Tocopherol stripped corn oil

CD-1 male and female mice were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were housed in the animal care facility at the University of Illinois at Urbana-Champaign under 12-hour light/dark cycles and allowed to acclimate to the facility for at least two weeks before use. The female dams were mated with male breeders at two months of age. Successful mating was confirmed by the presence of a vaginal sperm plug. The pregnant female mice were dosed with 20 µg/kg/day, 200 µg/kg/day, 200 mg/kg/day, or 500 mg/kg/day of the phthalate mixture in tocopherol-stripped corn oil (MP Bio Medicals, OH) from gestational day 10.5 to the day of birth. Doses of the phthalate mixture were chosen to cover a wide environmentally relevant range and to include some of the doses of individual phthalates that were shown to adversely affect reproductive health (Manikkam et al., 2013 (link); Rattan et al., 2017 (link); Zhou et al., 2017 (link)). The exposure window was chosen because it is a critical period for gonadal development in the mouse. The mice were dosed orally using a pipet tip into the mouth. All procedures involving animal care, euthanasia, and tissue collection were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign.

On postnatal day 35, animals (n=8) were randomly assigned to receive tocopherol-stripped corn oil (vehicle; MP Biomedicals, Solon, OH) or dibutyl phthalate (dissolved in vehicle, 99.6% purity, Sigma-Aldrich, St. Louis, MO) at 0.01, 0.1, and 1000 mg/kg/day. Animals were weighed and dosed daily for 10 consecutive days. All doses were administered orally by placing a pipette tip containing the dosing solution into the mouth past the incisors and into the cheek pouch. Doses were selected to approximate the tolerable daily intake (0.01 mg/kg/day; European Food Safety Agency) and the oral reference dose for DBP (0.3 mg/kg/day; [21 ]), as well as, a high dose (1000 mg/kg/day) to model toxicity at higher concentrations. DBP dosing did not cause overall toxicity as determined by the lack of differences in body and main organ weights between vehicle- and DBP-treated mice. A slight decrease in liver/body weight ratio in the absence of body weight change was observed in animals treated with DBP at 0.01 mg/kg/day (see Supplementary Table 1). Animals showed no other signs or symptoms indicative of overall toxicity. Most animals (all groups) were in the stage of diestrus at euthanasia, thus, only tissues from mice in diestrus were used for subsequent hormone and gene expression analyses.

All chemicals were obtained from Fisher Scientific (Hanover Park, IL) unless otherwise indicated. Acetyl tributyl citrate (ATBC, CAS 77-90-7), phosphate buffered saline (PBS), and sodium hydroxide (NaOH) were obtained from Sigma-Aldrich (St. Louis, MO). Tocopherol-stripped corn oil was obtained from MP Biomedicals (vehicle, Solon, OH).