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To address the urgent need for efficient and mechanistically informed toxicity screening, we developed a high-throughput screening, high-content analysis (HCA) platform using a 3D in vitro mini-testis model. This system was used to evaluate 87 structurally diverse compounds from the National Toxicology Program chemical library. The model incorporates murine-derived spermatogonia, Sertoli, and Leydig cells embedded in an extracellular matrix, providing a physiologically relevant environment for mechanistic toxicology. Each compound was tested across 10 phenotypic endpoints, including nuclear morphology, cytoskeletal integrity (F-actin), DNA damage (\u03b3H2AX), and cell viability by using high-content imaging. Quantitative Points of Departure (PODs) were calculated and integrated into a High-Content Assay Index. Toxicological Priority Index (ToxPi) scores, derived from the PODs, enabled compound ranking and clustering. Compared with existing in vivo reproductive toxicity data, the 3D model demonstrated 91.5% sensitivity, 93.8% specificity, and 93.6% concordance (n\u2009=\u200964 compounds). Notably, 22 compounds lacking reproductive toxicity data were identified as potentially reproductive toxicants. Mechanistic analyses revealed that nuclear morphology, F-actin intensity, and \u03b3H2AX were the most sensitive indicators of reproductive toxicity. Cluster and category-level analysis showed that flame retardants and pesticides ranked highest in toxicity. The integration of multi-parametric data via ToxPi facilitated high-resolution chemical prioritization. Given current ethical and technical challenges in sourcing human testicular tissue or differentiating stem cells into testicular cell types, murine cells provide a reproducible and practical alternative for complex multicellular testis modeling. Our results demonstrate that the HCA-integrated 3D mini-testis model offers a robust, scalable, and mechanistically insightful platform for male reproductive toxicity screening, supporting its adoption as New Approach Methodologies aligned with regulatory and ethical testing goals.<\/jats:p>","DOI":"10.1093\/toxsci\/kfaf136","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T12:06:10Z","timestamp":1758801970000},"page":"279-300","source":"Crossref","is-referenced-by-count":2,"title":["High-content toxicological profiling of 87 compounds using a 3D mouse mini-testis model: a New Approach Methodology (NAM) for prioritizing male reproductive toxicants"],"prefix":"10.1093","volume":"208","author":[{"given":"Lei","family":"Yin","sequence":"first","affiliation":[{"name":"Reprotox Biotech LLC , Albuquerque, NM 87106,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jamie Chelin","family":"Hu","sequence":"additional","affiliation":[{"name":"College of Nursing, University of New Mexico , Albuquerque, NM 87106,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7285-8469","authenticated-orcid":false,"given":"Menghang","family":"Xia","sequence":"additional","affiliation":[{"name":"National Center for Advancing Translational Sciences (NCATS), NIH , Bethesda, MD,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5523-9583","authenticated-orcid":false,"given":"Xiaozhong (John)","family":"Yu","sequence":"additional","affiliation":[{"name":"College of Nursing, University of New Mexico , Albuquerque, NM 87106,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2025,9,29]]},"reference":[{"key":"2025112517215049800_kfaf136-B1","doi-asserted-by":"crossref","first-page":"108857","DOI":"10.1016\/j.reprotox.2025.108857","article-title":"The biological effects of bisphenol AF in reproduction and development: what do we know so far?","volume":"132","author":"Alexander","year":"2025","journal-title":"Reprod Toxicol"},{"key":"2025112517215049800_kfaf136-B2","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1289\/ehp.1510456","article-title":"Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast high throughput data","volume":"124","author":"Auerbach","year":"2016","journal-title":"Environ Health Perspect"},{"key":"2025112517215049800_kfaf136-B3","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1093\/toxsci\/kfy278","article-title":"Screening for developmental neurotoxicity at the National Toxicology Program: the future is here","volume":"167","author":"Behl","year":"2019","journal-title":"Toxicol Sci"},{"key":"2025112517215049800_kfaf136-B4","doi-asserted-by":"crossref","first-page":"8804","DOI":"10.1021\/acs.est.5b02641","article-title":"Screening chemicals for estrogen receptor bioactivity using a computational model","volume":"49","author":"Browne","year":"2015","journal-title":"Environ Sci Technol"},{"key":"2025112517215049800_kfaf136-B5","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1136\/bmj.305.6854.609","article-title":"Evidence for decreasing quality of semen during past 50 years","volume":"305","author":"Carlsen","year":"1992","journal-title":"BMJ"},{"key":"2025112517215049800_kfaf136-B6","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1126\/science.1154619","article-title":"Toxicology. 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