Females were bred in a two to one ratio with males overnight and presence of a vaginal plug the next morning was designated gestational day (GD) 1. Pregnant females were housed together according to exposure group to which they were randomly designated. Dams were weighed and dosed on GDs 8, 10, 12, and 14 with 0 (n521), 5 (n510), 25 (n510), or 50 (n510) mg/ kg triphenyl phosphate (TPhP; Sigma-Aldrich, MO, USA) dissolved in MazolaTM corn oil via intraperitoneal (IP) injection. Dams were first weighed and then euthanized by cervical dislocation on GD19, one day prior to natural parturition. Fetuses were immediately removed, blotted, counted, and 484 | PHILBROOK ET AL. placed in a 37 8C incubator for 1 hr to test for viability. The placentas and other maternal organs including liver, kidneys, ovaries, spleen, thymus, and placentas were collected and weighed. Maternal liver was flash frozen in liquid nitrogen and stored at 280 8C. At this time, any resorptions were also noted.

Following 1 hr in the incubator, fetuses were weighed, and crown rump length and anogenital distance (AGD) were measured. Anogenital index (AGI) was calculated by dividing the AGD by the weight of the fetus. Gross morphological defects, with the exception of cleft palate, which was determined for each fetus following euthanasia, were also recorded at this time. AGD was used as a rough estimate of pup sex, which was later confirmed by PCR (described below). Two male and two female fetuses from each litter (wherever possible) were euthanized by decapitation and livers extracted, flash frozen in liquid nitrogen, and stored at 280 8C. All other fetuses were euthanized via CO2 asphyxiation. Following euthanasia, these fetuses were placed in Carnoy’s fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) and stored at room temperature until skeletal staining was performed. All handling and euthanasia of mice were performed in accordance with guidelines set by the Canadian Council on Animal Care and all experimental protocols were approved by the Queen’s University Animal Care Committee.

C57Bl/6 female mice dams (P0)