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Regarding long-term toxicity, few studies have investigated the genotoxic potential of NSOs, and no genotoxic data at all are available for the subclass of Brorphine-like benzimidazolone opioids. To deepen and broaden our understanding of their toxicological profile, this study was aimed at evaluating the genotoxicity of Brorphine and four of its analogues (Orphine, Fluorphine, Chlorphine and Iodorphine) on human lymphoblastoid TK6 cells employing a flow cytometric protocol of the \u201cIn Vitro Mammalian Cell Micronucleus (MN) test\u201d. The results show a statistically significant MNi increase for Fluorphine, Chlorphine and Iodorphine, but not for Brorphine and Orphine, demonstrating for the first three the ability to induce chromosomal damage. Afterwards, Brorphine and Orphine were tested on TK6 cells also in the presence of an exogenous metabolic activation system (S9 mix) to consider the possible genotoxic hazard posed by their metabolites as well. Also, under this experimental condition, no statistically significant increase in the MNi frequency was detected.<\/jats:p>","DOI":"10.3390\/ijms26115084","type":"journal-article","created":{"date-parts":[[2025,5,26]],"date-time":"2025-05-26T02:50:05Z","timestamp":1748227805000},"page":"5084","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["New Synthetic Opioids: What Do We Know About the Mutagenicity of Brorphine and Its Analogues?"],"prefix":"10.3390","volume":"26","author":[{"given":"Monia","family":"Lenzi","sequence":"first","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\/0000-0003-1933-3377","authenticated-orcid":false,"given":"Sofia","family":"Gasperini","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabrine","family":"Bilel","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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2973-2446","authenticated-orcid":false,"given":"Giorgia","family":"Corli","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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9686-1848","authenticated-orcid":false,"given":"Francesca","family":"Rombol\u00e0","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\/0000-0002-8415-3711","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"},{"name":"Clinical Pharmacology Unit, IRCCS AOU Bologna, 40138 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, Presidency of the Council of Ministers, 00186 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,26]]},"reference":[{"key":"ref_1","unstructured":"European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) (2025, March 15). European Drug Report 2021: Trends and Developments. Publications Office of the European Union, Available online: https:\/\/www.euda.europa.eu\/publications\/edr\/trends-developments\/2021_en."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1093\/jat\/bkaa094","article-title":"First Report on Brorphine: The Next Opioid on the Deadly New Psychoactive Substance Horizon?","volume":"44","author":"Verougstraete","year":"2020","journal-title":"J. Anal. Toxicol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"8895","DOI":"10.1021\/acs.jmedchem.8b01136","article-title":"Optimization of a Series of Mu Opioid Receptor (MOR) Agonists with High G Protein Signaling Bias","volume":"61","author":"Kennedy","year":"2018","journal-title":"J. Med. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.1007\/s00228-021-03136-7","article-title":"Severe Rhabdomyolysis and Acute Kidney Failure Due to Synthetic Opioid Brorphine Exposure in Combination with Chronic Sertraline Therapy","volume":"77","author":"Razinger","year":"2021","journal-title":"Eur. J. Clin. Pharmacol."},{"key":"ref_5","unstructured":"United Nations Office on Drugs and Crime (UNODC) (2025, February 26). CND Decision on International Control of Brorphine and Metonitazene Enters into Force. Available online: https:\/\/www.unodc.org\/LSS\/Announcement\/Details\/a56e0bd9-0da5-4152-a34d-7cff7746bf50."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1093\/jat\/bkab082","article-title":"The Rise and Fall of Isotonitazene and Brorphine: Two Recent Stars in the Synthetic Opioid Firmament","volume":"46","author":"Vandeputte","year":"2022","journal-title":"J. Anal. Toxicol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1072","DOI":"10.1021\/acschemneuro.1c00037","article-title":"DARK Classics in Chemical Neuroscience: Etonitazene and Related Benzimidazoles","volume":"12","author":"Christie","year":"2021","journal-title":"ACS Chem. Neurosci."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Gasperini, S., Bilel, S., Cocchi, V., Marti, M., Lenzi, M., and Hrelia, P. (2022). The Genotoxicity of Acrylfentanyl, Ocfentanyl and Furanylfentanyl Raises the Concern of Long-Term Consequences. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232214406"},{"key":"ref_9","unstructured":"OECD (2023). Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Publishing."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Basu, A.K. (2018). DNA Damage, Mutagenesis and Cancer. Int. J. Mol. 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