Environmental and Molecular Mutagenesis最新文献

{"title":"Consensus findings of an International Workshops on Genotoxicity Testing workshop on using transcriptomic biomarkers to predict genotoxicity.","authors":"Roland Froetschl, J Christopher Corton, Henghong Li, Jiri Aubrecht, Scott S Auerbach, Florian Caiment, Tatyana Y Doktorova, Yurika Fujita, Danyel Jennen, Naoki Koyama, Matthew J Meier, Roman Mezencev, Leslie Recio, Takayoshi Suzuki, Carole L Yauk","doi":"10.1002/em.22645","DOIUrl":"10.1002/em.22645","url":null,"abstract":"<p><p>Gene expression biomarkers have the potential to identify genotoxic and non-genotoxic carcinogens, providing opportunities for integrated testing and reducing animal use. In August 2022, an International Workshops on Genotoxicity Testing (IWGT) workshop was held to critically review current methods to identify genotoxicants using transcriptomic profiling. Here, we summarize the findings of the workgroup on the state of the science regarding the use of transcriptomic biomarkers to identify genotoxic chemicals in vitro and in vivo. A total of 1341 papers were examined to identify the biomarkers that show the most promise for identifying genotoxicants. This analysis revealed two independently derived in vivo biomarkers and three in vitro biomarkers that, when used in conjunction with standard computational techniques, can identify genotoxic chemicals in vivo (rat or mouse liver) or in human cells in culture using different gene expression profiling platforms, with predictive accuracies of ≥92%. These biomarkers have been validated to differing degrees but typically show high reproducibility across transcriptomic platforms and model systems. They offer several advantages for applications in different contexts of use in genotoxicity testing including: early signal detection, moderate-to-high-throughput screening capacity, adaptability to different cell types and tissues, and insights on mechanistic information on DNA-damage response. Workshop participants agreed on consensus statements to advance the regulatory adoption of transcriptomic biomarkers for genotoxicity. The participants agreed that transcriptomic biomarkers have the potential to be used in conjunction with other biomarkers in integrated test strategies in vitro and using short-term rodent exposures to identify genotoxic and non-genotoxic chemicals that may cause cancer and heritable genetic effects. Following are the consensus statements from the workgroup. Transcriptomic biomarkers for genotoxicity can be used in Weight of Evidence (WoE) evaluation to: determine potential genotoxic mechanisms and hazards; identify misleading positives from in vitro genotoxicity assays; serve as new approach methodologies (NAMs) integrated into the standard battery of genotoxicity tests. Several transcriptomic biomarkers have been developed from sufficiently robust training data sets, validated with external test sets, and have demonstrated performance in multiple laboratories. These transcriptomic biomarkers can be used following established study designs and models designated through existing validation exercises in WoE evaluation. Bridging studies using a selection of training and test chemicals are needed to deviate from the established protocols to confirm performance when a transcriptomic biomarker is being applied in other: tissues, cell models, or gene expression platforms. Top dose selection and time of gene expression analysis are critical and should be established during transcriptomic bi","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"31-46"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12988028/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142931001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the quality and making appropriate use of historical negative control data: A report of the International Workshop on Genotoxicity Testing (IWGT).","authors":"Stephen D Dertinger, Dingzhou Li, Carol Beevers, George R Douglas, Robert H Heflich, David P Lovell, Daniel J Roberts, Robert Smith, Yoshifumi Uno, Andrew Williams, Kristine L Witt, Andreas Zeller, Changhui Zhou","doi":"10.1002/em.22541","DOIUrl":"10.1002/em.22541","url":null,"abstract":"<p><p>Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as \"Criterion C\"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the workgroup are: When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect. HCD can provide useful context for interpreting study results, but this requires supporting evidence that (i) HCD were generated appropriately, and (ii) their quality has been assessed and deemed sufficiently high for this purpose. HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful. Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: (i) the stability of HCD over time, and (ii) the degree to which inter-study variation explains the total variability observed. When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results. When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results. The workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD test guidelines, we recommend a more flexible application that takes into consideration HCD quality. The workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"144-165"},"PeriodicalIF":2.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10598234/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9874572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).","authors":"Barbara L Parsons, Marc A Beal, Kerry L Dearfield, George R Douglas, Min Gi, B Bhaskar Gollapudi, Robert H Heflich, Katsuyoshi Horibata, Michelle Kenyon, Alexandra S Long, David P Lovell, Anthony M Lynch, Meagan B Myers, Stefan Pfuhler, Alisa Vespa, Andreas Zeller, George E Johnson, Paul A White","doi":"10.1002/em.22599","DOIUrl":"10.1002/em.22599","url":null,"abstract":"<p><p>Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An \"effect severity\" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a \"severe\" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"121-143"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12988043/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141199876","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Review of Transcriptomic Biomarkers That Predict In Vitro Genotoxicity in Human Cell Lines.","authors":"Heng-Hong Li, Jiri Aubrecht, Tatyana Y Doktorova, Danyel Jennen, J Christopher Corton, Roland Froetschl, Roman Mezencev, Carole L Yauk","doi":"10.1002/em.70004","DOIUrl":"10.1002/em.70004","url":null,"abstract":"<p><p>The current genotoxicity testing paradigm provides little mechanistic information, has poor specificity in predicting carcinogenicity in humans, and is not suited to assessing a large number of chemicals. Genomic technologies enable the characterization of genome-wide transcriptional changes in response to chemical treatments that can inform mechanisms or modes of action. These technologies provided an impetus to develop transcriptomic biomarkers that could transform genotoxicity hazard assessment for drugs, cosmetics, and environmental and industrial chemicals. In August 2022, the International Workshops on Genotoxicity Testing (IWGT) held a workshop to critically review progress in the development and application of transcriptomic biomarkers in genotoxicity testing. Here, we describe the findings of this workshop's subgroup that conducted a systematized review and analysis of in vitro transcriptomic biomarkers for evaluating genotoxicity. Although there is a multitude of published reports exploring transcriptomics in genetic toxicology, the working group identified only five in vitro transcriptomic biomarker candidates, of which three (GENOMARK, TGx-DDI, and MU2012) were independently developed with sufficiently defined context of use, validation data, and supporting case studies that warranted inclusion in the review. Although these in vitro biomarkers were developed independently and for different classes of chemicals (TGx-DDI for pharmaceuticals, GENOMARK for cosmetics, and MU2012 for medical and environmental chemicals), they all address the same shortfall of the standard in vitro genotoxicity testing battery, that is, lack of specificity by genotoxicity-induced stress response at the transcriptomic level. In this review, we discuss the development of these in vitro biomarkers, including challenges and progress toward achieving regulatory acceptance.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"47-57"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12353356/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143540637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Summary of Major Conclusions From the 8th International Workshop on Genotoxicity Testing (IWGT), Ottawa, Canada.","authors":"Hans-Joerg Martus, Carol Beevers, Stephen Dertinger, Roland Froetschl, Roger Godschalk, Bhaskar Gollapudi, Kiyohiro Hashimoto, David Kirkland, Silvana Libertini, Timothy McGovern, Kei-Ichi Sugiyama, Paul White, Andreas Zeller","doi":"10.1002/em.70043","DOIUrl":"https://doi.org/10.1002/em.70043","url":null,"abstract":"","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 Suppl 2 ","pages":"3-16"},"PeriodicalIF":2.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147456531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of the standard battery of in vitro genotoxicity tests to predict in vivo genotoxicity through mathematical modeling: A report from the 8th International Workshop on Genotoxicity Testing.","authors":"Mirjam Luijten, Jan van Benthem, Takeshi Morita, Raffaella Corvi, Patricia A Escobar, Yurika Fujita, Jennifer Hemmerich, Naveed Honarvar, David Kirkland, Naoki Koyama, David P Lovell, Miriam Mathea, Andrew Williams, Stephen Dertinger, Stefan Pfuhler, Jeroen L A Pennings","doi":"10.1002/em.22640","DOIUrl":"10.1002/em.22640","url":null,"abstract":"<p><p>In human health risk assessment of chemicals and pharmaceuticals, identification of genotoxicity hazard usually starts with a standard battery of in vitro genotoxicity tests, which is needed to cover all genotoxicity endpoints. The individual tests included in the battery are not designed to pick up all endpoints. This explains why resulting data can appear contradictory, thereby complicating accurate interpretation of the findings. Such interpretation could be improved through application of mathematical modeling. One of the advantages of mathematical modeling is that the strengths and weaknesses of each test are taken into account. Furthermore, the generated predictions are objective and convey the associated uncertainties. This approach was explored by the working group \"Predictivity of In Vitro Genotoxicity Testing,\" convened in the context of the 8th International Workshop on Genotoxicity Testing (IWGT). Specifically, we applied mathematical modeling to a database with publicly available in vitro and in vivo data for genotoxicity. The results indicate that a mammalian in vitro clastogenicity test and a mammalian cell gene mutation test together provide strong predictive weight-of-evidence for evaluating genotoxic hazard of a substance, although they are better in predicting absence of genotoxic potential than in predicting presence of genotoxic potential. Remarkably, the bacterial reverse mutation (Ames) test did not significantly change these predictions when used in combination with in vitro mutagenicity and clastogenicity tests using cells of mammalian origin. However, in case only data from a bacterial reverse mutation test are available for the assessment of genotoxic potential, these do bear weight of evidence and thus can be used. Genotoxicity assays are generally executed in tiers, in which the bacterial reverse mutation test often is the starting point. Thus, it is reasonable to suspect that early in development test results from the bacterial reverse mutation test have influenced the composition of the database studied here. We performed several tests on the robustness of the database used for the analyses presented here, and the forthcoming results do not indicate a strong bias. Further research comparing in vitro genotoxicity data with in vivo data for additional compounds will provide more insights whether it is indeed time to reconsider the composition of the standard in vitro genotoxicity battery.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"17-30"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12988027/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142767318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genotoxicity evaluation of gene therapies: A report from the International Workshop on Genotoxicity Testing (IWGT) 2022.","authors":"S Libertini, J K Jadlowsky, T A Lanz, L M Mihalcik, D M Pizzurro","doi":"10.1002/em.22633","DOIUrl":"10.1002/em.22633","url":null,"abstract":"<p><p>At the 8th International Workshop on Genotoxicity Testing meeting in Ottawa, in August 2022, a plenary session was dedicated to the genotoxicity risk evaluation of gene therapies, including insertional oncogenesis and off-target genome editing. This brief communication summarizes the topics of discussion and the main insights from the speakers. Common themes included recommendations to conduct tailored risk assessments based on a weight-of-evidence approach, to promote data sharing, transparency, and cooperation between stakeholders, and to develop state-of-the-art validated tests relevant to clinical scenarios.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"181-190"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142282303","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Review and meta-analysis of gene expression biomarkers predictive of chemical-induced genotoxicity in vivo.","authors":"J Christopher Corton, Scott S Auerbach, Naoki Koyama, Roman Mezencev, Carole L Yauk, Takayoshi Suzuki","doi":"10.1002/em.22646","DOIUrl":"10.1002/em.22646","url":null,"abstract":"<p><p>There is growing recognition across broad sectors of the toxicology community that gene expression biomarkers have the potential to identify genotoxic and nongenotoxic carcinogens through a weight-of-evidence approach, providing opportunities to reduce reliance on the 2-year bioassay to identify carcinogens. In August 2022, a workshop within the International Workshops on Genotoxicity Testing (IWGT) was held to critically review current methods to identify genotoxicants using various 'omics profiling methods. Here, we describe the findings of a workshop subgroup focused on the state of the science regarding the use of biomarkers to identify chemicals that act as genotoxicants in vivo. A total of 1341 papers were screened to identify those that were most relevant. While six published biomarkers with characterized accuracy were initially examined, four of the six were not considered further, because they had not been tested for classification accuracy using additional sets of chemicals or other transcript profiling platforms. Two independently derived biomarkers used in conjunction with standard computational techniques can identify genotoxic chemicals in vivo (rat liver or both rat and mouse liver) on different gene expression profiling platforms. The biomarkers have predictive accuracies of ≥92%. These biomarkers have the potential to be used in conjunction with other biomarkers in integrated test strategies using short-term rodent exposures to identify genotoxic and nongenotoxic chemicals that cause cancer.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"58-70"},"PeriodicalIF":2.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143002242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).","authors":"Carol Beevers, Yoshifumi Uno, Krista Meurer, Shuichi Hamada, Kiyohiro Hashimoto, David Kirkland, Matthew J LeBaron, Frank Le Curieux, Ludovic Le Hegarat, Hans-Joerg Martus, Kenichi Masumura, Wakako Ohyama, Daniel J Roberts, Marie Vasquez, James Whitwell, Kristine L Witt","doi":"10.1002/em.22578","DOIUrl":"10.1002/em.22578","url":null,"abstract":"<p><p>The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate \"sufficient\" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"81-100"},"PeriodicalIF":2.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71520850","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transferability and Reproducibility of the HepaRG CometChip Assay","authors":"Leslie Recio,&nbsp;Carol Swartz,&nbsp;Lincoln Martin,&nbsp;Emily Rottinger,&nbsp;Stefan Pfuhler,&nbsp;Erica Pinkus,&nbsp;Daniel J. Roberts,&nbsp;Leon F. Stankowski,&nbsp;Simran Kaushal,&nbsp;Norah A. Owiti,&nbsp;Bevin P. Engelward","doi":"10.1002/em.70037","DOIUrl":"10.1002/em.70037","url":null,"abstract":"<p>This interlaboratory evaluation of HepaRG CometChip was conducted to assess transferability and reproducibility of this new approach methodology (NAM) across four laboratories. Concentrations inducing up to ~70% relative cytotoxicity were determined by the organizing laboratory, and frozen chemical formulation blocks were sent to each participant. When noncytotoxic, 10 mM was the maximum dose. Cultures were exposed once daily for three consecutive days, and both cytotoxicity assessment, via ATP quantification, and comet analysis, commenced 3–4 h after initiation of the final exposure. Positive response was statistical pairwise significance (<i>p</i> &lt; 0.05) with concentration-related increases in %Tail DNA across ≥ 2 consecutive exposures. For 8 of 11 compounds, all four labs generated unanimous test results, with four negative compounds (2-acetylaminofluorene [2-AAF], 2,4-dichlorophenol, eugenol and hydroquinone) and four positive compounds (azidothymidine, benzo(a)pyrene [BP], cyclophosphamide [CP], ethyl methanesulfonate).For the remaining chemicals, three of four labs generated negative calls for amitrole, cadmium chloride, and DMBA. In cases where bulky lesions were anticipated, the magnitude of %Tail DNA was low, due to the inherent insensitivity of the alkaline comet assay (not the CometChip per se) to detect bulky adducts repaired by nucleotide excision repair. This is supported by the small magnitude in %Tail DNA induced by BP and CP. Taken together, for all compounds there was majority agreement in CometChip results across participating laboratories supporting that the endpoint is readily transferable to new labs. Overall, this platform is a promising human-relevant NAM, with a physiologically relevant detoxification process that could be incorporated into rodent replacement strategies.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"66 9","pages":"478-491"},"PeriodicalIF":2.3,"publicationDate":"2025-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/em.70037","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145631314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}