{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T17:34:57Z","timestamp":1791999297006,"version":"4.4.0"},"reference-count":83,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"22","content-domain":{"domain":["www.science.org"],"crossmark-restriction":true},"short-container-title":["Sci. Adv."],"published-print":{"date-parts":[[2026,5,29]]},"abstract":"<jats:p>\n                    The detection of\n                    <jats:italic toggle=\"yes\">N<\/jats:italic>\n                    -nitrosamines in pharmaceuticals has been a focus for industry and regulatory authorities since 2018. This has prompted extensive research into their mutagenic potential and mitigation strategies. We present a comprehensive evaluation of 25 structurally diverse\n                    <jats:italic toggle=\"yes\">N<\/jats:italic>\n                    -nitrosamines using in vitro (Ames test), in vivo (liver comet, Big Blue transgenic mutation, and duplex sequencing), and in silico methods [quantum mechanical modeling and cytochrome P450 (CYP)\u2013binding analysis]. Results show strong concordance across assays and reveal that complex active pharmaceutical ingredient (API)\u2013derived or process-related impurity (PRI)\u2013derived\n                    <jats:italic toggle=\"yes\">N<\/jats:italic>\n                    -nitrosamines are often not mutagenic or have low mutagenic potency. Quantitative in vivo data enabled the derivation of compound-specific acceptable intakes (AIs), frequently exceeding regulatory limits based on an assessment of structural features. Duplex sequencing demonstrated superior sensitivity over traditional assays, and quantum mechanical models proved effective for potency prediction. A weight-of-evidence decision tree to derive AIs that integrates experimental and computational data is proposed, which reduces animal testing and supports drug product remediation efforts that are commensurate with risk.\n                  <\/jats:p>","DOI":"10.1126\/sciadv.aee8680","type":"journal-article","created":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T18:02:22Z","timestamp":1779904942000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":1,"title":["Complex versus simple\n                    <i>N<\/i>\n                    -nitrosamines: Comprehensive genotoxicity and in silico carcinogenicity assessment toward future testing paradigms"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-5001-4632","authenticated-orcid":true,"given":"Xiaowen","family":"Sun","sequence":"first","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0189-0873","authenticated-orcid":true,"given":"Maik","family":"Schuler","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0169-4078","authenticated-orcid":true,"given":"Shaofei","family":"Zhang","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9727-0477","authenticated-orcid":true,"given":"Jakub","family":"Kostal","sequence":"additional","affiliation":[{"name":"DOT Consulting LLC, 2121 Eisenhower Ave Ste 301, Alexandria, VA 22314, USA."},{"name":"The George Washington University, 800 22nd ST NW, Washington, DC 20052, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer R.","family":"Cheung","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-1739-9433","authenticated-orcid":true,"given":"Michelle","family":"Kenyon","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1211-0229","authenticated-orcid":true,"given":"Eric","family":"Watt","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7129-1210","authenticated-orcid":true,"given":"Krista","family":"Dobo","sequence":"additional","affiliation":[{"name":"Pfizer Research, Development, and Medical, Groton, CT 06340, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_2_2_2","unstructured":"European Medicines Agency \u201cQuestions and answers for marketing authorisation holders\/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726\/2004 referral on nitrosamine impurities in human medicinal products\u201d (2025)."},{"key":"e_1_3_2_3_2","unstructured":"Health Canada \u201cNitrosamine impurities in medications: Guidance\u201d (2025)."},{"key":"e_1_3_2_4_2","unstructured":"U.S. Food and Drug Administration \u201cControl of nitrosamine impurities in human drugs guidance for industry\u201d (2024)."},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2019.01.007"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1208\/s12249-023-02523-w"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsps.2022.12.010"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.oprd.5c00097"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.comtox.2021.100186"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.2c00199"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.1c00369"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.3c00083"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.2c00217"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.4c00133"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.2c00380"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.4c00087"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2024.1415266"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.4c00482"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105640"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105762"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.oprd.3c00100"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.xphs.2023.10.001"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2025.105835"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105672"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00204-025-04103-2"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23094559"},{"key":"e_1_3_2_27_2","first-page":"241","article-title":"Mechanisms of action of N-nitroso compounds","volume":"8","author":"Archer M. C.","year":"1989","unstructured":"M. C. Archer, Mechanisms of action of N-nitroso compounds. Cancer Surv. 8, 241\u2013250 (1989).","journal-title":"Cancer Surv."},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1021\/tx980005y"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1021\/tx0502037"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.70007"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105681"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.molpharmaceut.4c01173"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2023.105410"},{"key":"e_1_3_2_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105709"},{"key":"e_1_3_2_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105730"},{"key":"e_1_3_2_36_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2025.105906"},{"key":"e_1_3_2_37_2","unstructured":"OECD \u201cTest no. 471: Bacterial reverse mutation test OECD Guidelines for the Testing of Chemicals Section 4\u201d (2020)."},{"key":"e_1_3_2_38_2","unstructured":"OECD \u201cTest no. 489: In vivo mammalian alkaline comet assay OECD Guidelines for the Testing of Chemicals Section 4\u201d (2016)."},{"key":"e_1_3_2_39_2","unstructured":"OECD \u201cTest no. 488: Transgenic rodent somatic and germ cell gene mutation assays OECD Guidelines for the Testing of Chemicals Section 4\u201d (2022)."},{"key":"e_1_3_2_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2025.105985"},{"key":"e_1_3_2_41_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.70006"},{"key":"e_1_3_2_42_2","doi-asserted-by":"crossref","unstructured":"J. Kostal \u201cQuantum mechanics approaches in computational toxicology\u201d in Computational Toxicology: Risk Assessment for Chemicals (John Wiley & Sons 2018) pp. 31\u201368.","DOI":"10.1002\/9781119282594.ch2"},{"key":"e_1_3_2_43_2","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21334"},{"key":"e_1_3_2_44_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.1c00203"},{"key":"e_1_3_2_45_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.8b00545"},{"key":"e_1_3_2_46_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.9b00778"},{"key":"e_1_3_2_47_2","doi-asserted-by":"publisher","DOI":"10.3390\/app9214538"},{"key":"e_1_3_2_48_2","doi-asserted-by":"publisher","DOI":"10.1016\/0027-5107(77)90151-8"},{"key":"e_1_3_2_49_2","doi-asserted-by":"publisher","DOI":"10.1016\/0027-5107(83)90058-1"},{"key":"e_1_3_2_50_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.toxrep.2024.01.012"},{"key":"e_1_3_2_51_2","unstructured":"International Council for Harmonisation (ICH) \u201cFinal Concept Paper Addendum to ICH M7 on Risk Assessment and Control of N-Nitrosamine Impurities\u201d (2025)."},{"key":"e_1_3_2_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2020.104749"},{"key":"e_1_3_2_53_2","unstructured":"International Council for Harmonisation (ICH) Assessment And Control Of DNA Reactive (Mutagenic) Impurities In Pharmaceuticals To Limit Potential Carcinogenicrisk M7(R2) (2023)."},{"key":"e_1_3_2_54_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2014.10.008"},{"key":"e_1_3_2_55_2","first-page":"e04658","article-title":"Update: Use of the benchmark dose approach in risk assessment","volume":"15","author":"Committee E. S.","year":"2017","unstructured":"E. S. Committee, A. Hardy, D. Benford, T. Halldorsson, M. J. Jeger, K. H. Knutsen, S. More, A. Mortensen, H. Naegeli, H. Noteborn, C. Ockleford, A. Ricci, G. Rychen, V. Silano, R. Solecki, D. Turck, M. Aerts, L. Bodin, A. Davis, L. Edler, U. Gundert-Remy, S. Sand, W. Slob, B. Bottex, J. C. Abrahantes, D. C. Marques, G. Kass, J. R. Schlatter, Update: Use of the benchmark dose approach in risk assessment. EFSA J. 15, e04658 (2017).","journal-title":"EFSA J."},{"key":"e_1_3_2_56_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.22446"},{"key":"e_1_3_2_57_2","first-page":"32","article-title":"The benchmark dose (BMD) approach for analysis and interpretation of polycyclic aromatic hydrocarbon (PAH) and PAH mixture mutagenicity dose-response data","volume":"65","author":"White P. A.","year":"2024","unstructured":"P. A. White, H. L. Battaion, A. S. Long, The benchmark dose (BMD) approach for analysis and interpretation of polycyclic aromatic hydrocarbon (PAH) and PAH mixture mutagenicity dose-response data. Environ. Mol. Mutagen. 65, 32\u201333 (2024).","journal-title":"Environ. Mol. Mutagen."},{"key":"e_1_3_2_58_2","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gew009"},{"key":"e_1_3_2_59_2","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/geae001"},{"key":"e_1_3_2_60_2","unstructured":"International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) \u201cImpurities in New Drug Products Q3B(R2)\u201d (2006)."},{"key":"e_1_3_2_61_2","doi-asserted-by":"crossref","unstructured":"T. K. Rao \u201cStructural basis for mutagenic activity of N-nitrosamines in the Salmonella histidine reversion assay\u201d in Genotoxicology of N-Nitroso Compounds (Springer 1984) pp. 45\u201358.","DOI":"10.1007\/978-1-4684-4595-4_3"},{"key":"e_1_3_2_62_2","doi-asserted-by":"publisher","DOI":"10.1093\/carcin\/13.10.1789"},{"key":"e_1_3_2_63_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrestox.3c00327"},{"key":"e_1_3_2_64_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.22618"},{"key":"e_1_3_2_65_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2016.10.001"},{"key":"e_1_3_2_66_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00204-025-04121-0"},{"key":"e_1_3_2_67_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00204-023-03527-y"},{"key":"e_1_3_2_68_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2023.503685"},{"key":"e_1_3_2_69_2","doi-asserted-by":"publisher","DOI":"10.1186\/s41021-023-00288-z"},{"key":"e_1_3_2_70_2","doi-asserted-by":"publisher","DOI":"10.1186\/s41021-025-00336-w"},{"key":"e_1_3_2_71_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.22615"},{"key":"e_1_3_2_72_2","doi-asserted-by":"publisher","DOI":"10.1002\/em.70012"},{"key":"e_1_3_2_73_2","doi-asserted-by":"publisher","DOI":"10.1124\/dmd.114.059717"},{"key":"e_1_3_2_74_2","doi-asserted-by":"publisher","DOI":"10.1002\/minf.201300132"},{"key":"e_1_3_2_75_2","doi-asserted-by":"publisher","DOI":"10.1002\/qsar.200390007"},{"key":"e_1_3_2_76_2","doi-asserted-by":"publisher","DOI":"10.1002\/minf.201000061"},{"key":"e_1_3_2_77_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1093-3263(01)00123-1"},{"key":"e_1_3_2_78_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00214-003-0518-3"},{"key":"e_1_3_2_79_2","doi-asserted-by":"publisher","DOI":"10.1002\/1873-3468.13489"},{"key":"e_1_3_2_80_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12860-019-0218-z"},{"key":"e_1_3_2_81_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2023.105415"},{"key":"e_1_3_2_82_2","doi-asserted-by":"publisher","DOI":"10.2903\/j.efsa.2023.7884"},{"key":"e_1_3_2_83_2","unstructured":"U.S. Food and Drug Administration \u201cCDER nitrosamine impurity acceptable intake limits\u201d (2026)."},{"key":"e_1_3_2_84_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yrtph.2024.105704"}],"container-title":["Science Advances"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/sciadv.aee8680","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T18:03:26Z","timestamp":1779905006000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/sciadv.aee8680"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,29]]},"references-count":83,"journal-issue":{"issue":"22","published-print":{"date-parts":[[2026,5,29]]}},"alternative-id":["10.1126\/sciadv.aee8680"],"URL":"https:\/\/doi.org\/10.1126\/sciadv.aee8680","relation":{},"ISSN":["2375-2548"],"issn-type":[{"value":"2375-2548","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,29]]},"assertion":[{"value":"2025-12-18","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-04-07","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-27","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"eaee8680"}}