Seventy-five Crl:CD®(SD)IGS VAF/Plus® rats per sex were assigned to five dosage groups (Groups I through V), 15 rats per sex per group. An additional three rats per sex per group were assigned to Groups I through V for toxicokinetic sample collection. The test substance, T-7706 [Perfluorohexane Sulfonate Potassium Salt (PFHS)], or vehicle, aqueous 0.5% carboxymethylcellulose (CMC), was administered via gavage to male rats once daily beginning 14 days before cohabitation and continuing through the day before sacrifice, after completion of the cohabitation period, after a minimum of 42 days of administration, and to female rats once daily beginning 14 days before cohabitation and continuing through the day before sacrifice, day 21 of lactation (DL 21) or day 25 of presumed gestation (DG 25, rats that did not deliver a litter). Dosages were 0, 0.3, 1, 3 and 10 mg/kg/day. The dosage volume, 10 mL/kg, was adjusted daily on the basis of the individual body weights recorded before intubation. Fl generation pups were not directly administered the test substance or vehicle.
Within each dosage group, rats were assigned to cohabitation, one male rat per female rat.
Rats were observed for viability at least twice each day of the study. Observations for clinical signs of effects of the test substance and deaths were made on the first day of dosage at approximately hourly intervals for the first four hours and at the end of the normal working day. Observations for clinical signs of effects of the test substance and deaths were made on subsequent days daily before dosage and approximately 60 ± 10 minutes after dosage administration and on the day of sacrifice. Once before the first dosage and at least once weekly thereafter, detailed clinical observations were conducted for all male and female rats assigned to the main study.
Body weights were recorded daily during the dosage period and at sacrifice. Feed consumption values for male rats assigned to the main study were recorded weekly during the dosage period. Feed consumption values for female rats assigned to the main study were recorded weekly to cohabitation, on DGs 0, 7, 10, 12, 15, 18, 20 and 25 (if necessary) and on DLs 1, 5, 8 and 15. During cohabitation, individual values were not recorded or tabulated.
Estrous cycling was evaluated in rats assigned to the main study by examination of vaginal cytology beginning with the day after the first administration and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Estrous cycling was evaluated in rats assigned to the toxicokinetic study during the cohabitation period until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ.
Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation, litter sizes and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.
Shortly before scheduled sacrifice, a functional observational battery (FOB) was conducted and motor activity was evaluated on 10 male and 10 female rats per group. On days 14 and 42 of study, blood samples were collected from each male rat assigned to the toxicokinetic sample collection portion of the study and on day 14 of study and DG 21, blood samples were collected from each female rat assigned to the toxicokinetic sample collection portion of the study.
Each litter was evaluated for viability at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily. Pup body weights were recorded on DLs 1, 8, 15 and 22.
Male and female rats assigned to the toxicokinetic study were sacrificed on day 42 of study and DG 21, respectively. Liver weights were recorded. The median liver lobe was shipped for analysis. Blood samples were collected from each fetus and pooled by litter and serum was shipped for analysis. The liver from each fetus was collected, pooled per litter and shipped for analysis. The number of implantation sites was recorded. Male and female rats assigned to the main study were sacrificed after a minimum of 42 days of dosage and on DL 22, respectively. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number of implantation sites were recorded. Gross lesions were examined histologically.
Ten rats per sex per group assigned to functional observational battery and motor activity tests were assigned to histological evaluations. The following organs were individually weighed: liver, kidneys, adrenals, thymus, testes, right epididymis, left epididymis (corpus and caput), seminal vesicles (with and without fluid), prostate, spleen, brain, heart, ovaries and uterus (with cervix). The following tissues or representative samples were retained: brain, small and large intestines, lungs, lymph nodes, peripheral nerve, stomach, kidneys, spleen, thymus, trachea, urinary bladder, spinal cord, liver, adrenals, heart, thyroid/parathyroid, bone marrow, testes, prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland (female rats only) and gross lesions. Histological examination of retained tissues, including reproductive organs, was conducted for the assigned ten rats per sex from the control and high dosage groups. A quantitative evaluation of primordial follicles was conducted for Fo generation female rats. Sperm evaluations (concentration, motility and morphology) were performed for 10 male rats in each dosage group.
At scheduled sacrifice, blood samples were collected from the 10 male and 10 female rats per group assigned to hematology and clinical chemistry sample collection. The following hematologic parameters were evaluated: erythrocyte count, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, total leukocyte count, differential leukocyte count, platelet count, mean platelet volume and cell morphology. Two blood smear slides were prepared for measurements of differential leukocyte count. Plasma samples evaluated for prothrombin time and activated partial thromboplastin time. Sera samples were evaluated for total protein, triglycerides, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, total bilirubin, urea nitrogen, creatinine, creatinine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, calcium, phosphorus, sodium, potassium and chloride.
On DL 22, pups were sacrificed and examined for gross lesions. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Blood samples were collected from five pups per sex per litter from the 10 female rats per group selected for FOB and motor activity assessment, blood sample collection for hematology and clinical chemistry and histological evaluations. Sera was shipped for analysis. The liver from each selected pup was weighed.