"All animals were observed for mortality and signs of overt toxicity twice each day (morning and afternoon), 7 days a week. During the study, detailed clinical observations were performed on days 1, 3, and 5 of week 1 and days 1 and 5 of week 2 during treatment on Phase A animals and on days

0, 6 (before exposure), 7, 10, 15, and 20 of gestation for Phase B animals. For Phase A, individual body weights were recorded on exposure days 1 (before initial exposure) and 5 of week 1 and exposure days 1 and 5 of week 2. Individual maternal body weights were recorded on gestation days 0, 6, 7, 10, 15, and 20, with day 0 being the day evidence of copulation (sperm in vagina or a vaginal plug) was found. Hematological tests were conducted at study termination on 5 male rat group, 5 nonpregnant female rats/group and 5 pregnant female rats/group. Blood samples were obtained via puncture of the orbital sinus plexus. Food and water were available before blood sample collection (i.e., animals were not fasted before bleeding). Hematological measurements were determined on whole blood and included total leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelets, reticulocyte count, and differential leukocyte count. Hematological indices, which included mean corpuscular hemoglobin concentration (MCHC), were automatically calculated by the analyzer. All surviving rats (pregnant and nonpregnant) were killed by intraperitoneal sodium pentobarbital anesthesia and exsanguination through the abdominal aorta. Body weights were measured and recorded at the time of killing. Each animal received a complete postmortem examination under the direct supervision of a pathologist. The following tissues were trimmed and weighed for all terminally sacrificed animals: lung and trachea, liver, kidneys (right and left), spleen, and both testes and epididymides. Representative samples of a complete list of tissues from all animals (pregnant and nonpregnant) were collected and placed in phosphate-buffered neutral formalin for fixation. Hematoxylin and eosin (H&E)-stained paraffin sections of the following tissues from all animals (pregnant and nonpregnant) were prepared and examined microscopically: lungs (all lobes), liver, both kidneys, trachea, ovaries, uteri, spleen, testes, and epididymides.

On gestation day 20, all surviving females were killed, and tissues and organs were preserved as described previously. The number and location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed, and the fetuses were removed. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for confirmation of pregnancy. The fetuses were individually examined for external malformations, including the palate and eyes. Fetal findings classified as malformations were recorded.

Statistical analysis

For the subacute and subchronic studies, data was analyzed by the method described by Gad and Wei. The results of measurements of all quantitative continuous variables, such as body weight, hematology, and clinical chemistry data (except for body weight data in the subacute study) were intercompared (exposure groups to controls) by the use of the following tests: Bartlett's homogeneity of variance, analysis of variance, and Duncan's procedure. The latter was used if F for analysis of variance was significantly high to delineate which groups differ from the controls. If Bartlett's test indicated heterogeneous variance, the Fmax test was used for each group versus the control. If these individual Fmax tests were not significant of N1 = N2, Student's t-test was used; if significant, the means were compared by the Cochran t-test or the Wilcoxon rank sum test. Body weights on the subacute study were compared using Dunnett's procedure."

F1 Male and Female CD Rats