"Materials and Methods

Animals and Husbandry

The methodology was comparable for each phase with the exception of the dosage levels. Eighty Crl: CD1(ICR; Charles River Laboratories, Inc, St Constant Canada or Kingston, New York) mice were used in each phase. Mice were approximately 61 to 63 days of age and weighed 24 to 33 g at arrival to the Testing Facility. They were bred with male mice of the same strain and source that were maintained as a breeding colony at the Testing Facility. Female F0-generation mice were given temporary numbers at receipt and given permanent identification numbers when assigned to the study on the basis of body weights obtained on the first day of gestation (day 0). Female mice were permanently identified using a tail tattoo. Pups were not individually identified during the lactation period; all parameters were evaluated in terms of the litter. At weaning, F1-generation mice were identified by tail tattoo. All husbandry, cage sizes, and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.12 The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% high-efficiency particulate absorption filters. Room temperature and humidity were monitored constantly throughout the studies. Room temperature was targeted at 64F to 79F (18C-26C); relative humidity was targeted at 30% to 70%. F0-generation mice were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female mice was housed in the male mouse’s cage. Each dam and delivered litter were housed in a common nesting box during the postpartum period. After weaning (postpartum day [PPD] 20), F1-generation mice were housed in nesting boxes. Mice were pair housed until at least PPD 27, after which they were individually housed. An automatically controlled 12-hour light–12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (+30 minutes). Cages were changed approximately every other week. Bedding was changed as often as necessary to keep the mice dry and clean. Mice were given ad libitum access to Certified Rodent Diet #5002 (PMI Nutrition International, Inc, St Louis, Missouri) in individual feeders. Local water that had been processed by passage through a reverse osmosis membrane (RO water) was available to the mice ad libitum from individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat. Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, Ohio) was used as the nesting material. Each lot was certified for contaminates by the Testing Facility.

Test Substance

PFHxA Ammonium Salt, a granular white solid, is also known as C-1500N and Ammonium Salt of Perfluorinated Hexanoic Acid. The test substance was supplied as a 50% aqueous solution in RO deionized water. The aqueous solution was a colorless liquid. Purity was 93.4%. Solutions of the test substance were prepared once weekly at the Testing Facility and stirred continuously for at least 24 hours prior to dosage administration and stored at room temperature. The vehicle (RO water) was available from a continuous source at the Testing Facility and maintained at room temperature. The study samples analyzed were within the acceptance criteria of +10% of their mean nominal concentrations. For homogeneity, the relative standard deviation for the formulation for the grand mean of the average value for the top, middle, and bottom formulations for each group was +5%. Homogeneity results showed that the formulation technique used produced homogenous preparations. Stability was demonstrated in a separate study for 10 days at room temperature from 7 to 70 mg/mL.

Mating and Treatment

After acclimation of approximately 1 week, 100 virgin female mice were cohabitated with 100 breeder male mice, 1 male mouse per female mouse. The cohabitation period consisted of a maximum of 5 days. Female mice with a copulatory plug observed in situ were considered to be at gestation day (DG) 0 and assigned to individual housing. F0-generation female mice were administered the test substance and/or vehicle once daily via oral gavage from DG 6 through DG 18. Dosages were adjusted daily for body weight changes and given at approximately the same time each day. Dams in the process of delivering pups were not adminstered the test substance or vehicle in order to preclude possible disruption to maternal behavior and/or cannibalization of the pups. F1-generation pups were not directly administered the test substance and/or vehicle but may have been possibly exposed to the test substance and/or vehicle during maternal gestation (in utero exposure) or via maternal milk during the lactation period. The treatment regimen for each of the 2 phases is summarized in Table 1. F0-generation mice were observed for viability at least twice each day of the study and for clinical observations and general appearance once weekly during acclimation and on DG 0. The mice were also examined for clinical observations, abortions,

premature deliveries, and deaths prior to dosage administration and between 1 and 2 hours after dosage administration and once daily during the postdosage period. Body weights were recorded once weekly during the acclimation period, on DG 0, and daily during the dosage and postdosage periods. F0-generation mice were evaluated for adverse clinical signs observed during parturition, duration of gestation, litter sizes (all pups delivered) and pup viability at birth, fertility index (percentage of matings that result in pregnancies), gestation index (percentage of pregnancies that result in birth of live litters), number of offspring per litter (live and dead pups), number of implantation sites, general condition of dam and litter during the postpartum period, viability indices (percentage of pups born that survive 4 and 7 days), and lactation index (percentage of pups born that survive 20 days). Maternal behavior was evaluated on PPDs 0, 4, 7, 14, and 20. Day 0 of lactation (postpartum) was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Each

litter was evaluated for viability and general appearance at least twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily during the preweaning period. Pup body weights were recorded on PPDs 0 (birth), 4, 7, 14, and 20. During the preweaning period, pups were evaluated for eye opening, beginning on PPD 10. F1-generation mice postweaning were observed for viability daily. These mice were also examined for clinical observations and general appearance once daily. Body weights were recorded weekly. Female mice were evaluated for the age of

vaginal patency, beginning on PPD 20. Male mice were evaluated for the age of preputial separation, beginning on PPD 26.

Gross Necropsy

All mice were necropsied and gross lesions were retained in neutral-buffered 10% formalin for possible future evaluation. Unless specifically cited subsequently, all other tissues were discarded. F0-generation mice were killed by carbon dioxide asphyxiation. Pups were killed by an intraperitoneal injection of sodium pentobarbital (pups 14 days of age) or by carbon dioxide asphyxiation (pups 15 days of age). At weaning of the F1-generation litters, 5 livers per group from the F0-generation mice were excised, weighed, and frozen on dry ice. Livers were maintained frozen (70C) for analysis. Mice that did not deliver a litter were killed on DG 23 and examined for gross lesions. The number and distribution of implantation sites were recorded after staining with 10% ammonium sulfide9 to confirm the absence of implantation sites. Dams with no surviving pups were killed after the last pup was found dead or missing, presumed cannibalized. A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed and implantation sites were recorded after staining with 10% ammonium sulfide.13 Mice that died before scheduled termination were examined for the cause of death as soon as possible after the observation was made. The mice were examined for gross lesions. The lungs, trachea, and esophagus were perfused and saved in neutral-buffered 10% formalin for possible future evaluation. The heart, kidneys, stomach, and spleen were retained in neutral-buffered 10% formalin for possible histological evaluation. Gravid uterine weights were recorded. Pregnancy status

and uterine contents of female mice were recorded. Conceptuses in utero were examined to the extent possible, using the same methods described for term fetuses/pups. The livers were excised, weighed, and frozen on dry ice. Livers were maintained frozen (70C) until shipped for analysis to Charles River Preclinical Services Montreal. Pups that died before initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Pups found dead were examined for gross lesions and for the cause of death as soon as possible. All pups found dead on PPD 1 to 3 were preserved in Bouin solution for possible future evaluation; all pups found dead on PPD 4 to 20 were preserved in neutral-buffered 10% formalin. On PPD 20, all pups not selected for continued evaluation were killed by carbon dioxide asphyxiation and examined for gross lesions. Necropsy of the pups included a single cut at the

suture of the frontal and parietal bones of the skull, and the cross-sectioned brain was examined for hydrocephaly. One male mouse that died before scheduled termination was examined for the cause of death as soon as possible after the observation was made. The mouse was examined for gross lesions. The heart, kidneys, lungs, stomach, and spleen were retained in neutral-buffered 10% formalin for possible histological evaluation. The liver was excised, weighed, and frozen on dry ice. Five F1-generation mice per sex per group (total 40 mice) in each phase were killed on PPD 41 for sample collection for determination of body burden. Blood samples (0.5-1.0 mL) and livers were collected from these mice. Blood samples were collected via the vena cava after killing. The blood samples were transferred into uncoated (red top) tubes and spun in a refrigerated (4C) centrifuge for 10 minutes at 3500 rpm. The resulting serum was transferred into appropriately labeled polypropylene tubes. All samples were frozen on dry ice as soon as possible and maintained frozen (70C) for the analysis. The remaining mice were killed by carbon dioxide asphyxiation on PPD 41. A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.

Statistical Analyses

Averages and percentages were calculated. Litter values were used where appropriate. Clinical observations and other proportional data were analyzed using the variance test for homogeneity of the binomial distribution.14 Continuous data, such as body weights, organ weights, percentage of litter reaching a developmental landmark, and percentage of mortality per litter, were analyzed as described under the parametric heading of the schematic. Bartlett test of homogeneity of variances15 was used to estimate the probability that the dosage groups had different variances. A nonsignificant result (P > 0.001) indicated that an assumption of

homogeneity of variance was not inappropriate, and the data were compared using the analysis of variance.16 If that test was significant (P 0.05), the groups given the test substance were compared with the control group using Dunnett test.17 If Bartlett test was significant (P > 0.001), the analysis of variance test was inappropriate and the data were analyzed as described under the nonparametric heading of the schematic. When 75% or fewer of the scores were tied, the Kruskal-Wallis test18 was used to analyze the data, and in the event of a significant result (P 0.05), Dunn method of multiple comparisons 19 was used to compare the groups given the test substance with the control group. When more than 75% of the scores were tied, Fisher exact test20 was used to compare the

proportion of ties in the dosage group. Variables with graded count scores, such as litter size, were analyzed using the nonparametric procedures described previously. The pup weights for all weigh days were covaried against litter size at birth.21

F1 Male Crl:CD1 Mice (Phase 2)