalate (DEHP) are positive for DNA damage induction in vitro using a repeat-dose (5 day) exposure system coupled to micronucleus detection. We now urgently need to better understand;

1. How generalisable is this phenomenon? - by testing further NGC's?

2. What are the specific mechanisms behind the DNA damage induced with repeat dosing.

By developing a novel in vitro testing approach for NGC's, we can remove a major barrier to replacing the 2-year rodent bioassay. This will have a major effect on reducing the numbers of animals used in safety testing in the years to come (12,000 per year in Europe).