{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:59:00Z","timestamp":1790441940667,"version":"4.1.0"},"reference-count":0,"publisher":"ALTEX Edition","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["ALTEX"],"abstract":"<jats:p>Cytochrome P450 (CYP)-mediated biotransformation of endogenous and xenobiotic substances can lead to altered exposure, toxicological impact, or adverse drug reactions. CYP induction data are fundamental to regulatory chemical hazard assessment because they directly affect the in vivo fate of xenobiotics, potentially influencing the safety and efficacy of pharmaceuticals, and impacting the safety assessment of industrial chemicals and environmental contaminants. Here we report the results on part 2 of the supplementary validation study of an established and previously validated in vitro method using human HepaRGTM cells able to detect CYP1A2, CYP2B6 and CYP3A4 induction to support the expansion of the method\u2019s chemical applicability domain beyond pharmaceuticals. This study addresses needs arising from part 1 of the study. We established the test method using ten previously validated pharmaceutical proficiency chemicals, then tested a further six proposed augmentation chemicals, tebuconazole, benfuracarb, chlorpyrifos, N, N-diethyl-meta-toluamide, fipronil, and permethrin (supporting part 1), and then four additional chemicals, prochloraz, atrazine, pyrimethanil and chlorpyrifos-methyl. LC-MS\/MS was utilized to measure the conversion of a cocktail mixture of prototypical selective CYP probe substrates to their metabolites, in parallel with mRNA measurements. We achieved high concordance with predicted activity for the proficiency and augmentation chemicals for CYP1A2 and CYP3A4, though with greater uncertainty for CYP2B6. Together with the part 1 study, these data were sufficient to support acceptance of the HepaRG\u2122 method for CYP induction testing and its adoption as OECD TG 445A. Tentative comparisons with mRNA based measurements suggested gene expression may serve as a complementary but not validated tool.\u00a0\nPlain language summaryCytochrome P450 (CYP) enzymes break down drugs, pesticides and other chemicals in the liver. Increases or decreases in CYP activity caused by certain chemicals can change internal levels of other chemicals, which can cause toxicity or impact drug effectiveness. Reliable non-animal methods are needed to identify chemicals with this activity for chemical safety assessment. This study describes results from a replacement second laboratory applying a previously validated human HepaRG\u2122 cell\u2011based method to assess induction of CYP1A2, CYP2B6, and CYP3A4 by ten reference pharmaceutical compounds and ten industrial chemicals. CYP induction was evaluated by measuring enzyme activity. These results strengthen confidence in the method\u2019s robustness and wider applicability and have supported its adoption as an OECD Test Guideline. The method showed a high level of agreement with expected outcomes particularly for CYP1A2 and CYP3A4. Additional exploration of changes in gene expression supported enzyme activity results but was not validated here.<\/jats:p>","DOI":"10.14573\/altex.2606132","type":"journal-article","created":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:27:06Z","timestamp":1790440026000},"source":"Crossref","is-referenced-by-count":0,"title":["HepaRG&lt;sup&gt;TM &lt;\/sup&gt;CYP induction test method augmentation part 2: Study supported by mRNA analysis"],"prefix":"10.14573","author":[{"given":"Emma","family":"Quartermain","sequence":"first","affiliation":[{"name":"UK Health Security Agency, Dept of Toxicology, Radiation, Chemical, Climate and Environmental Hazards (RCCE), Harwell Campus, Chilton, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinkang","family":"Zhang","sequence":"additional","affiliation":[{"name":"UK Health Security Agency, Dept of Toxicology, Radiation, Chemical, Climate and Environmental Hazards (RCCE), Harwell Campus, Chilton, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tim","family":"Marczylo","sequence":"additional","affiliation":[{"name":"UK Health Security Agency, Dept of Toxicology, Radiation, Chemical, Climate and Environmental Hazards (RCCE), Harwell Campus, Chilton, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy W.","family":"Gant","sequence":"additional","affiliation":[{"name":"UK Health Security Agency, Dept of Toxicology, Radiation, Chemical, Climate and Environmental Hazards (RCCE), Harwell Campus, Chilton, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4858-0118","authenticated-orcid":false,"given":"Miriam N.","family":"Jacobs","sequence":"additional","affiliation":[{"name":"UK Health Security Agency, Dept of Toxicology, Radiation, Chemical, Climate and Environmental Hazards (RCCE), Harwell Campus, Chilton, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"5675","published-online":{"date-parts":[[2026,9,25]]},"container-title":["ALTEX - Alternatives to Animal Experimentation"],"original-title":[],"link":[{"URL":"https:\/\/www.altex.org\/index.php\/altex\/article\/download\/3220\/3042","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.altex.org\/index.php\/altex\/article\/download\/3220\/3041","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.altex.org\/index.php\/altex\/article\/download\/3220\/3042","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:27:07Z","timestamp":1790440027000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.altex.org\/index.php\/altex\/article\/view\/3220"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,25]]},"references-count":0,"URL":"https:\/\/doi.org\/10.14573\/altex.2606132","relation":{},"ISSN":["1868-8551","1868-596X"],"issn-type":[{"value":"1868-8551","type":"electronic"},{"value":"1868-596X","type":"print"}],"subject":[],"published":{"date-parts":[[2026,9,25]]}}}