High-content Toxicological Profiling of 87 Compounds Using a 3D Mouse Mini-Testis Model: A New Approach Methodology (NAM) for Prioritizing Male Reproductive Toxicants.

Environmental exposure to industrial chemicals, endocrine disruptors, and pharmaceuticals has been increasingly linked to the global decline in male reproductive health. To address the urgent need for efficient and mechanistically informed toxicity screening, we developed a high-throughput, high-content screening (HTS/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 (NTP) 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 ten phenotypic endpoints, including nuclear morphology, cytoskeletal integrity (F-actin), DNA damage (γH2AX), and cell viability by using high-content imaging. Quantitative Points of Departures (PODs) were calculated and integrated into a High-content Assay Index (HCAI). Toxicological Priority Index (ToxPi) scores, derived from the PODs, enabled compound ranking and clustering. Compared to existing in vivo reproductive toxicity data, the 3D model demonstrated 91.5% sensitivity, 93.8% specificity, and 93.6% concordance (n = 64 compounds). Notably, 22 compounds lacking reproductive toxicity data were identified as potentially reproductive toxicants. Mechanistic analyses revealed that nuclear morphology, F-actin intensity, and γH2AX 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 (NAMs) aligned with regulatory and ethical testing goals.