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Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D\u00a0reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.<\/jats:p>","DOI":"10.1007\/s00204-022-03228-y","type":"journal-article","created":{"date-parts":[[2022,2,28]],"date-time":"2022-02-28T08:02:43Z","timestamp":1646035363000},"page":"1455-1471","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment\u00a0of orally ingested substances"],"prefix":"10.1007","volume":"96","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3510-5334","authenticated-orcid":false,"given":"Hui Kheng","family":"Lim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher Owen","family":"Hughes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle Jing Sin","family":"Lim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia\u2019En Jasmine","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moumita","family":"Rakshit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Calvin","family":"Yeo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kern Rei","family":"Chng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angela","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanne Sheot Harn","family":"Chan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kee Woei","family":"Ng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David Ian","family":"Leavesley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin Paul Chapman","family":"Smith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,2,28]]},"reference":[{"issue":"2","key":"3228_CR1","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/j.mrgentox.2010.05.017","volume":"701","author":"MJ Aardema","year":"2010","unstructured":"Aardema MJ, Barnett BC, Khambatta Z, Reisinger K, Ouedraogo-Arras G, Faquet B et al (2010) International prevalidation studies of the EpiDerm\u2122 3D human reconstructed skin micronucleus (RSMN) assay: transferability and reproducibility. 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