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However, animal tests are time-consuming and can hinder the rapid development of drugs needed to combat urgent health crises, such as the COVID-19 pandemic. Therefore, faster non-animal alternatives are critical to accelerating preclinical toxicity assessments. Molnupiravir, an antiviral medication authorized for emergency use to treat COVID-19, is an oral pro-drug that is metabolized into its active form, N4-hydroxycytidine (NHC). The developmental toxicity of molnupiravir was initially identified in preclinical animal studies. The present study aims to determine whether in vitro assays using gastruloids\u2014three-dimensional aggregates of pluripotent stem cells that mimic axial elongation morphogenesis of early embryos\u2014can effectively detect the developmental toxicity of molnupiravir in a clinically relevant context. In our experiments, NHC at 20\u2009\u03bcM significantly impaired the morphological progression and altered the gene expression profiles in gastruloids derived from mouse P19C5 stem cells. Similarly, in a human embryonic stem cell-based morphogenesis model, NHC reduced the aggregate size at 10\u2009\u03bcM and induced significant gene expression changes at concentrations as low as 2.5\u2009\u03bcM. Notably, these NHC concentrations are comparable to the plasma levels observed in humans (approximately 10.8\u2009\u03bcM) following administration of the clinically recommended dose of molnupiravir. These findings demonstrate that gastruloid-based assays can reliably detect the developmental toxicity of NHC at clinically relevant concentrations, supporting their utility as non-animal tools for expediting preclinical developmental toxicity assessments.<\/jats:p>","DOI":"10.1093\/toxsci\/kfaf093","type":"journal-article","created":{"date-parts":[[2025,7,19]],"date-time":"2025-07-19T11:56:20Z","timestamp":1752926180000},"page":"74-90","source":"Crossref","is-referenced-by-count":2,"title":["Detection of developmental toxicity of the anti-COVID-19 drug molnupiravir using gastruloid-based in vitro assays"],"prefix":"10.1093","volume":"207","author":[{"given":"Margaret Carrell","family":"Huntsman","sequence":"first","affiliation":[{"name":"Department of Anatomy, Biochemistry and Physiology, Yanagimachi Institute for Biogenesis Research, University of Hawaii John A. Burns School of Medicine , Honolulu, HI 96813,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5720-5806","authenticated-orcid":false,"given":"Yusuke","family":"Marikawa","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Biochemistry and Physiology, Yanagimachi Institute for Biogenesis Research, University of Hawaii John A. Burns School of Medicine , Honolulu, HI 96813,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,7,21]]},"reference":[{"key":"2025091914020106000_kfaf093-B1","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1101\/gad.855001","article-title":"The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures","volume":"15","author":"Abu-Abed","year":"2001","journal-title":"Genes Dev"},{"key":"2025091914020106000_kfaf093-B2","doi-asserted-by":"publisher","first-page":"iyad031","DOI":"10.1093\/genetics\/iyad031","article-title":"The gene ontology knowledgebase in 2023","volume":"224","author":"Aleksander","year":"2023","journal-title":"Genetics"},{"key":"2025091914020106000_kfaf093-B3","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1007\/s12015-022-10462-5","article-title":"Gastruloids: a novel system for disease modelling and drug testing","volume":"19","author":"Amel","year":"2023","journal-title":"Stem Cell Rev Rep"},{"key":"2025091914020106000_kfaf093-B4","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1016\/j.ydbio.2022.05.002","article-title":"Gastruloids: pluripotent stem cell models of mammalian gastrulation and embryo engineering","volume":"488","author":"Arias","year":"2022","journal-title":"Dev Biol"},{"key":"2025091914020106000_kfaf093-B5","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1038\/75556","article-title":"Gene ontology: tool for the unification of biology. 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