Qualification of the human Reconstructed Intestine Micronuclei Cytome assay for site-of-contact genotoxic hazard identification

• First in vitro assay to address site-of-contact genotoxic hazard identification for oral or GI route of exposure. • Assay acceptance criteria have been established to determine/ classify genotoxic status of test articles. • Assay accuracy established for genotoxic risk identification. • Can potentially reduce the unnecessary usage of animal experimentation once sufficiently validated. While valuable for hazard identification, in vitro genotoxicity tests that are conducted in 2D monolayer cell cultures possess limitations to accurately envisage in vivo outcomes and may result in unnecessary follow-up in vivo studies. One limitation is their relevance to actual human exposure and the assessment of genotoxicity in tissues representative of “sites-of-first-contact” with genotoxic agents. The revised OECD testing guidelines had emphasized the need for considering site-of-first-contact effect when carrying out genotoxic hazard investigations. To date, only in vivo studies can adequately address the oral/gastrointestinal route of exposure and there exists no validated in vitro assay for ingestible substances. Previously, we described the development of a new human Reconstructed Intestine Micronuclei Cytome (RICyt) assay using EpiIntestinal™ microtissues for site-of-contact genotoxic hazard identification of orally ingested materials. The human small intestine is where approximately 90 % of the digestion and adsorption of food occurs and a major exposure site of ingested genotoxicants, thus a key site for evaluation. Here, we have investigated on the qualification of the RICyt assay in genotoxic hazard identification. The assay platform was challenged with a training set of 16 test materials which include standard reference genotoxins, non-genotoxins and food relevant substances with various mode of actions. The overall RICyt accuracy was 93.3 % with sensitivity of 83.3 % and specificity of 100 %. Majority of the test materials were correctly identified though yielded one false negative (KBrO 3 ) and one equivocal outcome (EUG). MCT, a known genotoxin, with no known effect on the GI tract, did not induce significant MN, showcasing the ability of the assay to detect tissue specific genotoxicity. These results emphasize that the RICyt assay is promising in vitro tool for genotoxic hazard identification of orally ingested substances.