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.

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).

Continuing harmonization of terminology and innovations for methodologies in developmental toxicology: Report of the 8th Berlin Workshop on Developmental Toxicity, 14-16 May 2014.

This article is a report of the 8th Berlin Workshop on Developmental Toxicity held in May 2014. The main aim of the workshop was the continuing harmonization of terminology and innovations for methodologies used in the assessment of embryo- and fetotoxic findings. The following main topics were discussed: harmonized categorization of external, skeletal, visceral and materno-fetal findings into malformations, variations and grey zone anomalies, aspects of developmental anomalies in humans and laboratory animals, and innovations for new methodologies in developmental toxicology. The application of Version 2 terminology in the DevTox database was considered as a useful improvement in the categorization of developmental anomalies. Participants concluded that initiation of a project for comparative assessments of developmental anomalies in humans and laboratory animals could support regulatory risk assessment and university-based training. Improvement of new methodological approaches for alternatives to animal testing should be triggered for a better understanding of developmental outcomes.

Investigation for methods of anesthesia and euthanasia for rat fetuses in developmental toxicity studies.

Japan Association for Laboratory Animal Medicine (JALAM) recommends humane handling of rat fetuses. However, it is a challenge to accept proposed euthanizing methods such as cervical dislocation, decapitation and/or intracardiac injection of potassium chloride, because these methods would damage fetal specimens for skeletal and visceral examinations in developmental toxicity studies. The present study aimed at seeking better methodologies for fetal euthanasia and anesthesia. We were unable to accomplish fetal euthanasia directly, but instead, we could euthanize fetuses under pain-controlled anesthesia. It is recommended that hypothermia by immersion in cold physiological saline is an appropriate method for anesthesia. Moreover, we recommend that the anesthetized fetuses should be euthanized immediately by removal of the vital organs or immersion in appropriate fixatives.

Effects of maternal exposure to thiamazole on behavioral development in infant cynomolgus monkeys.

Thiamazole, an anti-hyperthyroidism agent, was administered orally to pregnant cynomolgus monkeys at doses of 2.0 and 3.5 mg/kg per day from GD 120 to GD 150 to investigate effects on behavioral development of their infants. Swelling of the throat region due to enlargement of the thyroid glands was observed at birth in thiamazole-treated infants, and it returned to normal around postnatal day (PND) 30. At necropsy of infants at 12 months of age, thyroidal weight in the thiamazole groups was increased. This finding suggested the likelihood that administration of thiamazole to maternal animals during the late gestational period induced thyroid goiter in fetal/infant monkeys through placental transfer of thiamazole. No clear changes were noted in thyroid histopathology or serum thyroid hormone levels in maternal animals or infants, but goiter formation might have been indicative of exposure to high thyroid stimulating hormone (TSH) and low T3 or T4 in utero from maternal treatment with thiamazole. Age-related changes were observed in the control in behavioral development tests, while infants at 3.5 mg/kg showed no age-related decrease in contact behavior and no increase in exploratory activity on PND 90 or PND 170. In addition, the number of eye contacts between PND 210 and PND 240 was less frequent. This indicated that maternal exposure to thiamazole induced mental retardation-like behaviors in infants. Thiamazole may directly inhibit thyroid hormone synthesis in the fetus by placental transfer. From these results, it was speculated that oral administration of thiamazole to maternal animals during the late gestational period induced retardation of behavioral development in their infants.

Testicular development in cynomolgus monkeys.

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.

Morphometric examination for development of reproductive organs in male cynomolgus monkeys.

We previously reported on a histological classification of cynomolgus monkey testis into six grades (1, immature; 2, prepuberty; 3, onset of puberty; 4, puberty; 5, early adult; 6, adult) based on spermatogenesis development. In this investigation, the accessory reproductive organs from the same animals underwent histomorphometric examination, in addition to being examined histologically and weighed, to evaluate relationships between these parameters and the six grades. Seminiferous tubule diameter increased corresponding to the testicular maturity grade and was notably increased at grade 6. Beginning from grade 3, increases in the areas of the ductus epididymis were noted, and reserved sperm was visible in the lumen. In the prostate, the glandular lumen area per unit area showed an increase beginning from grade 3 but no clear differences between grades 4 and 6; advanced development of epithelial height was observed at grade 6. In the seminal vesicle, development of the epithelial cell layer was markedly increased at grade 6. It was concluded that development of the male accessory reproductive organs began after reserved sperm was observed in the lumen of the ductus epididymis (grade 3) and that these organs were developed notably when the testis reached sexual maturity (grade 6).

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces disruption of glands of the prostate and fibrosis in rhesus monkeys.

We investigated the effects that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure has on the prostate in rhesus monkey offspring. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and examined histopathologically. Dose-dependent decreases in glands of the prostate and widespread fibrosis were observed in offspring. It is noteworthy that 7 years from the final lactational TCDD exposure, inflammatory cell infiltration and disruption of glands of the prostate were still observed. Differential mRNA expression associated with fibrosis, inflammatory response and disruption of cell components were demonstrated by microarray analysis, with up-regulation of TGM4, TGFB1, COL1A1 and MMP2 confirmed. In conclusion, in utero and lactational exposure to TCDD induced dose-related prostatic fibrosis, indicating prostatic dysfunction and inducible semen quality reduction in second-generation rhesus monkeys.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) incorporates improvements and enhancements to both content and organization of the terminology to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e. rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, 'malformation' or 'variation' remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis or interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Embryo-fetal developmental toxicity study design for pharmaceuticals.

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the International Conference on Harmonization (ICH) S5(R2) document (available at http://www.ich.org). The studies that assess developmental hazard are generally conducted in rodents and rabbits. Based on the authors' collective experience, adequate designs (including range-finding studies) and the presentation of data for these studies are described in detail. In addition, the suggested initiation and then total duration of these studies in relation to clinical studies that enroll women of childbearing potential are described. Optional parameters that may be included in the studies are discussed, as are study designs that combine assessments of fertility and developmental toxicity. New methods that may replace or enhance current procedures are outlined. The details described herein will assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agencies that ultimately review these studies.

Developmental and reproductive toxicology studies in nonhuman primates.

Developmental and reproductive toxicology testing in nonhuman primates (NHPs) has become more common due to the increasing number of biopharmaceuticals in drug development, since NHPs are frequently the only species to express pharmacologic responses similar to humans. NHPs may also be used to help resolve issues associated with small-molecule reproductive toxicology in traditional species (rodents and rabbits). Adequate designs in NHP are presented for developmental toxicity (embryo-fetal development, pre-postnatal development, enhanced pre-postnatal development), reproductive toxicity (male and female), and juvenile toxicity studies. Optional parameters that may be included in these studies are discussed, as are new study designs that consolidate multiple aspects of the reproductive assessment and thereby conserve the limited supply of sexually mature NHPs available for testing. The details described will assist scientists in pharmaceutical, regulatory, and contract research organizations who are involved in conducting these unique studies to optimize their design based on case-by-case considerations.

Juvenile animal toxicity study designs to support pediatric drug development.

The objective of juvenile animal toxicity studies of pharmaceuticals is to obtain safety data, including information on the potential for adverse effects on postnatal growth and development. Studies in juvenile animals may assist in identifying postnatal developmental toxicities or other adverse effects that are not adequately assessed in the routine toxicity evaluations and that cannot be safely or adequately measured in pediatric clinical trials. Unlike the traditional reproductive and developmental toxicology studies that have been discussed in the accompanying reports, the design requirements for toxicity studies in juvenile animals are not explicitly defined in regulatory guidance. However, studies in juvenile animals can be useful in providing safety information necessary to enable pediatric clinical trials in pediatric patients or when there are special concerns for toxicities that cannot be safely or adequately measured in clinical trials. These juvenile animal toxicity studies are designed on a case-by-case basis. General design considerations and examples of study designs for assessment of juvenile animal toxicity are discussed.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (version 2) of the Terminology of developmental abnormalities in common laboratory mammals (version 1) by Wise et al. [Wise LD, Beck SL, Beltrame D, Beyer BK, Chahoud I, Clark RL, Clark R, Druga AM, Fueston MH, Guittin P, Henwood SM, Kimmel CA, Lindstrom P, Palmer AK, Petrere JA, Solomon HM, Yasuda M, York RG. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997;55:249-92] incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a reduction in epididymal and ejaculated sperm number in rhesus monkeys.

A long-term developmental toxicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure was performed in rhesus monkeys and the effect on male reproductive organs was determined in the second generation. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and evaluated by semen analysis, and histopathology of the testes and epididymides. Ejaculated sperm concentration was severely reduced at 300 ng/kg, and sperm viability and activity were dose-proportionally reduced, although effects on spermatogenesis were slight. Histomorphometry revealed markedly reduced area of the ductus epididymis accompanying decreased reserved sperm in the 30 and 300 ng/kg groups. In conclusion, in utero and lactational exposure to TCDD induced a reduction of sperm quality in rhesus monkeys.

Terminology of developmental abnormalities in common laboratory mammals (Version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) by Wise et al. (1997) incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Improved technique for fetal heart examination in developmental toxicity studies in rats.

Bouin's or a 10% formalin solution has been used to fixate internal fetal observations for developmental toxicity studies in rats. However, these fixatives are known to cause contraction of the ventricle of the heart and arteries, which makes dissection and observation more difficult. Fetuses on day 20 of gestation from pregnant Crj:CD(SD)IGS rats were injected with 10 w/v% magnesium chloride/10 vol% neutral buffered formalin solution into the thoracic cavity, and then fixed in 10 vol% neutral buffered formalin. After fixation, the heart was dissected using a modified Staples technique. In treated fetuses, the membranous region of the ventricular septum and the valves were clearly observed in an expanded state. We conclude that this method increases the ability to detect heart anomalies and decreases the chance of a false-positive finding.

Comments from the Behavioral Teratology Committee of the Japanese Teratology Society on OECD guideline for the testing of chemicals, proposal for a new guideline 426, developmental neurotoxicity study, draft document (September 2003).

In September 2003, a new revision of the draft guideline (Organization for Economic Co-operation and Development [OECD] Guideline for the Testing of Chemicals, Proposal for a New Guideline 426, Developmental Neurotoxicity Study) was distributed. The draft guideline consists of 51 paragraphs and an appendix. The National Coordinators were requested to arrange national expert reviews of the guideline proposal in their member countries. The member of the Behavioral Teratology (BT) Committee of the Japanese Teratology Society (JTS) reviewed, discussed and commented on the draft Test Guideline proposal. The BT Committee of the JTS also commented that the International Collaborative Study to validate this protocol should be definitely performed. These comments were sent to the OECD Secretariat. The BT Committee of the JTS expects that the comments are useful for further discussion.

Defective balancing ability and hyperactivity in the CLX (circling behavior linked to the X-chromosome) mutant rat.

We have reported that the recently described circling behavior rat (CLX) is a hereditary mutant controlled by a single sex-linked recessive gene (gene symbol: clx). This mutant shows intermittent circle walking and/or running and head tossing with the neck twisted. The abnormal behavior begins to appear around weaning and continues throughout life. In the present study, behavioral tests were performed during the suckling and post-weaning periods and when the rats reached maturity, and the following peculiar abnormalities were revealed: (1) in the righting reflex test, the CLX young show a tendency to take a longer time to revert to normal posture; (2) in the negative geotaxis test, they had difficulty moving upward at 12 days of age; (3) in the air righting reflex test, they frequently fell on their backs or shoulders even after weaning; (4) almost none of the CLX rats showed nystagmus, which is invariably observed in normal rats after rotating stimulation, at 20 weeks of age; and (5) they showed hyperactivity in the open field test at the age of 5 or 6 weeks and a higher degree of locomotor activity in the home cage at the age of 7 and 15 weeks. These results suggest that CLX mutant rats may have some defect in vestibular function (balance sense) or abnormalities in an area of the central nervous system responsible for posture control, e.g., in the dopaminergic or GABAergic neurons.

New behavioral mutant rat exhibiting circling behavior (clx) controlled by a sex-linked recessive gene.

A male rat showing intermittent circling behavior was discovered among the Jcl:Wistar rats in our laboratories, and among its backcross offspring individuals showing the same behavior were found. The abnormalities in these animals were characterized by intermittent circling behavior (walking and/or running in circles) and head tossing with the neck twisted. No abnormalities were observed in fertility, delivery or pup mortality. The results of mating experiments indicated that the circling behavior phenotype is controlled by a single sex-linked recessive gene, and the mutant was named "circling behavior linked to X-chromosome (gene symbol: clx)." This circling behavior mutant is considered to be different from the previously reported mutants, the behavior in all of which has been found to be autosomally inherited. Sib-mating is continuing to produce an inbred strain with this newly discovered circling behavior mutant gene.