{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T14:34:24Z","timestamp":1791038064206,"version":"4.1.0"},"reference-count":91,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,8,28]],"date-time":"2025-08-28T00:00:00Z","timestamp":1756339200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union\u2019s Horizon 2020 research","award":["964537"],"award-info":[{"award-number":["964537"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["101057014"],"award-info":[{"award-number":["101057014"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["161L0243B"],"award-info":[{"award-number":["161L0243B"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["016LW0146K"],"award-info":[{"award-number":["016LW0146K"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["964537"],"award-info":[{"award-number":["964537"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["101057014"],"award-info":[{"award-number":["101057014"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["161L0243B"],"award-info":[{"award-number":["161L0243B"]}]},{"name":"European Union\u2019s Horizon 2020 research","award":["016LW0146K"],"award-info":[{"award-number":["016LW0146K"]}]},{"DOI":"10.13039\/501100002347","name":"BMBF","doi-asserted-by":"publisher","award":["964537"],"award-info":[{"award-number":["964537"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"BMBF","doi-asserted-by":"publisher","award":["101057014"],"award-info":[{"award-number":["101057014"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"BMBF","doi-asserted-by":"publisher","award":["161L0243B"],"award-info":[{"award-number":["161L0243B"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"BMBF","doi-asserted-by":"publisher","award":["016LW0146K"],"award-info":[{"award-number":["016LW0146K"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Compounds, which rely on metabolism to exhibit toxicity, pose a challenge for next-generation risk assessment (NGRA). Since many of the currently available non-animal new approach methods (NAMs) lack metabolic activity, their use may lead to an underestimation of the true hazard to humans (false negative predictions). We explored here strategies to deal with metabolite-mediated toxicity in assays for developmental neurotoxicity. First, we present an overview of substances that may serve as potential positive controls for metabolite-related neurotoxicity. Then, we demonstrate, using the MitoMet (UKN4b) assay, which assesses the adverse effects of chemicals on neurites of human neurons, that some metabolites have a higher toxic potency than their parent compound. Next, we designed a strategy to integrate elements of xenobiotic metabolism into assays used for (developmental) neurotoxicity testing. In the first step of this approach, hepatic post-mitochondrial fractions (S9) were used to generate metabolite mixtures (\u201cmetabolisation module\u201d). In the second step, these were applied to a NAM (exemplified by the UKN4b assay) to identify metabolite-mediated toxicity. We demonstrate the applicability and transferability of these approaches to other assays, by an exemplary study on the basis of the cMINC (UKN2) assay, another NAM of the developmental neurotoxicity in vitro battery. Based on the experience gained from these experiments, we discuss key issues to be addressed if this approach is to be used more broadly for NAM in the NGRA context.<\/jats:p>","DOI":"10.3390\/ijms26178338","type":"journal-article","created":{"date-parts":[[2025,8,28]],"date-time":"2025-08-28T10:29:18Z","timestamp":1756376958000},"page":"8338","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Testing Strategies for Metabolite-Mediated Neurotoxicity"],"prefix":"10.3390","volume":"26","author":[{"given":"Julian","family":"Suess","sequence":"first","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4596-4161","authenticated-orcid":false,"given":"Moritz","family":"Reinmoeller","sequence":"additional","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viktoria","family":"Magel","sequence":"additional","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baiba","family":"Gukalova","sequence":"additional","affiliation":[{"name":"Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2213-8337","authenticated-orcid":false,"given":"Edgars","family":"Liepinsh","sequence":"additional","affiliation":[{"name":"Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iain","family":"Gardner","sequence":"additional","affiliation":[{"name":"Certara Predictive Technologies, Level 2-Acero, 1 Concourse Way, Sheffield S1 2BJ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7103-0990","authenticated-orcid":false,"given":"Nadine","family":"Dreser","sequence":"additional","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"},{"name":"Center for Alternatives to Animal Testing in Europe (CAAT-Europe), University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1453-1019","authenticated-orcid":false,"given":"Anna-Katharina","family":"Holzer","sequence":"additional","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3778-8693","authenticated-orcid":false,"given":"Marcel","family":"Leist","sequence":"additional","affiliation":[{"name":"In Vitro Toxicology and Biomedicine, Dept Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, 78457 Konstanz, Germany"},{"name":"Center for Alternatives to Animal Testing in Europe (CAAT-Europe), University of Konstanz, 78457 Konstanz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.comtox.2018.06.001","article-title":"Principles Underpinning the Use of New Methodologies in the Risk Assessment of Cosmetic Ingredients","volume":"7","author":"Dent","year":"2018","journal-title":"Comput. Toxicol."},{"key":"ref_2","first-page":"419","article-title":"Next-Generation Risk Assessment of Chemicals\u2014Rolling Out a Human-Centric Testing Strategy to Drive 3R Implementation: The RISK-HUNT3R Project Perspective","volume":"39","author":"Pallocca","year":"2022","journal-title":"ALTEX"},{"key":"ref_3","first-page":"323","article-title":"Developmental Neurotoxicity (DNT): A Call for Implementation of New Approach Methodologies for Regulatory Purposes: Summary of the 5th International Conference on DNT Testing","volume":"42","author":"Celardo","year":"2025","journal-title":"ALTEX"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S1359-6446(05)03681-0","article-title":"Role of Pharmacologically Active Metabolites in Drug Discovery and Development","volume":"11","author":"Fura","year":"2006","journal-title":"Drug Discov. Today"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/0163-7258(95)02013-6","article-title":"The Role of Cytochrome P450 Enzymes in Hepatic and Extrahepatic Human Drug Toxicity","volume":"68","author":"Park","year":"1995","journal-title":"Pharmacol. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Rendic, S.P., and Guengerich, F.P. (2021). Human Family 1\u20134 Cytochrome P450 Enzymes Involved in the Metabolic Activation of Xenobiotic and Physiological Chemicals: An Update, Springer.","DOI":"10.1007\/s00204-020-02971-4"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/s002800050863","article-title":"Use of V79 Cells with Stably Transfected Cytochrome P450 CDNAs in Studying the Metabolism and Effects of Cytotoxic Drugs","volume":"43","author":"Philip","year":"1999","journal-title":"Cancer Chemother. Pharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1115","DOI":"10.1007\/s00204-013-1012-x","article-title":"HepG2 Cells Simultaneously Expressing Five P450 Enzymes for the Screening of Hepatotoxicity: Identification of Bioactivable Drugs and the Potential Mechanism of Toxicity Involved","volume":"87","author":"Tolosa","year":"2013","journal-title":"Arch. Toxicol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1016\/S0006-2952(98)00035-5","article-title":"Inhibition of Acetylcholinesterase and Butyrylcholinesterase by Chlorpyrifos-Oxon","volume":"56","author":"Amitai","year":"1998","journal-title":"Biochem. Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4055","DOI":"10.1007\/s00204-020-02927-8","article-title":"Acetylcholinesterase Inhibition in Electric Eel and Human Donor Blood: An in Vitro Approach to Investigate Interspecies Differences and Human Variability in Toxicodynamics","volume":"94","author":"Kasteel","year":"2020","journal-title":"Arch. Toxicol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"047008","DOI":"10.1289\/EHP6993","article-title":"Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition","volume":"129","author":"Li","year":"2021","journal-title":"Environ. Health Perspect."},{"key":"ref_12","first-page":"306","article-title":"Recommendation on Test Readiness Criteria for New Approach Methods in Toxicology: Exemplified for Developmental Neurotoxicity","volume":"35","author":"Hogberg","year":"2018","journal-title":"ALTEX"},{"key":"ref_13","first-page":"152","article-title":"Revolutionizing Developmental Neurotoxicity Testing\u2014A Journey from Animal Models to Advanced In Vitro Systems","volume":"41","author":"Smirnova","year":"2024","journal-title":"ALTEX"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"137035","DOI":"10.1016\/j.chemosphere.2022.137035","article-title":"Establishment of a Human Cell-Based in Vitro Battery to Assess Developmental Neurotoxicity Hazard of Chemicals","volume":"311","author":"Blum","year":"2023","journal-title":"Chemosphere"},{"key":"ref_15","first-page":"49","article-title":"Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: Example lists and criteria for their selection and use","volume":"34","author":"Aschner","year":"2017","journal-title":"ALTEX-Altern. Anim. Exp."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1007\/s00204-015-1464-2","article-title":"International STakeholder NETwork (ISTNET): Creating a Developmental Neurotoxicity (DNT) Testing Road Map for Regulatory Purposes","volume":"89","author":"Crofton","year":"2015","journal-title":"Arch. Toxicol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1007\/s00204-013-1072-y","article-title":"Evaluation of a Human Neurite Growth Assay as Specific Screen for Developmental Neurotoxicants","volume":"87","author":"Krug","year":"2013","journal-title":"Arch. Toxicol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"235","DOI":"10.14573\/altex.1712182","article-title":"A High-Throughput Approach to Identify Specific Neurotoxicants\/Developmental Toxicants in Human Neuronal Cell Function Assays","volume":"35","author":"Delp","year":"2018","journal-title":"ALTEX"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1585","DOI":"10.1007\/s00204-019-02473-y","article-title":"Development of a Neurotoxicity Assay That Is Tuned to Detect Mitochondrial Toxicants","volume":"93","author":"Delp","year":"2019","journal-title":"Arch. Toxicol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Holzer, A.K., Suciu, I., Karreman, C., Goj, T., and Leist, M. (2022). Specific Attenuation of Purinergic Signaling during Bortezomib-Induced Peripheral Neuropathy In Vitro. Int. J. Mol. Sci., 23.","DOI":"10.1101\/2022.02.17.479688"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3613","DOI":"10.1007\/s00204-017-1977-y","article-title":"Combination of Multiple Neural Crest Migration Assays to Identify Environmental Toxicants from a Proof-of-Concept Chemical Library","volume":"91","author":"Nyffeler","year":"2017","journal-title":"Arch. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Magel, V., Blum, J., Dolde, X., Leisner, H., Grillberger, K., Khalidi, H., Gardner, I., Ecker, G.F., Pallocca, G., and Dreser, N. (2024). Inhibition of Neural Crest Cell Migration by Strobilurin Fungicides and Other Mitochondrial Toxicants. Cells, 13.","DOI":"10.3390\/cells13242057"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1038\/s41419-024-06998-8","article-title":"Drug-Induced Oxidative Stress Actively Prevents Caspase Activation and Hepatocyte Apoptosis","volume":"15","author":"Lambrecht","year":"2024","journal-title":"Cell Death Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.toxlet.2023.06.005","article-title":"Exposure to Cis- and Trans-Regioisomers of S-(1,2-Dichlorovinyl)-L-Cysteine and S-(1,2-Dichlorovinyl)-Glutathione Result in Quantitatively and Qualitatively Different Cellular Effects in RPTEC\/TERT1 Cells","volume":"383","author":"Capinha","year":"2023","journal-title":"Toxicol. Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1089\/ars.2015.6297","article-title":"Preferential Extracellular Generation of the Active Parkinsonian Toxin MPP+ by Transporter-Independent Export of the Intermediate MPDP+","volume":"23","author":"Schildknecht","year":"2015","journal-title":"Antioxid. Redox Signal."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1007\/s00204-002-0353-7","article-title":"Effects of the Dithiocarbamate Fungicide Propineb in Primary Neuronal Cell Cultures and Skeletal Muscle Cells of the Rat","volume":"76","author":"Schmuck","year":"2002","journal-title":"Arch. Toxicol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1410","DOI":"10.1021\/acs.chemrestox.6b00079","article-title":"Prediction of Estrogenic Bioactivity of Environmental Chemical Metabolites","volume":"29","author":"Pinto","year":"2016","journal-title":"Chem. Res. Toxicol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/S0303-7207(02)00094-1","article-title":"Phenylphenols, Biphenols, Bisphenol-A and 4-Tert-Octylphenol Exhibit \u03b1 and \u03b2 Estrogen Activities and Antiandrogen Activity in Reporter Cell Lines","volume":"193","author":"Paris","year":"2002","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Koenig, L., Ramme, A.P., Faust, D., Mayer, M., Fl\u00f6tke, T., Gerhartl, A., Brachner, A., Neuhaus, W., Appelt-Menzel, A., and Metzger, M. (2022). A Human Stem Cell-Derived Brain-Liver Chip for Assessing Blood-Brain-Barrier Permeation of Pharmaceutical Drugs. Cells, 11.","DOI":"10.3390\/cells11203295"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Oleaga, C., Bernabini, C., Smith, A.S.T., Srinivasan, B., Jackson, M., McLamb, W., Platt, V., Bridges, R., Cai, Y., and Santhanam, N. (2016). Multi-Organ Toxicity Demonstration in a Functional Human in Vitro System Composed of Four Organs. Sci. Rep., 6.","DOI":"10.1038\/srep20030"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.jbiotec.2015.02.002","article-title":"A Multi-Organ Chip Co-Culture of Neurospheres and Liver Equivalents for Long-Term Substance Testing","volume":"205","author":"Materne","year":"2015","journal-title":"J. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1016\/S0887-2333(02)00085-1","article-title":"Short-Term Assay for the Identification of Neurotoxic Compounds and Their Liver Derived Stable Metabolites","volume":"16","author":"Bruinink","year":"2002","journal-title":"Toxicol. Vitr."},{"key":"ref_33","first-page":"22","article-title":"Identification of Neurotoxic Chemicals in Cell Cultures","volume":"24","author":"Bruinink","year":"2007","journal-title":"ALTEX"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1007\/s00204-018-2249-1","article-title":"High-Throughput Metabolism-Induced Toxicity Assays Demonstrated on a 384-Pillar Plate","volume":"92","author":"Yu","year":"2018","journal-title":"Arch. Toxicol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.vascn.2018.03.002","article-title":"MRNA Transfection Retrofits Cell-Based Assays with Xenobiotic Metabolism","volume":"92","author":"DeGroot","year":"2018","journal-title":"J. Pharmacol. Toxicol. Methods"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"389","DOI":"10.14573\/altex.1611021","article-title":"Incorporation of a Metabolizing System in Biodetection Assays for Endocrine Active Substances","volume":"34","author":"Mollergues","year":"2017","journal-title":"ALTEX"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.reprotox.2017.11.005","article-title":"Incorporation of Metabolic Enzymes to Improve Predictivity of Reporter Gene Assay Results for Estrogenic and Anti-Androgenic Activity","volume":"75","author":"Man","year":"2018","journal-title":"Reprod. Toxicol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1177\/026119290603400113","article-title":"Metabolism: A Bottleneck in in Vitro Toxicological Test Development","volume":"34","author":"Coecke","year":"2006","journal-title":"ATLA Altern. Lab. Anim."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1093\/mutage\/10.6.517","article-title":"Evaluation of Phenobarbital\/\u03b2-Naphthoflavone as an Alternative S9-Induction Regime to Aroclor 1254 in the Rat for Use in in Vitro Genotoxicity Assays","volume":"10","author":"Callander","year":"1995","journal-title":"Mutagenesis"},{"key":"ref_40","first-page":"173","article-title":"Revised Methods for the Salmonella Mutagenicity Test","volume":"113","author":"Maron","year":"1983","journal-title":"Mutat. Res.\/Environ. Mutagen. Relat. Subj."},{"key":"ref_41","unstructured":"OECD (2025, August 25). Test No. 471: Bacterial Reverse Mutation Test. Available online: https:\/\/www.oecd.org\/en\/publications\/test-no-471-bacterial-reverse-mutation-test_9789264071247-en.html."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/j.taap.2010.10.029","article-title":"Use of External Metabolizing Systems When Testing for Endocrine Disruption in the T-Screen Assay","volume":"250","author":"Taxvig","year":"2011","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1093\/toxsci\/kfaa147","article-title":"The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay with Metabolic Competence","volume":"178","author":"Deisenroth","year":"2020","journal-title":"Toxicol. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Hopperstad, K., and Deisenroth, C. (2023). Development of a Bioprinter-Based Method for Incorporating Metabolic Competence into High-Throughput in Vitro Assays. Front. Toxicol., 5.","DOI":"10.3389\/ftox.2023.1196245"},{"key":"ref_45","first-page":"643","article-title":"An Intact Insect Embryo for Developmental Neurotoxicity Testing of Directed Axonal Elongation","volume":"36","author":"Bergmann","year":"2019","journal-title":"ALTEX"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1007\/s00204-022-03237-x","article-title":"Inhibition of Neurite Outgrowth and Enhanced Effects Compared to Baseline Toxicity in SH-SY5Y Cells","volume":"96","author":"Lee","year":"2022","journal-title":"Arch. Toxicol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.taap.2018.02.003","article-title":"Rotenone Exerts Developmental Neurotoxicity in a Human Brain Spheroid Model","volume":"354","author":"Pamies","year":"2018","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1016\/j.taap.2011.06.002","article-title":"Effects of Sub-Lethal Neurite Outgrowth Inhibitory Concentrations of Chlorpyrifos Oxon on Cytoskeletal Proteins and Acetylcholinesterase in Differentiating N2a Cells","volume":"256","author":"Flaskos","year":"2011","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"5418","DOI":"10.1038\/s41467-021-25698-x","article-title":"Large Scale Enzyme Based Xenobiotic Identification for Exposomics","volume":"12","author":"Liu","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2653","DOI":"10.1038\/s41467-022-30204-y","article-title":"Next-Generation Biomonitoring of the Early-Life Chemical Exposome in Neonatal and Infant Development","volume":"13","author":"Jamnik","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1111\/cbdd.13445","article-title":"Computational Methods and Tools to Predict Cytochrome P450 Metabolism for Drug Discovery","volume":"93","author":"Tyzack","year":"2019","journal-title":"Chem. Biol. Drug Des."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1038\/nrd4581","article-title":"Predicting Drug Metabolism: Experiment and\/or Computation?","volume":"14","author":"Kirchmair","year":"2015","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1007\/s13318-013-0139-8","article-title":"An LC-MS Based Study of the Metabolic Profile of Primaquine, an 8-Aminoquinoline Antiparasitic Drug, with an in Vitro Primary Human Hepatocyte Culture Model","volume":"39","author":"Jin","year":"2014","journal-title":"Eur. J. Drug Metab. Pharmacokinet."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1007\/s00228-003-0636-9","article-title":"Identification of Human Cytochrome P450s That Metabolise Anti-Parasitic Drugs and Predictions of in Vivo Drug Hepatic Clearance from in Vitro Data","volume":"59","author":"Li","year":"2003","journal-title":"Eur. J. Clin. Pharmacol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1111\/j.2042-7158.1998.tb06173.x","article-title":"The Pharmacokinetics and Bioavailability of Dihydroartemisinin, Arteether, Artemether, Artesunic Acid and Artelinic Acid in Rats","volume":"50","author":"Li","year":"1998","journal-title":"J. Pharm. Pharmacol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1006\/taap.1996.8042","article-title":"The Role of the Benomyl Metabolite Carbendazim in Benomyl-Induced Testicular Toxicity","volume":"142","author":"Lim","year":"1997","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1021\/jf60193a046","article-title":"Fate of Benomyl in Animals","volume":"22","author":"Gardiner","year":"1974","journal-title":"J. Agric. Food Chem."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.envint.2017.10.005","article-title":"Presence of Benzophenones Commonly Used as UV Filters and Absorbers in Paired Maternal and Fetal Samples","volume":"110","author":"Krause","year":"2018","journal-title":"Environ. Int."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/S0300-483X(00)00329-2","article-title":"Metabolism and Toxicity of Benzophenone in Isolated Rat Hepatocytes and Estrogenic Activity of Its Metabolites in MCF-7 Cells","volume":"156","author":"Nakagawa","year":"2000","journal-title":"Toxicology"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1289\/ehp.1408642","article-title":"Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds That Acutely Decrease Mitochondrial Membrane Potential","volume":"123","author":"Huang","year":"2015","journal-title":"Environ. Health Perspect."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"1487","DOI":"10.1351\/pac199567081487","article-title":"Detection and Significance of Active Metabolites of Agrochemicals and Related Xenobiotics in Animals (Technical Report)","volume":"67","author":"Hollingworth","year":"1995","journal-title":"Pure Appl. Chem."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1016\/0272-0590(88)90129-7","article-title":"The Toxicity and Mechanism of Action of Bromethalin: A New Single-Feeding Rodenticide","volume":"11","author":"Cherry","year":"1988","journal-title":"Fundam. Appl. Toxicol."},{"key":"ref_63","first-page":"e8444","article-title":"Targeted Review of Maximum Residue Levels (MRLs) for Chlorfenapyr","volume":"21","author":"Bellisai","year":"2023","journal-title":"EFSA J."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"3107","DOI":"10.1016\/0006-2952(87)90619-8","article-title":"In Vitro Febantel Transformation by Sheep and Cattle Ruminal Fluids and Metabolism by Hepatic Subcellular Fractions from Different Animal Species","volume":"36","author":"Beretta","year":"1987","journal-title":"Biochem. Pharmacol."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1186\/s43094-020-00138-7","article-title":"Characterization and Toxicity Evaluation of Degradation Products of Febantel","volume":"6","author":"Mahajan","year":"2020","journal-title":"Future J. Pharm. Sci."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1016\/S0090-9556(24)02977-5","article-title":"Identification of an N-Methyl-4-Phenylpyridinium-like Metabolite of the Antidiarrheal Agent Loperamide in Human Liver Microsomes: Underlying Reason(s) for the Lack of Neurotoxicity despite the Bioactivation Event","volume":"32","author":"Kalgutkar","year":"2004","journal-title":"Drug Metab. Dispos."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1021\/tx960001y","article-title":"Studies on the Conversion of Haloperidol and Its Tetrahydropyridine Dehydration Product to Potentially Neurotoxic Pyridinium Metabolites by Human Liver Microsomes","volume":"9","author":"Usuki","year":"1996","journal-title":"Chem. Res. Toxicol."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"113894","DOI":"10.1016\/j.expneurol.2021.113894","article-title":"Neurotoxicity of MDMA: Main Effects and Mechanisms","volume":"347","author":"Costa","year":"2022","journal-title":"Exp. Neurol."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1038\/311467a0","article-title":"Protection against the Dopaminergic Neurotoxicity of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine by Monoamine Oxidase Inhibitors","volume":"311","author":"Heikkila","year":"1984","journal-title":"Nature"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"141468","DOI":"10.1016\/j.scitotenv.2020.141468","article-title":"Optimization of a Pre-Metabolization Procedure Using Rat Liver S9 and Cell-Extracted S9 in the Ames Fluctuation Test","volume":"749","author":"Shao","year":"2020","journal-title":"Sci. Total Environ."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.2165\/00003088-200544110-00003","article-title":"Clinical Pharmacokinetics of Cyclophosphamide","volume":"44","author":"Huitema","year":"2005","journal-title":"Clin. Pharmacokinet."},{"key":"ref_72","doi-asserted-by":"crossref","unstructured":"Feng, X., Wang, K., Cao, S., Ding, L., and Qiu, F. (2020). Pharmacokinetics and Excretion of Berberine and Its Nine Metabolites in Rats. Front. Pharmacol., 11.","DOI":"10.3389\/fphar.2020.594852"},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"1414","DOI":"10.2337\/db07-1552","article-title":"A Mechanism for the Action of Berberine to Activate","volume":"57","author":"Turner","year":"2008","journal-title":"Diabetes"},{"key":"ref_74","doi-asserted-by":"crossref","unstructured":"Suciu, I., Delp, J., Gutbier, S., Suess, J., Henschke, L., Celardo, I., Mayer, T.U., Amelio, I., and Leist, M. (2024). Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors. Antioxidants, 13.","DOI":"10.3390\/antiox13010049"},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"114876","DOI":"10.1016\/j.taap.2019.114876","article-title":"Bioactivity Screening of Environmental Chemicals Using Imaging-Based High-Throughput Phenotypic Profiling","volume":"389","author":"Nyffeler","year":"2020","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_76","first-page":"7794E","article-title":"EFSA Pilot Project on New Approach Methodologies (NAMs) for Tebufenpyrad Risk Assessment. Part 2. Hazard Characterisation and Identification of the Reference Point","volume":"20","author":"Alimohammadi","year":"2023","journal-title":"EFSA Support. Publ."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"476","DOI":"10.5966\/sctm.2015-0108","article-title":"Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants","volume":"5","author":"Hoelting","year":"2016","journal-title":"Stem Cells Transl. Med."},{"key":"ref_78","first-page":"155","article-title":"Determination of Benchmark Concentrations and Their Statistical Uncertainty for Cytotoxicity Test Data and Functional In Vitro Assays","volume":"37","author":"Krebs","year":"2020","journal-title":"ALTEX"},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1023\/B:MODI.0000006562.93049.36","article-title":"In Silico Prediction of Aqueous Solubility, Human Plasma Protein Binding and Volume of Distribution of Compounds from Calculated PKa and AlogP98 Values","volume":"7","author":"Lobell","year":"2003","journal-title":"Mol. Divers."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1080\/00498254.2022.2132426","article-title":"Non-Specific Binding of Compounds in in Vitro Metabolism Assays: A Comparison of Microsomal and Hepatocyte Binding in Different Species and an Assessment of the Accuracy of Prediction Models","volume":"52","author":"Gardner","year":"2022","journal-title":"Xenobiotica"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"6263","DOI":"10.1021\/es049285w","article-title":"Toward More Useful in Vitro Toxicity Data with Measured Free Concentrations","volume":"38","author":"Heringa","year":"2004","journal-title":"Environ. Sci. Technol."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.pharmthera.2012.12.007","article-title":"Cytochrome P450 Enzymes in Drug Metabolism: Regulation of Gene Expression, Enzyme Activities, and Impact of Genetic Variation","volume":"138","author":"Zanger","year":"2013","journal-title":"Pharmacol. Ther."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1021\/tx500444e","article-title":"Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals","volume":"28","author":"Rendic","year":"2015","journal-title":"Chem. Res. Toxicol."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/0041-008X(84)90039-5","article-title":"Teratogenicity in Vitro of Two Deacetylated Metabolites of N-Hydroxy-2-Acetylaminofluorene","volume":"76","author":"Greenaway","year":"1984","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1093\/carcin\/7.5.697","article-title":"N -Acetyltransferase Multiplicity and the Bioactivation of N -Arylhydroxamic Acids by Hamster Hepatic and Intestinal Enzymes","volume":"7","author":"Smith","year":"1986","journal-title":"Carcinogenesis"},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1007\/s00204-020-02939-4","article-title":"Cross-Species Analysis of Hepatic Cytochrome P450 and Transport Protein Expression","volume":"95","author":"Hammer","year":"2020","journal-title":"Arch. Toxicol."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1093\/mutage\/gev082","article-title":"The Utility of Metabolic Activation Mixtures Containing Human Hepatic Post-Mitochondrial Supernatant (S9) for in Vitro Genetic Toxicity Assessment","volume":"31","author":"Cox","year":"2016","journal-title":"Mutagenesis"},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"83","DOI":"10.2174\/1872312810666160223121836","article-title":"Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability","volume":"10","author":"Richardson","year":"2016","journal-title":"Drug Metab. Lett."},{"key":"ref_89","doi-asserted-by":"crossref","unstructured":"Mishra, N., and Srivastava, R. (2023). Bacterial Worth in Genotoxicity Assessment Studies. J. Microbiol. Methods, 215.","DOI":"10.1016\/j.mimet.2023.106860"},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"142269","DOI":"10.1016\/j.scitotenv.2020.142269","article-title":"Using a High-Throughput Method in the Micronucleus Assay to Compare Animal-Free with Rat-Derived S9","volume":"751","author":"Brendt","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1111\/j.1365-2125.1990.tb03793.x","article-title":"Bioactivation of Dapsone to a Cytotoxic Metabolite: In Vitro Use of a Novel Two Compartment System Which Contains Human Tissues","volume":"30","author":"Riley","year":"1990","journal-title":"Br. J. Clin. Pharmacol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/17\/8338\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:34:20Z","timestamp":1760034860000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/17\/8338"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,28]]},"references-count":91,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["ijms26178338"],"URL":"https:\/\/doi.org\/10.3390\/ijms26178338","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,28]]}}}