Risk assessment for the reproductive and developmental (R/D) toxicity is particularly challenging since R/D system is highly complex, involving interactions between multiple organs and systems at different time points and life stages.The report "Toxicity Testing in the 21st Century" by the NAS (2007) envisioned that the animal testing in vivo 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.There is a great need to develop in vitro assays targeting on the processes such as spermatogenesis.Tissue engineering techniques could conceivably be immediately implemented to improve existing models.However, it is likely that in vitro testis models which use single or multiple cell types will be needed to address such endpoints as accurate prediction of chemically-induced testicular toxicity in humans, elucidation of mechanisms of toxicity and identification of possible biomarkers of testicular toxicity.We have developed a three dimensional 3D) testicular cells co-culture system (3D-TCS) including germ, Sertoli and Leydig cells to mimic the process of spermatogenesis and applied this model to examine the mechanism of testicular toxicants.We have applied this model to compare the traditional cytotoxic endpoints as well as genomic responses among the developmentally toxic and non-toxic phthalates esters (PEs).Our studies demonstrated that this 3D-TCS successfully discriminates the reproductive toxic and non-toxic phthalates, toxic metals and also provides an insight of the mechanism.Therefore, further integration of genomic approach to develop pathways-based assays is undergoing to make this 3D-TCS model more efficient and mechanistically-based.It's of great importance to build-up such a test battery for the R/D toxicity to fulfill the goal set by the NAS in the report of"Toxicity Testing in the 21st Century".