4 2.5. Statistical Analyses. Statistical analysis for the repro- ductive and developmental toxicity study was conducted using several diﬀerent tests, using the toxicological data processing system (MiTOX, Mitsui Zosen Systems Research, Inc., Japan). The data of oﬀspring obtained before weaning were analyzed on the basis of litter mean values except for the sex ratio. The normality of the data was assumed, unless otherwise indicated. Homogeneity of variance was tested by Bartlett’s test; when the data were homogeneous, a one-way analysis of variance (ANOVA) was performed for statistical comparison. Heterogeneous data were tested by the Kruskal- Wallis test. When a signiﬁcant intergroup diﬀerence was found, Dunnett’s or the Dunnett-type multiple comparison test was used. For some of the data (e.g., days until copula- tion, number of estrus stages without copulation, gestation length, sperm motility, birth index, viability index on PND 0, 4, and 21, incidence of oﬀspring with external anomalies, postnatal physical development, reﬂex and sensory function, as well as sexual maturation (vaginal opening and cleavage of balanopreputial gland)), the Kruskal-Wallis test was applied ﬁrst, and when a signiﬁcant intergroup diﬀerence was found, the Dunnett-type multiple comparison test was used. The multiple comparison test was utilized to evaluate statistical signiﬁcance of changes in body weight, body weight gain, food consumption, organ weights, number of implantations, number of oﬀspring, number of live oﬀspring, and estrous cycle lengths. However, for the spermatid count and sperm count, the homogeneity of variance was tested by the F test, and when the variance was homogeneous, Student’s t-test was performed for the statistical comparison. When heterogeneous, the Aspin-Welch t-test was used. Moreover, Wilcoxon’s rank sum test was used for comparison of the incidence of abnormal sperm (morphologically abnormal sperm and tailless sperm), visceral examination, and skeletal examinations. The categorical data (e.g., copulation index, fertility index, gestation index, sex ratio (male/female), incidence of females with irregular estrous cycles, and dams with external abnormal oﬀspring) were analyzed by Fisher’s exact probability test. All statistical tests used two-tailed signiﬁcance level of 5%. 3. Results 3.1. Developmental Toxicity Study 3.1.1. Eﬀects on Dams. With the exception of a transient lack of weight gain (see Figure 2), no abnormal clinical signs were observed in any of the dams. This included observations for death, moribundity, abortion, or premature delivery (data not shown). In dams exposed to 2000 mg/kg, signiﬁcant decreases in body weight gain were noted on GD 8–16 but these values had returned to control levels by GD 20. The same transient lack of weight gain was noted for dams in the 1000 mg/kg exposure group, but only on GD 8. No treatment-related eﬀects were seen for food consumption, gravid uterine weights, corrected body weight, and corrected body weight gain; and there were no abnormalities seen at necropsy (data not shown). 160 140 ) sm120 a r (g 100 n i tag 80 h ieg 60 w yd 40 o B 20 0 6 Journal of Toxicology ∗ 8 ∗ ∗∗ ∗∗ ∗∗ ∗∗ 10 12 14 Time (gestational days) 16 18 20 Dose = 0 mg/kg/day Dose = 200 mg/kg/day Dose = 1000 mg/kg/day Dose = 2000 mg/kg/day Figure 2: Changes in body weight gain (grams) observed in dams exposed to SE5-OH during gestational days 0–20 during the developmental toxicity study in rats; signiﬁcant when compared to control: ∗P < .05; ∗∗P < .01 3.1.2. Eﬀects on Embryo-Fetal Development. In all treated groups, there were no signiﬁcant diﬀerences in number of corpora lutea, implantations, dead embryos, postimplanta- tion losses, live fetuses, fetal deaths, fetal weight, or sex ratio. Several isolated embryo-fetal eﬀects were noted during the placental observations, and external, visceral, and skeletal examinations. These changes are summarized in Table 2. 3.2. Reproductive Toxicity Study 3.2.1. Parental Animals. Transient decrease of weight gain and reduced food intake in the F0 and F1 females of 2000 mg/kg/day, and histological change of adrenal cortex (a diﬀuse increase in lipid droplets in the zona glomeru- losa) in the F0 females of 2000 mg/kg/day were noted (see Table 3). Food intake was reduced in the F0 females in the 1000 mg/kg/day group. In addition, increased liver weight in the F0 female of 1000 and 2000 mg/kg/day dose groups, increased adrenal weight in the F0 males at the 2000 mg/kg/day dose group, as well as increased thyroid weight in the F1 males (see Table 4) was noted. No adverse clinical signs (including death, moribundity, abortion, or premature delivery), necropsy ﬁndings, or organ weights were observed in any of the F0 and F1 parental animals. There were no adverse reproductive eﬀects observed in any treatment group of F0 and F1 animals in terms of estrous cycle, copulation index, fertility index, or precoital period (see Tables 5 and 6). No adverse eﬀects were seen in gestation period, gestation index, or parturition. No eﬀect on implantation number was found in the F0 generation although the number of implantations was slightly, but signiﬁcantly, reduced in the F1 dams at 2000 mg/kg/day, there were no eﬀects on implantation in the 200 and 1000 mg/kg/da F1 generation (see Tables 7 and 8). The decrease in implantation number in the high dose did not signiﬁcantly aﬀect the birth index or the number of