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Among New Psychoactive Substances, nitazenes are potent non-fentanyl opioids associated with severe cases of intoxication. This study evaluated the genotoxic potential of metodesnitazene and etodesnitazene in the human TK6 cell line. Cells were exposed to increasing concentrations of studied compounds, with and without S9 metabolic activation system. Preliminary assessments and micronuclei frequency analyses were performed by flow cytometry in at least three independent experiments. Metodesnitazene induced an increase in micronuclei frequency starting from 12.5 \u03bcM (p &lt; 0.05), whereas etodesnitazene induced an effect only at 50 \u03bcM. Metabolic activation increases micronuclei formation at higher concentrations of metodesnitazene 25 \u03bcM, but did not substantially affect the response to etodesnitazene. Both compounds also induced intracellular reactive oxygen species production, measured through a chemiluminescent-based bioassay, suggesting oxidative stress as a potential contributing mechanism. These findings highlight the need for compound-specific toxicological profiling to better anticipate the acute and long-term risks associated with nitazene consumption.<\/jats:p>","DOI":"10.3390\/ijms27125360","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T08:57:58Z","timestamp":1781513878000},"page":"5360","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genotoxic Potential of Metodesnitazene and Etodesnitazene: Insights with and Without S9 Metabolic Activation"],"prefix":"10.3390","volume":"27","author":[{"given":"Francesca","family":"Rombol\u00e0","sequence":"first","affiliation":[{"name":"Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dalila","family":"Maurizzi","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2085-2334","authenticated-orcid":false,"given":"Alessia","family":"Silla","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6740-5331","authenticated-orcid":false,"given":"Cristiana","family":"Caliceti","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabrine","family":"Bilel","sequence":"additional","affiliation":[{"name":"Department of Environmental and Prevention Sciences, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8415-3711","authenticated-orcid":false,"given":"Patrizia","family":"Hrelia","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0349-7772","authenticated-orcid":false,"given":"Marco","family":"Malaguti","sequence":"additional","affiliation":[{"name":"Department for Life Quality Studies, Alma Mater Studiorum University of Bologna, 47921 Rimini, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monia","family":"Lenzi","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8751-2882","authenticated-orcid":false,"given":"Matteo","family":"Marti","sequence":"additional","affiliation":[{"name":"Department of Translational Medicine, Section of Legal Medicine, LTTA Center and University Center of Gender Medicine, University of Ferrara, 44121 Ferrara, Italy"},{"name":"Collaborative Center for the Italian National Early Warning System (NEWS-D), Department of Anti-Drug Policies and Other Addictions, Presidency of the Council of Ministers, 00186 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/s40199-025-00572-z","article-title":"New Psychoactive Substances: A Multidisciplinary Review of Challenges and Their Diverse Character","volume":"33","author":"Marandiuc","year":"2025","journal-title":"DARU J. 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