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Studies Division Genomics Laboratory, Health Canada, Government of Canadahttps://www.ebi.ac.uk/ena/browser/view/PRJNA880874Homo sapiensxref:PubMed:36756349The conventional battery for genotoxicity testing is not well suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. New Approach Methodologies (NAMs) aim to accelerate the pace of hazard assessment and reduce reliance on in vivo tests that are time-consuming and resource-intensive. As such, high-throughput transcriptomic and flow cytometry-based assays have been developed for modernized in vitro genotoxicity assessment. This includes: the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mode-of-action (MoA) information). The objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated NAM-based testing strategy for screening data-poor compounds prioritized by Health Canada’s New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Gene expression profiling was conducted using the targeted TempO-SeqTM assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five of the data-poor compounds were genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one via a pan-genotoxic MoA. Two compounds were likely irrelevant positives in the MN test two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. BMC modeling revealed nearly identical potency rankings for each assay. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that the integration of TGx-DDI, MicroFlow®, and MultiFlow® endpoints is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds. Overall design: A total of 276 samples were sequenced for this toxicogenomics study on 10 data-poor compounds, including technical controls (reference RNA media only cells with no treatment). The solvent controls were comprised of 0.1% DMSO. 6 concentrations of each data-poor chemical were tested in duplicate. Three reference chemicals, etoposide, 7,12-dimethylbenz[a]anthracene, and d-mannitol were also included in this study. Six chemicals (NSACB #1,12,13,14,18,D-mannitol) were tested both with and without metabolic activation.ENAcongenital myopathy - cleft palate - malignant hyperthermia, congenital myopathy cleft palate and malignant hyperthermia., myopathy, F14G24.15, NAC DOMAIN PROTEIN NAM, ANAC018, congenital, NO APICAL MERISTEM, techniques, with cleft palate and malignant hyperthermia, F14G24_15, procedures, NARS2, NAC-REGULATED SEED MORPHOLOGY 2, Arabidopsis NAC domain containing protein 18, NAM, ATNAM, methodologycongenital myopathy - cleft palate - malignant hyperthermia, congenital myopathy cleft palate and malignant hyperthermia., myopathy, F14G24.15, NAC DOMAIN PROTEIN NAM, ANAC018, congenital, NO APICAL MERISTEM, techniques, with cleft palate and malignant hyperthermia, F14G24_15, procedures, NARS2, NAC-REGULATED SEED MORPHOLOGY 2, Arabidopsis NAC domain containing protein 18, NAM, ATNAM, methodologyfalseApplication of a New Approach Methodology (NAM)-based Strategy for Genotoxicity Assessment of Data-poor CompoundsApplication of a New Approach Methodology (NAM)-based Strategy for Genotoxicity Assessment of Data-poor Compounds2025-09-242022-11-19PRJNA880874GSE213454960636756349