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.

G.4. Frequency of Administration

Appropriate dosages of the test substance or vehicle were administered orally (via gavage) once daily to rats on DGs 6 through 20. The dosage volume was adjusted daily on the basis of the individual body weights recorded before intubation. The rats were intubated once daily at approximately the same time each day.

G.5. Method of Study Performance

After acclimation, 140 healthy virgin female rats were placed into cohabitation with 140 breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum of five days. Mating performance was evaluated daily during the cohabitation period. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug in situ were considered to be at DG 0 and assigned to individual

housing. Rats were observed for viability at least twice each day of the study. Rats were also examined for clinical observations and general appearance weekly during the acclimation period and on DG 0. Observations for clinical signs of effects of the test substance, abortions, premature deliveries and deaths were also made daily before and approximately 60 ± 10 minutes after dosage administration and on the day of scheduled

sacrifice. Body weights were recorded weekly during the acclimation period, on DG 0, daily during the dosage period and prior to sacrifice. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18, 20 and 21.

G.6. Gross Necropsy

In order to minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group. All surviving rats were sacrificed by carbon dioxide asphyxiation on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The gravid uterus was excised and weigheda. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses and early and late resorptions. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Each fetus was removed from the uterus, placed in an individual container and identified with a tag noting the study number, litter number, uterine distribution and fixative. Each fetus was subsequently weighed and examined for sex and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Late

resorptions were examined to the extent possible. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection technique of Staples03>. These fetuses were fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning; head sections were retained in alcohol. The decapitated carcasses were discarded. The remaining fetuses (approximately one-half of the fetuses in each litter) were examined for skeletal alterations (bone and cartilage) after staining with alizarin

red s<14)_ The fetuses were initially fixed in alcohol; skeletal preparations were retained in glycerin with thymol added as a preservative.

Rats that died or were sacrificed because of premature delivery were examined for the cause of death on the day the observation was made. The rats were examined for gross lesions. Pregnancy status and uterine contents of female rats were recorded. Gravid uterine weights were recorded unless precluded by autolysis. Delivered pups and fetuses in uteri were examined to the extent possible, using the methods described for term

fetuses . Uteri of apparently nonpregnant rats will be examined while being pressed between glass plates to confirm the absence of implantation sites.a. See APPENDIX D, item 2.

G.7. Data Collection and Statistical Analyses

Data generated during the course of this study were recorded either by hand or using the Primedica Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized and/or statistically analyzed using the Primedica Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel [part of Microsoft Office 97 (version SR-2)] and/or the SAS System (version 6.12).Averages and percentages were calculated. Litter values were used where appropriate.The following schematic represents the statistical analyses of data:

Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution<15>_

Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances06> and the Analysis of Variance<l7), when appropriate [i.e., Bartlett's Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p~0.05), Dunnett's Test08> was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p~0.001)], the Kruskal-Wallis Test09> was used,when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p~0.05), Dunn's Method of Multiple Comparisons<20> was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test<21> was used to analyze the data. Count data obtained at Caesarean-sectioning of the darns were evaluated using the procedures described above for the Kruskal-Wallis TestO9>.

Ossification Sites, Sternum Xiphoid

Endpoint Details

Dataset

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