Methods

G.1. Dosage Administration

Dosage

Dosage Dosage• Concentration Volume Number

Group (mg/kg/day) (mg/mL) (mUkg) of Rats Assigned Rat Numbers

I 0 (Vehicle) 0 IO 25 19I01 - 19125

II 100 IO 10 25 19126 - 19150

III 300 30 10 25 19151 - 19175

IV IOOO 100 10 25 19176- 19200

a. The test substance was considered I00% pure for the purpose of dosage calculations.

G.2. Rationale for Dosage Selection

Dosages were selected on the basis of a dosage-range study (Argus Research, Protocol 418-023P). It was anticipated that the highest dosage level selected would induce some overt developmental and/or maternal mortality, the intermediate dosage levels would produce minimal observed toxic effects and the lowest dosage level would not produce any evidence of either maternal or developmental toxicity.In the 418-023P study, all rats survived to scheduled sacrifice. Clinical observations considered test substance-related were limited to urine-stained abdominal fur in four rats in the 2000 mg/kg/day dosage group. Rats in the 2000 mg/kg/day dosage group lost weight on DGs 6 to 9 and body weight gains were reduced at several intervals tabulated during the dosage period. Reflecting these effects of the test substance, body weight gain was reduced in the 2000 mg/kg/day dosage group for the entire treatment (DGs 6 to 21) and gestation (DGs Oto 21) periods. Absolute and relative feed consumption values were reduced in the 2000 mg/kg/day dosage group on DGs 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21, and for the entire treatment and gestation periods. Fetal body weights were reduced in the 2000 mg/kg/day dosage group, as compared with the control group. No other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 2000 mg/kg/day. No fetal gross alterations occurred.

G.3. Route and Rationale for Route of Administration

The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure.

Test Substance and Vehicle Potassium Perfluorobutane Sulforate (PFBS or T-7485). The sponsor will provide to the testing Facility documentation or certification of the identity, composition, method of synthesis, strength and purity of the test substance. Vehicle: Aqueous 0.1% carboxymethylcellulose (CMC) (medium viscosity). Neither sponsor nor the study director is aware of any potential contaminants likely to be present in the vehicle that would interfere with the results of this study.

Test System The Crl:CD (SD) IGs BR VAR/Plus rat was selected as the test system: because 1) it is one mammalian species accepted and widely used throughout industry for non clinical studies of developmental toxicity; 2) this strain has been demonstrated to be sensitive to developmental toxins; and 3) historical data and experience exist at the Testing Facility.

Number: Initial population acclimated:140 virgin female rats, Population selected for study: 100 mated female rats (25 per dosage group) The rats will be shipped from Charles River Laboratories, Inc., to the Testing Facillity

Body weight and age: Female rats will be ordered to have body weights of 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights the day after receipt will be recorded.

Sex: Female rats will be given the test substance. male rats of the same source and strain will be used only as breeders and are not considered part of the Test System

Randomization and Cohabitation Upon arrival male and female rats will be assigned to individual housing on the basis of computer-generated random units. After acclimation, virgin female rats will be cohabited with breeder male rats, one male per female rat. The cohabitation period will consist of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Healthy mated female rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures.

Administration The oral gavage route was selected for use because : 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure. Female rats will be given the test substance once daily on days 6 through 20 of presumed gestation. Dosages will be adjusted daily for body weight changes and given at approximately the same time each day. The highest dose should induce some overt developmental and /or maternal mortality. The intermediate dosage levels should produce minimal observed toxic effects. The lowest dosage level should not produce any evidence of either maternal or developmental toxicity. Doses: 0, 100, 300, 1000 mg/kg-day. Animals were checked for viability at least twice daily, general appearance weekly during the acclimatin period and the daily during treatment. Body weight were accessed according to the same schedule. Feed consumption was also monitored on Days 6, 9,12,15, 18 and 20 of gestation.

Mating Performance: Mating will be evaluated daily during the cohabitation period and confirmed by vaginal smear and/or copulatory plug observation

Caesarean-sectioning Observations: The rats will be examined for the number and distribution of: Corpora Lutea, Implantation sites, Live and dead fetuses, early and late resportions

Fetal Observations: Gross external Alterations and sex, body weights and identification (study number litter number, uterine distribution and fixative), soft tissue examination and skeletal examinations (approximately one-half of the fetuses in each litter)

Method of Sacrifice: Rats will be sacrificed by carbon dioxide asphyxiation. Live fetuses will be sacrificed by an i.p. injection of sodium pentobarbital

Necropsy: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation

Scheduled sacrifice: On day 21 of presumed gestation, female rats will be C-sectioned and a gross necropsy of the thoracic, abdominal , and pelvic visera will be performed. The gravid uterus will be excised and weighed.

Rats found dead or moribund: Rats that die or are sacrificed because of moribund condition, abortion or premature delivery will be examined for cause of death.

Thoracic Vertabrae Centrum BIFID

Endpoint Details

Dataset

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