Summary Occupational exposures during manufacture and application of these chemicals represent the highest level of exposure that could cause potential adverse health effects. There remains a considerable knowledge gap regarding the relationship between workplace exposure of industrial chemicals and possible adverse health effects. Current test methods set by regulatory agencies predominately rely on animal testing. The cost and time with the animal testing for risk assessment seriously limit efforts for extensive chemical screening. These data gaps necessitate the implementation of a new vision for toxicity testing as reflected in the National Research Council’s report “Toxicity Testing in the 21st Century”. Under the support of NIOSH R21 OH010473 grant, we established a rodent testicular cell co-culture model and found this in vitro co-culture model could identify the reproductive toxicants. While results from these in vivo or in vitro rodent models are informative, these models could not recapitulate the whole process of human spermatogenesis as rodent spermatogenesis proceeds in a distinctly different fashion compared to humans. Thus, developing in vitro human models for reproductive toxicity testing is paramount for advancing the field and for understanding the cellular and molecular mechanisms that may underlie these disruptions in human spermatogenesis. In this proposal, we will develop a human testicular cell co-culture model, and demonstrate the reliability and sensitivity of this model in high-throughput and multi-parametric high-content analytical screening for workplace chemicals on the reproductive and developmental systems. The specific aims are (1) to establish testicular cell co-culture model from human spermatogonial stem cells (hSSCs), Sertoli cell and Leydig cells; (2) to develop integrated assays based on the adverse outcome pathways; (3) to apply the multi-parametric high-throughput and high- content analysis (HT/HCA) to examine and compare reproductive toxic and non-toxic compounds to validate the in vitro testicular cell co-culture model. Through this hypothesis-driven research, we will test the hypothesis that an in vitro 3D human testicular cell co-culture model creates a proper microenvironment that mimics a human testis to promote the growth of germ cells in vitro and generate reproducible results for predicting reproductive toxicity. The research proposed will result in publications and generate toxicity data of the chemicals in the workplace, and provide data for risk assessment. Therefore, this proposal meets the mission of Cancer, Reproductive, and Cardiovascular Research Program (CRC). The identification of reproductive toxicants will reduce hazardous exposures and lower the risk of reproductive malfunction. Therefore, our proposed project is within the NIOSH CRC’s Research to Practice (r2p) strategy to promote the transfer and translation of research findings into prevention practices.