"2. Materials and methods

2.1. Animals

Crl:CD(SD)IGS BR VAF/Plus® (Sprague Dawley) rats were supplied by Charles River Laboratory (Kingston, NY – male rats; and Raleigh, NC – female rats). Rats were housed individually except during mating and lactation. Rats were maintained on a 12-h light/12-h dark cycle and fed Certified Rodent Diet® #5002 (PMI Nutrition International, St. Louis, MO) ad libitum. Water was provided ad libitum from individual bottles and/or from an automatic watering access system. Bed-o’cobs® bedding (Anderson’s Industrial Products, Maumee, OH) was used as the nesting material.

The study was conducted in a facility accredited with by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Procedures involving the use of laboratory ratswere reviewed and approved by the facility Institutional Animal Care and Use Committee and conformed to the “Guide for the Care and Use of Laboratory Animals” (ILAR, 1996).

2.2. Test substance

The potassium salt of PFBS (K+PFBS, Lot 2, 97.90% pure) was provided by 3M Company (St. Paul, MN). The sample was 97.86% normal chain with 0.04% branched as determined by quantitative 19F NMR. Solutions of K+PFBS were prepared using 0.1% carboxymethyl cellulose in reverse osmosis processed deionized water.

2.3. Study design

The study was performed based on the following guidelines: Organisation for Economic Cooperation and Development (OECD) 416 and USEPA OPPTS 870.3800. The parental-generation (P) consisted of five treatment groups, 30 rats per sex per group. At least one first generation (F1) pup per sex per litter per groupwas selected for continued evaluation at weaning. K+PFBS or vehicle (0.1% carboxymethyl cellulose) was administered by daily oral gavage to the P-generation rats, beginning at 6 weeks of age and at least 70 days before cohabitation, at daily dosages of 0, 30, 100, 300 and 1000 mg/kg using a constant volume of 10mL/kg. The oral route was presumed to be most relevant, and doses were selected based on the results of a

previous oral toxicity study in rats with K+PFBS (Lieder et al., 2009). In addition, gavage dosing delivers amore accurate quantitative dose than other non-parenteral methods. The F1-generation rats were individually identified and given the same dosage level and volume of the test substance as their respective sires and dams beginning at weaning on lactation day (LD) 22. The P- and F1-generation rats were observed for clinical observations, abortions or premature deliveries, and deaths before and 60 min post-dose. Body weights and food consumption values of the male and female rats were recorded weekly and on gestation day (GD) 0, 7, 10, 14, 18, 21 and 25 (if females did not give birth), on LD 1, 5, 8, 11, 15 and 22 for the dams

(terminal sacrifice), and for F1-generation rats on attainment of sexual maturation. Food consumption was recorded weekly but was not recorded during cohabitation and after LD 15, because pups begin to consume maternal food on or about this time.

F1-generation male and female rats were examined for age of vaginal patency or balanopreputial separation beginning on day 28 or 29 postpartum, respectively. Estrous cycling was evaluated by examination of vaginal cytology beginning 21 days before cohabitation and until mating. The cohabitation period was a maximum of 14 days. Evidence of mating was determined by spermatozoa observed in a vaginal smear or a copulatory plug observed in situ, and this was considered GD 0. On the day of sacrifice, stage of estrous was again determined to ascertain if there was a treatment effect and to aid the pathologist in the microscopic evaluation of uterine tissues.

The P- and F1-generation female rats were evaluated for duration of gestation, fertility and gestation indices, number and sex of offspring, number of implantation sites, condition of dam and litter, litter size, viability index, lactation index, percent survival and sex ratio. Second generation (F2) pup body weights were recorded on LD 1, 5, 8, 15 and 22.

At scheduled termination (after cohabitation formales and on LD 22 for females) P- and F1-generation rats were euthanized by carbon dioxide (CO2) asphyxiation, necropsied and examined for gross lesions. The brain, kidneys, spleen, ovaries, testes, thymus, liver, adrenal glands, pituitary, uterus with oviducts and cervix, left epididymis, right epididymis, prostate and seminal vesicles with coagulating glands and fluid were individually weighed. The liver, kidneys, pituitary, adrenal glands, vagina, uterus with oviducts, cervix and ovaries, right testis (initially fixed in Bouin’s solution), seminal vesicles (with coagulating glands), right epididymis and prostate were retained in formalin for histology.

All F2-generation pups culled on LD 22 were euthanized by CO2 asphyxiation and three randomly selected pups per sex per litter were examined for gross lesions. The necropsy included a single cross-section of the head at the level of the frontal–parietal suture and examination for apparent hydrocephaly. The brain, spleen, liver, thymus, and kidneys and thymus from the first of the three randomly selected pups per litter were weighed.

Histological evaluation was performed by a Board Certified Veterinary Pathologist. While liver and kidneys collected from all of the control and treatment groups in P-, F1-, and F2-generation rats were examined microscopically, only the reproductive tissues from 10 randomly selected rats per sex fromthe control and high dosage groups in P- and F1-generations were assessed. Histopathological examination of the testis was conducted in order to identify retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis (including the caput, corpus and cauda)was conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or absence of clear cells in the cauda epididymal epithelium.

Sperm parameters were evaluated in male rats at sacrifice. The P-generation male rats were 106–110 days of age and the F1-generation male rats were 109–120 days of age. A portion of the left cauda epididymis was used for evaluation of cauda epididymal sperm concentration andmotility using computer assisted sperm analysis (CASA). The motility was evaluated by the Hamilton Thorne IVOS. The remaining left cauda epididymis was used to manually evaluate sperm morphology. Sperm morphology evaluations included both the determination of the percentage of normal sperm (in a sample of at least 200) and qualitative evaluation of abnormal sperm. The left testis was used for evaluation of testicular spermatid concentration (10 fields) using the CASA (Hamilton Thorne Biosciences Inc., Beverly, MA). The left testis was weighed both before and after removal of the tunica albuginea

and then homogenized; a sample from the homogenate was stained with an IDENT stain (Hamilton Thorne Biosciences Inc., Beverly,MA) before IVOSanalysis (Hamilton Thorne Biosciences Inc., Beverly, MA).

Uteri of female rats were evaluated at sacrifice. The number and distribution of implantation sites were recorded. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. A quantitative evaluation of ovarian follicles was conducted for F1 female rats.

2.4. Statistical analyses

Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochron, 1967). Continuous data (bodyweights, bodyweight changes, food consumption data, organ weights, duration of gestation, litter averages for pup body weights, percent male pups, pup viability and cumulative survival)were analyzed using Bartlett’s Test of Homogeneity of Variances (Sokal and Rohlf, 1969) and the Analysis of Variance (Snedecor and Cochran, 1967), when Bartlett’s Test was not significant (p > 0.001). If the Analysis of Variance was significant (p < 0.05), Dunnett’s test (Dunnett, 1955) was used to identify the statistical significance of the individual groups as compared to the control. If Bartlett’s Test was significant (p≤0.001), the Kruskal–Wallis Test (Sokol and Rohlf, 1969) was used, when 75% or fewer ties were present. When more than 75% tieswere present, Fisher’s Exact Test (Siegel, 1956)was used. In cases where the Kruskal–Wallis was statistically significant (p < 0.05), Dunn’s Method of Multiple Comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. All other natural delivery data involving discrete data were evaluated using the Kruskal–Wallis Test procedures."

≥6 Days of Estrus

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