Developmental Toxicity Screening in Rats

All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee (IACUC approval # 02568-026). The University Department of Environmental Health and Safety approved all the protocols for the use of hazardous chemicals in this experiment. Sprague Dawley SD female and male rats of an outbred strain (Harlan) at about 70 and 100 days of age were maintained in ventilated (up to 50 air exchanges/hour) isolator cages (cages with dimensions of 10 ¾″ W×19 ¼″ D×10 ¾″ H, 143 square inch floor space, fitted in Micro-vent 36-cage rat racks; Allentown Inc., Allentown, NJ) containing Aspen Sani chips (pinewood shavings from Harlan) as bedding, and a 14 h light: 10 h dark regimen, at a temperature of 70 F and humidity of 25% to 35%. The mean light intensity in the animal rooms ranged from 22 to 26 ft-candles. Rats were fed ad lib with standard rat diet (8640 Teklad 22/5 Rodent Diet; Harlan) and ad lib tap water for drinking. During the procedures, rats were held in an animal transfer station (AniGard 6VF, The Baker Company, Sanford, ME) that provided an air velocity of about 0.5 inch.
At proestrus as determined by daily vaginal smears, the female rats, (90 days) were pair-mated with male rats (120 days). On the next day, the females were separated and their vaginal smears were examined microscopically and if they were sperm-positive (day 0) the rats were tentatively considered pregnant and then weighed with a digital animal weighing balance to monitor increases in body weight. Vaginal smears were continued for monitoring diestrus status in these rats until day 7. On embryonic day 7 (E-7) these females were weighed to determine if there was a significant increase in (greater than about 10 g) body weight, to confirm pregnancy in sperm-positive females. These pregnant rats were then given daily intraperitoneal injections of any one of the following single chemicals or mixtures with an equal volume of sesame oil (Sigma) on days E-8 through E-14 of gestation [43] (link). Treatment groups were Control, Pesticide (Permethrin+DEET), Plastics (Bisphenol-A, DBP and DEHP), Dioxin (TCDD), and Jet Fuel (JP8 hydrocarbon). The pregnant female rats treated with various mixtures were designated as the F0 generation. When there was a drop in the litter size and the sex ratio of pups in F1 generation of Plastics group, another treatment group was included with only half the dose of Bisphenol-A, DBP and DEHP and this group was designated ‘Low Dose Plastics’ group. Doses, percent of oral LD50, and sources of chemicals for the compounds are given in Table S1A.