Risk assessment for reproductive and developmental (R/D) toxicity is particularly challenging since the R/D system is highly complex, involving interactions between multiple organs and systems at different time points and life stages. Since the report Toxicity Testing in the 21st Century by the NAS (2007) envisioned that in vivo animal testing can eventually be replaced by a combination of in silico and in vitro approaches, the need to identify in vitro systems for R/D toxicity test has grown. Over the years, many alternative methods have been developed as a part of a test battery for assessing R/D toxicity, but the majority are focused on embryotoxicity testing. There is a great need to develop in vitro assays targeting processes such as spermatogenesis. We have developed a three-dimensional testicular cells co-culture system (3D-TCS) including germ, Sertoli and Leydig cells. Our initial validation studies demonstrated that this 3D-TCS can discriminate known developmentally toxic phthalate esters from non-toxic phthalate esters. Therefore, further validation of our established 3D-TCS system will be critical to allow this promising model to become part of an integrated testing battery for R/D toxicity assessment. In this proposal, we will build a list of gold standard testing compounds and blindly test the predictability of these assays in our 3D-TCS system. We propose to integrate genomic and metabolomic approaches to develop pathways-based assays to make this 3D-TCS more efficient and mechanistically based. These assays will not only be directly linked to the endpoint of male reproductive toxicity, but also linked to developmental toxicity since these pathways proposed also play critical roles during normal development for both males and females. The specific aims (SAs) are to (1) establish the 3D-TCS model for R/D toxicity evaluation; (2) to quantitatively compare toxicogenomic and metabolomic effects of potential R/D toxicants by using our established systems based GO-Quant analysis approach to identify genomic and metabolomic signatures for R/D toxicity; and (3) to develop an integrated pathway-based high content (HCS) and high throughput (HTS) screening assay in the 3D-TCS model for R/D toxicity evaluation. The milestones for SA1 are to (1) optimize and finalize a Standard Operating Procedure for the 3D-TCS; (2) create a database of gold standard testing compounds; (3) obtain dose and time dependent data on cytotoxicity and cell viability for 70 compounds; and (4) calculate the predictivity, precision, and accuracy for three classes of compounds tested in this 3D-TCS model. For SA2, the milestones are to (1) generate genomic profiles and (2) metabolomic profiles for the 40 compounds in the 3D-SGC model. For SA3, the milestones are to (1) establish the HCS/HTS assay in the 3D-SGC model; (2) generate and model R/D toxicity for evaluating the R/D toxic and non-R/D toxic compounds; and (3) design metrics for acceptance or rejection of R/D toxic versus non-R/D toxic compounds based on dose and time-dependent pathway-based R/D toxicity signatures.