Malformation spectrum induced by ketoconazole after single administration to pregnant rats during the critical period - comparison with vitamin A-induced malformation spectrum.

Azole derivatives have teratogenic effects in rodents. In the present study, malformations and their sensitive windows induced by high-dose ketoconazole (KCZ), an azole derivative, without maternal toxicity were investigated. In addition, the malformation spectrum determined was compared with that induced by vitamin A palmitate (VAP). Pregnant rats were administered a single dose of KCZ by oral gavage on specific individual days from gestational days 8 to 15 (GDs 8-15). Maternal animals were subjected to necropsy on GD 20, and the obtained fetuses were examined for external, visceral and skeletal malformations. The malformation spectrum of VAP was identified from available published data (Noda, Sato, and Udaka, 1982) and a complementary study (single administration of VAP at 1 200 000 IU kg(-1) ). Embryonic lethality was observed in dams given KCZ on GDs 9-12 with peak incidence on GDs 10 and 11 with complete resorption. KCZ induced major malformations included cleft palate, digital anomalies, misshapen limbs and unique discontinuous ribs, and the sensitive window for each was identified. Compared with the malformations induced by VAP, unique malformations (e.g. discontinuous ribs by KCZ, neural tube defects by VAP), similar malformations with similar sensitive windows (e.g. digital and limb malformations) and similar malformations with different sensitive windows (e.g. embryonic lethality and cleft palate) were distinguished, suggesting that the mechanisms of several of the types of KCZ-induced malformation are related to excessive vitamin A.

Comparative study on detectability of learning and memory disorder between two water maze tests commonly used in juvenile rat toxicity studies using isoflurane inhaled rat model.

Evaluation of learning and memory is crucial in juvenile animal toxicity studies (JAS) during the development of CNS active drugs, but there are no currently recommended test methods. We compared the ability of the Morris water maze (MWM) and the Biel water maze (BWM) to detect learning and memory disorder (LMD) using rats inhaled isoflurane (IFN). Rats were treated with 1% IFN using inhalation on postnatal day (PND) 7 for 6 h. All rats were subjected to the MWM on PND 33 and the BWM on PND 55/57 (Experiment 1), or the BWM on PND 32/33 and the MWM on PND 54/55 (Experiment 2). On PND 70, the brain was weighed and then neurohistopathology was conducted. There were no IFN-related changes in clinical signs and body weight. In the tests beginning on PND 32/33, the MWM clearly detected IFN-related LMD in both sexes whereas the BWM detected LMD only in males. With an additional benefit of a simpler procedure, the MWM was considered superior to the BMW for JAS. LMD was not detected in both mazes tested from PND 54/55/57, which was considered due to weak effect and/or recovery of brain function with growth. Single IFN inhalation on PND 7 was considered useful as positive control to induce LMD caused by postnatal exposure in rats, but stronger treatment regimens was recommended.

A comparative investigation of fetal skeletal anomalies in rats induced by acetylsalicylic acid with single- and double-staining techniques.

Acetylsalicylic acid (ASA) at single doses of 125, 250, and 500mg/kg was administered to pregnant rats on Gestation Day (GD) 10, and skeletal changes in fetuses harvested on GD 20 and pups on post-natal (PN) Day 21 were evaluated. Changes in cartilage and ossified bones identified by Alizarin Red S single-staining were compared with Alizarin Red S and Alcian Blue double-staining. By the single-staining technique, skeletal abnormalities including fused rib, incomplete ossification of the cervical arch, absent/hemicentric body of thoracic or lumbar vertebra, deformation of lumbar arch, and absent sacral arch were demonstrated in at 250 and 500mg/kg ASA on GD 20. The double-staining technique facilitated identification of additional cartilaginous changes in the vertebrae, paws, and ribs: including discontinuous rib cartilage, fused carpus, and split cartilage of thoracic centrum at same doses. Discontinuous rib cartilage and fused carpus persisted in pups until PN Day 21 demonstrating that these changes were irreversible. With use of the double-staining technique, the incidence of abnormalities at 250mg/kg were dramatically increased, thus this technique was more sensitive for identifying fetal cartilaginous and ossified skeletal changes.

Historical control data on developmental toxicity studies in rats.

Historical control data from prenatal developmental toxicity studies in rats have been used to evaluate whether toxicology outcomes were induced by exposure to a chemical or were within the range of spontaneous variation. These data are also important for monitoring animal characteristics. As a follow-up to historical control data from 1998 to 2010, this study analyzed control data from prenatal developmental studies performed in rats from 2011 to 2015. Data were collected from studies performed by 24 Japanese laboratories, including 15 pharmaceutical and chemical companies and nine contract research organizations, in Sprague-Dawley and two-sub-strains of Wistar Hannover rats. The data included maternal reproductive findings at terminal cesarean section and fetal findings, including incidences of spontaneous external, visceral, and skeletal anomalies. No noticeable differences in maternal reproductive data were observed among laboratories. The inter-laboratory variations in the incidences of fetal anomalies seemed to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, as well as to differences in terminology of fetal alterations. These historical control data may be helpful for adequate interpretation of experimental results and for evaluating the reproductive and developmental toxicities of various chemicals.

The effects of ethylene glycol monomethyl ether on female reproductive system in juvenile rats.

Ethylene glycol monomethyl ether (EGME), which is widely used in various industrial products, is known for adverse effects on the reproductive system in adult rats. However, the effects of EGME on reproductive development in juvenile rats have not been demonstrated. In order to investigate the effects of EGME on the female reproductive system and pubertal development in juvenile rats, EGME was administered to female Sprague Dawley rats from postnatal day 21 to 41 at a dose level of 0, 50, 100, or 300 mg/kg. The animals were examined for general condition, body weight, vaginal opening (VO), estrous cyclicity, and histopathology of reproductive organs. EGME treatment resulted in a prolonged estrous cycle interval characterized by persistent diestrus at 50 mg/kg without effects on body weight, timing of VO, or histology of the reproductive organs. EGME at 100 mg/kg induced decreases in body weight gain, a delay of VO, and irregular estrous cycle with absence of corpora lutea and hypertrophy of uterine epithelium indicating disturbance of the ovulatory process associated with hormonal effect. At 300 mg/kg, there was significant delay of puberty due to severe growth retardation. The present results revealed that irregular estrous cycle is a first indicator of the effects of EGME on the female reproductive system in juvenile rats, with delayed pubertal onset and ovulatory process disturbance at a higher dose.

Impact of globalization under the ICH guidelines on the conduct of reproductive toxicity studies--report on current status in Japan, Europe and the U.S. by questionnaire survey.

We surveyed interpretation of the ICH guidelines concerning reproductive toxicology. Valid responses were obtained from Japan (JPN), Europe (EUR) and the U.S. The results obtained were compared to those at the time of a previous survey targeted at JPN facilities in 1995-1996 as well as compared among all three regions. Compared to the previous survey in Japan, the number of facilities performing toxicokinetics (TK) in rats has slightly increased. This result was considered to represent changes of attitude toward TK in reproductive toxicity studies. Differences in interpretation of the guidelines between JPN, EUR and the US were widely seen. Clear differences were noted in sperm examinations, postnatal tests, fetal examinations, some examinations for F1 animals after culling and TK. Researchers in the West seemed to be interpreting the ICH guidelines more flexibly from the scientific point of view. JPN researchers appeared to interpret the guidelines, including notes, as rigid requirements. Most of the parts which produced different interpretations were the notes in the guidelines. The force of mention in the notes should be defined in the future. In addition, there were doubts about some parts, including notes, which had been found to have become unsuitable for the implementation of studies because of scientific progress or from long experience in using the guidelines. Therefore, updates of the guidelines may be needed in the future as well as the remedy of interpretation by JPN researchers. In JPN, the number of reproductive toxicity studies has decreased. The scanty experience in JPN therefore raises apprehension of appropriate selection and stagnating development of methodology, and might hinder the maintenance of the guidelines. In the future, the cooperation of CROs as well as global collaboration will be essential not only to scientific developments of reproductive toxicology but also updates of the guidelines.

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.

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.

Collaborative work on evaluation of ovarian toxicity. 11) Two- or four-week repeated-dose studies and fertility study of ethylene glycol monomethyl ether in female rats.

The objective of this study was to determine the optimal period of administration for detection of ovarian toxicity in rat repeated-dose toxicity studies. A well-known ovarian toxicant, ethylene glycol monomethyl ether (EGME), was administered to female rats at dose levels of 0, 30, 100, or 300 mg/kg for 2 or 4 weeks (repeated-dose toxicity studies). The same doses were administered to female rats for 2 weeks prior to mating, during mating, and until Day 6 of pregnancy (fertility study). In the repeated-dose toxicity studies, continuous diestrus was observed at > or = 100 mg/kg regardless of period of administration. The alterations of ovarian morphology observed at > or = 100 mg/kg after 2 or 4 weeks of administration were characterized by hypertrophy of the corpora lutea with decreased cellular debris indicating apoptosis, and increased proliferating cell nuclear antigen (PCNA)-negative large atretic follicles. The finding that newly-formed basophilic corpora lutea were scarce in affected animals exhibiting continuous diestrus suggested suppression of ovulation due to hypertrophic corpora lutea. In the fertility study, irregular estrous cycles, prolonged mating periods, lower pregnancy rates and decreased corpora lutea of pregnancy were observed at > or = 100 mg/kg. The irregularities of estrous cycle observed in some animals at 30 mg/kg were minimal. The ovarian histopathological changes in repeated-dose toxicity studies correlated well with impairment of female fertility found in the fertility study. It is concluded that a repeated-dose toxicity study with a treatment period for 2 weeks or longer is sufficient for evaluation of ovarian toxicity induced by EGME.