Journal of Toxicology Laboratory for Ethics in Animal Studies in compliance with “Guidelines for Animals Studies (Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Kamisu-shi, Ibaraki, Japan).” 2.4. Experimental Design 2.4.1. Developmental Toxicity Study (1) Exposure and Observations. On gestational days (GDs) 6–19, three groups of 22 pregnant females were administered doses of SE5-OH in 1% aqueous HPMC by gavage at levels of 200, 1000, and 2000 mg/kg/day, respectively; the control group (22 pregnant females) was administered 1% HPMC vehicle only. Animals were observed twice daily for clinical signs during the dosing period, and once daily following the dosing regimen. Body weights and gross weights of the diets were measured on GD 0, 6, 8, 10, 12, 14, 16, 18, and 20. Body weight gain was calculated on the basis of body weights measured on GD 6. (2) Necropsy. On GD 20, dams were euthanized, the gravid uterus was collected from each dam and weighed to allow for calculation of corrected body weight and corrected body weight gain. Corrected body weight was determined by subtracting the gravid uterine weight from the body weight on GD 20, while the corrected body weight gain was calculated by subtracting the body weight on GD 6 from the corrected body weight. At necropsy of the dams, thoracic and abdominal organs and tissues were examined macro- scopically. Any organs or tissues showing abnormalities were collected and preserved. If such collections were conducted, the same tissues were collected from three control dams and also preserved. Ovaries and uterus were removed and examined for the number of corpora lutea, implantations, live fetuses, and dead embryos. Early embryonic deaths were considered to be represented by the number of implantation sites plus placental remnants; late embryonic deaths were represented by macerated embryos plus dead fetuses. Live fetuses were weighed, euthanized, sexed, and exam- ined (external and oral cavity examinations). Approximately half of the live fetuses in each litter from the control and the 2000 mg/kg dose dams were subjected to visceral examination, which included the head, neck, thorax, and abdomen. The remaining fetuses were examined for skeletal anomalies and degrees of ossiﬁcation in cervical vertebral bodies, sternebrae, metacarpi, metatarsi, and sacral/caudal vertebral bodies. 2.4.2. Reproductive Toxicity Study. Groups of 52 rats (26/sex/ group) served as the F0 generation and were administered the same SE5-OH test article at the same doses via gavage as in the developmental toxicity study (i.e., 0, 200, 1000, and 2000 mg/kg/day). Dosing commenced at six weeks of age and lasted for ten weeks before mating and then throughout the mating period. The mating procedure involved the placement of one male and one female in the same cage for a maximum of two weeks, with reproductive capacity 3 judged by copulation index, fertility index, precoital period, gestation period, and gestation index. F0 females that became pregnant continued on this exposure regimen through gestation and lactation until weaning of the F1 animals. The F1 generation males and females were also administered the same dosing regimen of SE5-OH, beginning on postnatal day (PND) 21, for ten to eleven weeks before mating and throughout the mating period, with pregnant females continuing on the dosing schedules through gestation and lactation until weaning of the F2 generation. Throughout this entire process, animals were observed twice daily for clinical signs; body weights and food consumption were measured weekly until the start of gestation in both F0 and F1 generations. Estrous cycle was examined for three weeks before the start of mating in both F0 and F1 females. During gestation, females in both generations were weighed on GD 0, 3, 7, 10, 14, 17, and 20 and then on lactation day (LD) 0, 4, 7, 10, 14, 17, and 21 during lactation. Food consumption was also measured during GD 0, 7, 14, and 20 and on LD 0, 4, 7, 14, and 21. On the day after the animals reached the end of their respective exposure periods, they were euthanized for gross necropsy. The following tissues were weighed and preserved: adrenals, brain, kidney, liver, female repro- ductive tissues (uterus, ovaries), male reproductive tissues (testes, epididymis, prostate, and seminal vesicles including coagulating glands), pituitary, spleen, thymus (neonatal only), and thyroid gland. Histological examinations were conducted on tissues from control and high- (2000 mg/kg) dose groups only and included adrenals (F0 females were examined in all dose groups), male and female reproductive organs, pituitary, and liver tissues from F0 females. Sperm was also collected from the cauda epididymis of all F0 and F1 males and evaluated for motility, and the sperm from the control and 2000 mg/kg dose group were also evaluated for spermatid cells (testis), sperm number, and morphology. Clinical parameters for oﬀspring included examination for external anomalies, sex ratios, live birth index (i.e., number of live pups/number of total pups born), as well as viability index on PND 4 and weaning index on PND 21; pup weights were measured at birth as well as PND 4, 7, 14, and 21. On PND 4, litter sizes were adjusted randomly to eight pups per dam. Pups were also examined for physical and functional developmental parameters, which included incisor eruption, eye opening, reﬂexes for righting, auditory startle, pain, corneal, and pupillary reactions. Sexual maturation was determined in F1 rats by recording the age and weight on the day of vaginal opening after PND 27 was reached or when balanopreputial separation from PND 35 was recorded. At gross necropsy of F1 and F2 weanlings, brain, spleen, and thymus were collected from selected animals for preservation (one male and one female selected from each litter in numerical order of the oﬀspring number in each litter, which was randomly assigned). Ovaries, vagina, and uterus were collected from females, while testes, epididymis, seminal vesicles, and prostate were collected from males. Organ weights were determined from one animal of each sex per litter for brain, thymus, spleen, and uterus.