Hitoshi Hojo

Historical control data on reproductive abilities and incidences of spontaneous fetal malformations in Wistar Hannover GALAS rats.

ABSTRACT  Wistar Hannover rats, which have recently been introduced into Japan, are expected to be used in reproductive and developmental toxicity studies, yet the accumulation of background data is insufficient. This paper describes our historical data on the reproductive ability of this strain of rat Three lots of sexually matured females (40 each) were received from CLEA JAPAN, Inc. with males of the same strain (30 or 36 each) and mated. A total of 47 dams were killed on gestation day 20 to examine their fetuses. The remaining 71 pregnant females were allowed to deliver spontaneously and observed for common reproductive parameters. The mating and fertility indices of females were both 99.2%. Overall mean numbers of implants and live fetuses at cesarean sectioning were 12.5 and 11.5, respectively. Fetal resorptions and deaths occurred at an incidence of 8.6%. Morphological examinations of fetuses revealed low incidences of spontaneous malformations (each one case of double aortic arch and absent cervical vertebral arch) and a variety of common variations. The followings are overall means of major reproductive parameters obtained from females with live birth: no. of implants, 12.5; no. of pups delivered, 11.8; viability index of pups at birth, 99.8%; and days of age at sexual maturation (vaginal opening and preputial separation), 30.3 and 42.8, respectively. Our present observations confirmed a minimal deviation among 3 lots of animals in terms of reproductive abilities. These results suggest that this strain of rat can be used in reproductive and developmental toxicity studies, although the sensitivity to toxicants remains to be elucidated.

Two‐generation reproduction toxicity study in rats with methoxychlor

A two‐generation reproduction toxicity study was conducted in rats with a reference estrogenic pesticide, methoxychlor, to validate the sensitivity and competency of current guidelines recommended by the United States Environmental Protection Agency; Japanese Ministry of Agriculture, Forestry and Fisheries; and Organisation for Economic Co‐operation and Development for predicting reproductive toxicity of the test compound based on estrogenic endocrine disrupting effects. Both sexes of SD rats were exposed to methoxychlor in the diet at concentrations of 0, 10, 500 and 1500 ppm for two successive generations. The present study has successfully detected estrogenic activities and reproductive toxicities of methoxychlor, as well as its systemic toxicity. Body weights, body weight gains and food consumption of both sexes of animals were suppressed significantly in the 500 and 1500 ppm groups. Typical reproductive toxicities observed in females of these groups included, but were not limited to, prolonged estrous cycle, reduced fertility, decreased numbers of implantation sites and newborns, decreased ovary weights and/or increased incidences of cystic ovary. Uterine weights of weanlings increased significantly in these groups, suggesting that the sensitivity of this parameter for predicting estrogenic ability of the test compound is comparable to that of the uterotrophic assay. Reproductive toxicities of methoxychlor seemed less potent in males than in females. Methoxychlor delayed preputial separation and significantly reduced sperm counts and reproductive organ weights of males of the 500 and/or 1500 ppm groups; however, most males that failed to impregnate females in the same group showed normal fertility when they were re‐mated with untreated females. Neither systemic nor reproductive toxicities appeared in the 10 ppm group.

Two-generation reproduction toxicity study in rats with 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT).

ABSTRACT  DDT, an organochlorine pesticide, has been cited as a representative chemical suspected of having endocrine disrupting effects. In this study, the potential endocrine disrupting activities of p,p′‐DDT, a major component of DDT, were investigated in rats in a 2‐generation reproduction toxicity study in accordance with the most current test guidelines of the Ministry of Agriculture, Forestry and Fisheries in Japan, Organization for Economic Cooperation and Development (OECD) and United States Environmental Protection Agency (USEPA) with some modifications and additions. p,p′‐DDT was given to parental rats at dietary levels of 0, 5, 50 or 350 ppm. Systemic toxicities in the parental animals consisted of tremors and subsequent deaths (females only) and/or pathological alterations of the liver (both sexes of animals) of the 2 higher dose groups. Reproductive and postnatal developmental toxicities were not evident up to the highest dose level except for the decreased pup viability index on postnatal day 21 in the 350 ppm group. Changes in serum estradiol and progesterone levels and/or a delay in male sexual maturation were noted in the 2 higher dose groups in a dose‐dependent fashion, suggesting alterations of endogenous endocrine functions. However, these changes never resulted in substantial reproductive disorders.

Historical control data on prenatal developmental toxicity studies in rabbits

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994–2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter‐laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Historical control data on developmental toxicity studies in rodents.

Historical control data on rodent developmental toxicity studies, performed between 1994 and 2010, were obtained from 19 laboratories in Japan, including 10 pharmaceutical and chemical companies and nine contract research organizations. Rats, mice, and hamsters were used for developmental toxicity studies. Data included maternal reproductive findings at terminal cesarean sections and fetal findings including the spontaneous incidences of external, visceral, and skeletal anomalies. No noticeable differences were observed in maternal reproductive data between laboratories. Inter‐laboratory variations in the incidences of fetuses with anomalies appeared to be due to differences in the selection of observation parameters, observation criteria, classification of the findings, and terminology of fetal alterations. Historical control data are useful for the appropriate interpretation of experimental results and evaluation of the effects of chemical on reproductive and developmental toxicities.

Observation of X-Y chromosomal configuration in spermatocytes of infertile PD strain male rats

ABSTRACT  PD strain male rats that carry an autosomal recessive gene, preaxial duplication (gene symbol: pd), are sterile in the homozygous condition (pd/pd) due to a spermatogenic breakdown in the process of spermatogenesis at the spermatocyte and/or spermatid stage(s), although heterozygotes (pd/+) are normal. In this study, pd/pd males were examined for the presence of abnormal association of the sex chromosomes that might lead to spermatogenic breakdown. Light and electron microscopic observations of the chromosomes at meiotic prophase and metaphase in primary spermatocytes revealed several types of abnormal X‐Y association and configurations in pd/pd males. However, the incidences of the abnormal configuration were comparable to those in pd/+ males. These results suggest that abnormal X‐Y chromosome association in the germ cells is not a significant cause of spermatogenic breakdown in pd/pd males.

Prenatal developmental toxicity studies of 1,1,1‐trichloro‐2,2‐bis(4‐methoxyphenyl)ethane (methoxychlor) in rats and rabbits

ABSTRACT  The studies were conducted in rats and rabbits to elucidate the potential developmental toxicity of methoxychlor in general accordance with the improved Japanese MAFF guidelines (12‐Nousan‐No. 8147, 2–1–18, 2000). Methoxychlor dissolved in corn oil was administered orally to pregnant Jcl:SD rats on gestational days (GD) 6–19 at a dose of 0,1,50, or 150 mg/kg/day and to pregnant Kbl:JW rabbits on GD 6–27 at a dose of 0,1, 15, or 45 mg/kg/day. Maternal animals were killed on the day after the last day of administration for morphological examination of their fetuses with special attention to the reproductive organs.

Lack of Teratogenicity of 3-Chloro-4-(Dichloromethyl)-5-Hydroxy-2 (5H)-Furanone (MX) in Embryonic Mouse Palate Culture in vitro

3‐Chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX) is known as a by‐product of wood pulp manufacture and a contaminant of chlorinated drinking water. Since our previous studies (Teramoto et al., 1998, 1999) demonstrated in a micromass in vitro test a strong inhibitory effect of MX on rat embryo cell differentiation, the potential teratogenicity was investigated in this study by using a suspension organ culture system. Twelve‐day mouse embryo palatal explants were cultivated for 72 hr in the MX‐containing medium at a concentration of 0, 1, 10, 100 or 300 μg/ml and examined for closure of the palatal shelves. All control explants showed almost complete closure of the palatal shelves. Similar results were also obtained in the MX‐treated explants at concentrations up to and including 100 μg/ml. Immunohistochemistry revealed no difference between the control and MX‐treated explants in distribution of PCNA‐and TUNEL‐positive cells in the palatal mesenchyme and medial edge epithelium, respectively. When the MX concentration was raised to 300 μg/ml, palatal shelves remained wide open. However, histopathology revealed extensive pyknosis of the mesenchymal cells and loss of the epithelium. These results may indicate that MX is cytotoxic against the mouse palate at a high concentration, and that it has no cleft‐palate inducing effects in mice.

Fused pulmonary lobes is a rat model of human Fraser syndrome.

Fused pulmonary lobes (fpl) is a mutant gene that is inherited in an autosomal recessive manner and causes various developmental defects, including fusion of pulmonary lobes, and eyelid and digit anomalies in rats. Since these developmental defects closely resemble those observed in patients with Fraser syndrome, a recessive multiorgan disorder, and its model animals, we investigated whether the abnormal phenotypes observed in fpl/fpl mutant rats are attributable to a genetic disorder similar to Fraser syndrome. At the epidermal basement membrane in fpl/fpl mutant neonates, the expression of QBRICK, a basement membrane protein whose expression is attenuated in Fraser syndrome model mice, was greatly diminished compared with control littermates. Quantitative RT-PCR analyses of Fraser syndrome-related genes revealed that Frem2 transcripts were markedly diminished in QBRICK-negative embryos. Genomic DNA sequencing of the fpl/fpl mutant identified a nonsense mutation that introduced a stop codon at serine 2005 in Frem2. These findings indicate that the fpl mutant is a rat model of human Fraser syndrome.

Immunohistochemical Evaluation of Glucocorticoid Receptors in Developing Eyelids of NC-eob Mouse Fetuses with Genetically Determined Open-Eyelid Malformation

NC‐eob mice are mutants having open eyelids at birth. Previous studies have revealed that this defect is related to the absence of epidermal differentiation at the tip of the developing eyelids on gestation day (GD) 16, and that maternal treatment with cortisone acetate (CA), a glucocorticoid compound, which has an ability to accelerate epidermal differentiation, is not effective against the epithelium in NC‐eob fetuses although the eyelid growth is slightly enhanced.