Some plasticizers are endocrine-disrupting chemicals that cause reproductive toxicity in both males and females. Several chemicals already approved by the Food and Drug Administration have been proposed as substitutes for some of these plasticizers, one example is acetyl tributyl citrate (ATBC). However, no studies have tested whether ATBC causes direct toxicity to the ovary. Ovarian antral follicles are essential for female fertility because they are the major producers of ovarian steroids and are the only follicle type that can ovulate in response to gonadotropin stimulation. Previous studies have used in-vitro ovarian follicle culture as a screening tool to demonstrate that EDCs can cause direct ovarian toxicity. Therefore, we designed this study to test whether exposure to in vitro treatment with ATBC causes ovarian toxicity in CD-1 mice. We mechanically isolated antral follicles from the ovaries of adult CD-1 mice (35-39 days old) and individually exposed them (n=5 cultures with n≥8 follicles per treatment) to supplemented media alone (NT), dimethyl sulfoxide (DMSO, vehicle for ATBC), and ATBC (0.001-100 µg/mL) for 24-72 h. Follicle growth and survival were monitored by measuring follicle diameter and cytotoxicity (compromised membrane integrity; CellTox Green) every 24 h, and assessing number of metabolically active cells (ATP concentration; Promega CellTiterGlo) at the end time point. The DNA synthesis inhibitor hydroxyurea (HU, 100 mM) was used as a positive control for the viability assays. Exposure to ATBC did not affect the ability of antral follicles to increase their diameter over time at all concentrations tested. When stratified by growth pattern, there was not a significant difference in the proportion of follicles growing normally, growing slowly, or not growing following ATBC exposure at all concentrations tested. ATBC treatment did not cause compromised membrane integrity and did not inhibit ATP production at any of the concentrations tested. The positive control, HU, inhibited follicle growth (24-72 h), decreased follicle cell membrane integrity (72 h), and inhibited ATP production (24-72 h). The purpose of this experiment is to evaluate the effects of oral exposure to ATBC in female CD-1 mice. For the in vivo experiments, the female mice (n=22; PND 81) were randomly divided into treatment groups and dosed according to daily body weight with one of the following treatments: corn oil (vehicle, n=7), 5 mg/kg/day ATBC (n=8), or 10 mg/kg/day (n=7) ATBC for 15 consecutive days. Vaginal smears were performed and analyzed daily to measure any change in estrous cyclicity. After the 15th day of dosing the female mice were introduced into an individually housed proven breeder male’s cage. Daily body weight measurements continued and plug checks were performed every morning. Pregnancy and time to conception data did not statistically differ from the vehicle (oil) for all ATBC treatments (days to conception: vehicle 2.43 ± 0.65, 5 mg/kg/day ATBC 2 ± 0.33, 10 mg/kg/day ATBC 2.5 ± 0.22; gestation length: vehicle 19.71 ± 0.18, 5 mg/kg/day ATBC 20 ± 0.19, 10 mg/kg/day ATBC 20 ± 0.00). On the day of parturition the dams and pups were sacrificed; organ weight and gross morphology data was collected for: uterus, kidneys, adrenals, spleen, liver, and ovaries. The data analyzed includes: estrous cyclicity, pre-dosing body weight % gain, dosing body weight % gain, pregnant body weight % gain, organ weight, gestation length, litter size (live vs. dead), litter weight, implantation sites, time to conception, and pup sex ratio. Interestingly, there was an increase in spleen weight at the 5 mg/kg/day treatment when compared to vehicle control-treated spleen weights (spleen weight, Oil: n=7; 5 mg/kg ATBC: n= 8). Treatment with 5 mg/kg/day ATBC caused a significant decrease in average estrous cycle length in days compared to the pre-dosing average estrous cycle length. Animals exposed orally to 10 mg/kg/day ATBC showed a significant decrease in total follicle number (10 mg/kg/day ATBC, 313 ± 20.37) when compared to vehicle (oil) treated mice (vehicle, 433.71 ± 34.85). Also treatment with 10 mg/kg/day ATBC resulted in significant reduction in secondary (101.67 ± 5.7) and late antral (4.17 ± 1.45) follicle numbers when compared to the vehicle-treated mice (secondary follicles: 141.14 ± 14.79, late antral follicles: 7.00 ± 1.54). ATBC treatment with 10 mg/kg/day showed a significantly decreased mean body weight percent gain during pregnancy on days 3, 5, and 14 when compared to animals treated with vehicle (oil), while animals treated with 5 mg/kg/day ATBC showed a significant decrease in weight gained on only day 13 when compared to the vehicle (oil). These novel findings show that ATBC could disrupt ovarian function in mice when exposed to low-dose ATBC.