Current activities between the DevTox Berlin workshops and the Japanese Teratology Society Terminology Committee in harmonizing the terminology for classifying anomalies in laboratory animals in developmental toxicity studies: Report from the Satellite Workshop of the 60th Annual Meeting of the Japanese Teratology Society.

In recent years, the Japanese Teratology Society has worked with the DevTox Berlin Workshops project to provide internationally consistent terminology for teratogenic effects. This paper summarizes a satellite workshop of the 60th Annual Meeting of the Japanese Teratology Society, which was entitled "Current activities between DevTox Berlin Workshops to develop a harmonized terminology for classifying anomalies in laboratory animals in developmental toxicity studies." The Japanese Teratology Society - Laboratory Animal Terminology Project (JTS-LATP) reviewed "gray zone" anomalies and focused on developing criteria for reclassifying a large number of gray zone anomalies to clarify them and to make it easier to judge fetal categories. This effort will lead to international agreement, based on shared conceptions. The present article aimed to provide the reader with a summary of the issues discussed at the 2020 satellite meeting, which included discussions on open issues from the DevTox Berlin Workshops, ongoing work by the JTS-LATP on gray zone (GZ) anomalies, current industrial concerns, and future challenges.

Initial hazard assessment of 1,4-dichlorobutane: Genotoxicity tests, 28-day repeated-dose toxicity test, and reproductive/developmental toxicity screening test in rats.

1,4-Dichlorobutane (1,4-DCB) is used as raw materials for drugs, pesticides, fragrances, and chemical fibers, and being used as a solvent. Its toxicity data was insufficient for screening assessment under the Japanese Chemical Substances Control Law. We conducted toxicity tests and hazard classification for screening assessment 1,4-DCB showed negative in the Ames test, positive in the in vitro chromosomal aberrations test with metabolic activation, and negative in the in vivo mouse bone-marrow micronucleus test. The 28-day repeated-dose toxicity study, where male and female rats were administered 1,4-DCB by gavage at 0, 12, 60, and 300 mg/kg/day, showed significant effects on the liver and pancreas from 12 mg/kg/day and kidney at 300 mg/kg/day. Based on periportal hepatocellular hypertrophy and decreased zymogen granules in pancreas, the lowest observed adverse effect level (LOAEL) of 12 mg/kg/day was obtained. The reproductive/developmental toxicity screening study, in which male and female rats were administered 1,4-DCB by gavage at dose of 0, 2.4, 12, and 60 mg/kg/day for 42-46 days, showed that the delivery index was decreased at 60 mg/kg/day without maternal toxicity. Based on the general toxicity, we classified this chemical as hazard class 2, with a D-value (Derived No Effect Level) of 0.002 mg/kg/day.

Categorization of fetal external findings in developmental toxicology studies by the Terminology Committee of the Japanese Teratology Society.

Categorization of fetal external findings in common laboratory animals, intended to make the agreement at Berlin Workshop in 2014 more practical, was proposed by the Terminology Committee of the Japanese Teratology Society at the Workshop in the 55th Japanese Teratology Society Annual Meeting in 2015. In the Workshop, 73 external findings, which had been categorized as "Gray zone" anomalies but not as "Malformation" or "Variation" in the 2014 Berlin Workshop, were discussed and classified as Malformation, "Non-structural abnormality," Variation, and "Not applicable." The proposal was based on the results of a survey conducted in 2014, where 20 facilities (including pharmaceutical, chemical, and pesticide companies and contract laboratories) and 2 selected expert teratologists in Japan were asked for their opinions on the categorization of these findings. Based on the discussion, Japanese Teratology Society members have agreed that 42 out of the 73 findings can be classified as Malformations (38), Non-structural abnormalities (3), Malformations/Non-structural abnormalities (1), and Variations (0), while the remaining 31 findings were recommended to be categorized as Not applicable for fetuses. The details of the classification are shown on the website of the Japanese Teratology Society (http://www.umin.ac.jp/cadb/External.pdf).

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.

Morphological features of mutant rat, IS-Tlk/Kyo, fetuses with caudal vertebral anomalies.

IS-Tlk/Kyo, a rat mutant strain derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to a congenital anomaly of the lumbar vertebrae that is a hallmark of IS/Kyo rats. Homozygotes (Tlk/Tlk) of Tlk dominant gene are known to die during embryonic development. The present report deals with the morphological features of heterozygous IS-Tlk/Kyo rat fetuses in comparison with those of IS/Kyo rat fetuses. One of the morphological features was a high incidence of tail vertebral anomalies in IS-Tlk rats (81.6% versus 0% in IS/Kyo rats). Significantly low values in number of live fetuses and ossified 5th sternebra and sacral and caudal vertebrae were observed in IS-Tlk/Kyo rats compared with those in IS/Kyo rats as well as a low incidence of fetuses with ventral septal defects in IS-Tlk/Kyo (0% versus 54.4% in IS rats). These results suggest that the Tlk gene may be involved in the formation of the vertebral centra and the ventral septum when it expresses on the genetic background of the IS rat.

Studies of the toxicological potential of capsinoids: III. A two-generation reproduction study of CH-19 Sweet extract in rats.

CH-19 Sweet extract, containing 66.5 to 75.05 mg/ml capsinoids, was administered once daily by gavage, to two generations of male and female Sprague-Dawley rats, at dose levels of 0 (vehicle), 1.25, 2.5, and 5.0 ml/kg/day (83.13 to 93.81, 166.25 to 187.63, and 332.50 to 375.25 mg/kg as capsinoids, respectively) in order to determine its potential reproductive effects. In the first generation (F(0)) males and females, there were no test substance-related deaths, toxic changes, gross pathological findings, or adverse findings in clinical signs, body weight, or food consumption. There were no test substance-related effects on estrous cycles, copulation index, days required for copulation, fertility index, number of implantations, gestation period, number of liveborn pups, delivery index, stillbirth index, livebirth index, or lactation or nursing. In the second generation (F(1)), there were no test substance-related changes observed in clinical signs, body weights, sex ratios at birth, external abnormalities, differences in survival at any point from birth to weaning, and no deaths after weaning. There were no changes suggestive of adverse test substance-induced effects on body weight, food consumption, or external differentiation after birth, and there was no test substance-related damage on sensory/reflex functions. As with the first generation, there were no test substance-related effects on reproductive indices, in the offspring, no untoward effects on development, viability during the lactation period, body weight, external differentiation, or sensory/reflex functions, and there were no gross morphological abnormalities. Based on these results, the no observed adverse effect level (NOAEL) of CH-19 Sweet extract on the reproductive function and growth of offspring in this two generation study was judged to be 5.0 ml/kg/day (332.50 to 375.25 mg/kg as capsinoids).

Studies of the toxicological potential of capsinoids: IV. Teratology studies of CH-19 Sweet extract in rats and rabbits.

In order to evaluate the safety of CH-19 Sweet extract that contains capsinoids, teratology studies were conducted in pregnant Sprague-Dawley rats (20 rats per group) and pregnant New Zealand white rabbits (17 to 22 animals per group). The test substance was administered to rats by gavage for 11 days on gestation days 7 to 17 at doses of 0 (vehicle), 1.25, 2.5, and 5.0 ml/kg and to rabbits for 13 days on gestation days 6 to 18 at doses of 0 (vehicle), 0.25, 0.5, and 1.0 ml/kg. As the concentration of capsinoids in CH-19 Sweet extract was 72.2 to 75.05 mg/ml, the resulting dose of capsinoids administered to rats was 90.25, 180.5, and 361 mg/kg, and to rabbits was 18.76, 37.53, and 75.05 mg/kg in the vehicle, low-, mid-, and high-dose groups, respectively. In the rat study, no deaths occurred in any group and there were no test substance-related changes or abnormalities in clinical signs, body weight, food consumption, or gross pathological findings. There were no test substance-related changes in the number of corpora lutea, number or index of implantations, index of embryofetal deaths, number of live fetuses, sex ratio, fetal body weight at the end of the gestation period, or abnormalities in the placenta of live fetuses. There were no test substance-related abnormalities or variations in the external, skeletal, or visceral examinations of live fetuses. It was concluded that the test article caused neither teratogenic effects nor abnormalities in the progression of ossification. In the rabbit study, there were no test substance-related effects on clinical signs, body weight, food consumption, or necropsy findings. There were neither test substance-related abortions nor test substance-related effects on the number of corpora lutea, or number or index of implantations. There were no test substance-related effects on the number of dead embryos/fetuses, the number of live fetuses, sex ratio, body weight of live fetuses, or gross pathological finding in the placentas. There were no test substance-related external abnormalities or incidences of visceral or skeletal abnormalities or variations, and there were no test substance-related effects on the progress of ossification in any group. The authors concluded the no observed adverse effect level (NOAEL) of CH-19 Sweet extract containing capsinoids on pregnant animals and fetal development/growth was > 5.0 ml/kg/day (> 361 mg/kg/day as capsinoids) in rats and > 1.0 ml/kg/day (> 75.05 mg/kg/day as capsinoids) in rabbits.

Tissue oxygenation in a murine SCC VII tumor after X-ray irradiation as determined by EPR spectroscopy.

PURPOSE: The goal of this study was to clarify the dynamics of oxygenation (partial pressure of oxygen, pO(2)) in SCC VII murine tumors in mice after X-ray irradiation. MATERIALS AND METHODS: Changes in pO(2) in tumors were measured by 1.2-GHz electron paramagnetic resonance (EPR) spectroscopy after they were exposed to various doses of irradiation. The pO(2) in tumors was followed for up to six days after irradiation at doses of 0, 5, 10, 15, and 20 Gy. Paramagnetic crystals were used as an oximetry probe and implanted into normal or tumor tissues in mice for prolonged periods. RESULTS: The pattern of tumor oxygen after a single dose of radiation with the 5-Gy dose was different from those with other doses (10, 15, and 20 Gy). After 5 Gy, pO(2) increased rapidly (P<0.01, Student's t test) and then returned to the level observed before irradiation by 12h (P<0.01). In contrast, after 10, 15, or 20 Gy, pO(2) increased rapidly by 6h after irradiation, continued to increase until at least 24h (P<0.01), and then gradually decreased. CONCLUSIONS: In tumors that received 5 Gy, post-irradiation increases in pO(2) at 4h after irradiation were detected by EPR oximetry (P<0.01) noninvasively.