Broad Spectrum epidemiological contribution of cannabis and other substances to the teratological profile of northern New South Wales: geospatial and causal inference analysis.

BACKGROUND: Whilst cannabis commercialization is occurring rapidly guided by highly individualistic public narratives, evidence that all congenital anomalies (CA) increase alongside cannabis use in Canada, a link with 21 CA's in Hawaii, and rising CA's in Colorado indicate that transgenerational effects can be significant and impact public health. It was therefore important to study Northern New South Wales (NNSW) where cannabis use is high. METHODS: Design: Cohort. 2008-2015. SETTING: NNSW and Queensland (QLD), Australia. PARTICIPANTS: Whole populations. Exposures. Tobacco, alcohol, cannabis. SOURCE: National Drug Strategy Household Surveys 2010, 2013. MAIN OUTCOMES: CA Rates. NNSW-QLD comparisons. Geospatial and causal regression. RESULTS: Cardiovascular, respiratory and gastrointestinal anomalies rose with falling tobacco and alcohol but rising cannabis use rates across Queensland. Maternal age NNSW-QLD was not different (2008-2015: 4265/22084 v. 96,473/490514 > 35 years/total, Chi.Sq. = 1.687, P = 0.194). A higher rate of NNSW cannabis-related than cannabis-unrelated defects occurred (prevalence ratio (PR) = 2.13, 95%C.I. 1.80-2.52, P = 3.24 × 10- 19). CA's rose more potently with rising cannabis than with rising tobacco or alcohol use. Exomphalos and gastroschisis had the highest NNSW:QLD PR (6.29(2.94-13.48) and 5.85(3.54-9.67)) and attributable fraction in the exposed (84.11%(65.95-92.58%) and 82.91%(71.75-89.66%), P = 2.83 × 10- 8 and P = 5.62 × 10- 15). In multivariable geospatial models cannabis was significantly linked with cardiovascular (atrial septal defect, ventricular septal defect, tetralogy of Fallot, patent ductus arteriosus), genetic (chromosomal defects, Downs syndrome), gastrointestinal (small intestinal atresia), body wall (gastroschisis, diaphragmatic hernia) and other (hypospadias) (AVTPCDSGDH) CA's. In linear modelling cannabis use was significantly linked with anal stenosis, congenital hydrocephalus and Turner syndrome (ACT) and was significantly linked in borderline significant models (model P < 0.1) with microtia, microphthalmia, and transposition of the great vessels. At robust and mixed effects inverse probability weighted multivariable regression cannabis was related to 18 defects. 16/17 E-Values in spatial models were > 1.25 ranging up to 5.2 × 1013 making uncontrolled confounding unlikely. CONCLUSIONS: These results suggest that population level CA's react more strongly to small rises in cannabis use than tobacco or alcohol; cardiovascular, chromosomal, body wall and gastrointestinal CA's rise significantly with small increases in cannabis use; that cannabis is a bivariate correlate of AVTPCDSGDH and ACT anomalies, is robust to adjustment for other substances; and is causal.

Noncoding RNAs in development and teratology, with focus on effects of cannabis, cocaine, nicotine, and ethanol.

Completion of the Human Genome Project has led to the identification of a large number of transcription start sites that are not paired with protein-coding genes, supporting the growing recognition of the abundance of encoded nonprotein-coding RNAs (ncRNAs) and their importance for speciation and species-specific development. Present in both plants and animals, ncRNAs vary in size, function, primary sequence, and secondary structure. While microRNAs (miRNAs) are the best known, there are a number of other ncRNAs (long[er] nonprotein-coding RNA, pseudogenes, circular RNAs, and so on) that have been shown to play an important role in the development either directly or via networks of proteins and other ncRNAs, including modulating the impact of miRNAs. Furthermore, these ncRNAs and their developmental regulatory networks are sensitive to teratogens such as ethanol, cannabis, cocaine, and nicotine. A better understanding of the developmental role of ncRNAs and their capacity to mediate teratogenesis is a necessary step in efforts to minimize the long-term consequences of developmental exposures to drugs-of-abuse. Moreover, with increasing awareness of the prevalence of polydrug use, experimental models will need to incorporate more complex drug exposure paradigms into meaningful assessments of developmental ncRNA function.

Reversal of neurobehavioral teratogenicity in animal models and human: Three decades of progress.

Early studies of behavioral teratology were mostly descriptive, fulfilling the necessary first requirement in a new field. The next obvious stage was put forward in the 80's as mechanism driven science enabled reversal of the teratogens-induced deficits. Three decades later a plethora of studies have been published demonstrating the success of the new direction. Complete and long-term (ostensibly permanent) reversal has been demonstrated in numerous animal models representing the realization of the ultimate goal of the field. Perhaps less sought after, but still significant, are the studies on recovery which needs consistent treatment for its persistence The studies reviewed here have been summarized in Tables 1 and 2. Clinically, the field is only in its incipient stage because of the paucity in translational findings for complete reversal or even complete alleviation. Human findings are emerging but in partial alleviation, noteworthy were the demonstration of FASD children who showed improvement after choline treatment while others showed no effect. Consequently, while further studies in an animal model on the mechanism by which the teratogen exerts its deleterious effects and the reversal procedure action are important, the main thrust of the research should now be translation of the animal model findings into a standard clinical routine. Indeed, first steps towards these goals are being made in children with various neurodevelopmental disorders via the application of a variety of rehabilitation programs by physiotherapists, occupational therapists and speech and language therapists, but the results are partial and may not be long-lasting.

First use of anatomical networks to study modularity and integration of heads, forelimbs and hindlimbs in abnormal anencephalic and cyclopic vs normal human development.

The ill-named "logic of monsters" hypothesis of Pere Alberch - one of the founders of modern evo-devo - emphasized the importance of "internal rules" due to strong developmental constraints, linked teratologies to developmental processes and patterns, and contradicted hypotheses arguing that birth defects are related to a chaotic and random disarray of developmental mechanisms. We test these hypotheses using, for the first time, anatomical network analysis (AnNA) to study and compare the musculoskeletal modularity and integration of both the heads and the fore- and hindlimbs of abnormal cyclopic trisomy 18 and anencephalic human fetuses, and of normal fetal, newborn, and adult humans. Our previous works have shown that superficial gross anatomical analyses of these specimens strongly support the "logic of monsters" hypothesis, in the sense that there is an 'order' or 'logic' within the gross anatomical patterns observed in both the normal and abnormal individuals. Interestingly, the results of the AnNA done in the present work reveal a somewhat different pattern: at least concerning the musculoskeletal modules obtained in our AnNA, we observe a hybrid between the "logic of monsters" and the "lack of homeostasis" hypotheses. For instance, as predicted by the latter hypothesis, we found a high level of left-right asymmetry in the forelimbs and/or hindlimbs of the abnormal cyclopic trisomy 18 and anencephalic human fetuses. That is, a network analysis of the organization of/connection between the musculoskeletal structures of these fetuses reveals a more "chaotic" pattern than that detected by superficial gross anatomical comparisons. We discuss the broader developmental, evolutionary, and medical implications of these results.

Approach to evaluating pregnancy safety of anti-rheumatic medications in the OTIS MotherToBaby pregnancy studies: what have we learned?

For the last 30 years, pregnancy exposure studies, with varying methodologies, have been the mainstay of post-marketing surveillance for new drugs likely to be used by women of reproductive age. While they provide valuable data to inform use during pregnancy, they have limitations that render them necessary but not sufficient in supplying timely information to patients and prescribers. The Organization of Teratology Information Specialists MotherToBaby Pregnancy Studies' collaborative research group operates to help fill this gap. This paper provides an overview of the research that has been and is currently being conducted, as well as best practices determined over the past two decades. The Organization of Teratology Information Specialists MotherToBaby studies can provide earlier signaling with regard to concerns following possible teratogenic exposures, which when examined in conjunction with larger database studies and case-control designs, can move us closer to developing a fuller picture of drug safety for women of reproductive age.

A shortened study design for embryo-fetal development studies in the minipig.

The minipig is accepted from scientific and regulatory perspectives for the safety evaluation of drug candidates on embryo-fetal development. The relative size and the duration of gestation (112-115 days) in the minipig is, however, considered a drawback compared with routine smaller species. We evaluated if study duration and cost could be optimized without impacting scientific validity by performing all terminal procedures around mid-gestation (60 days). At this stage, minipig fetal size is not too dissimilar to full term rabbit and therefore better suited to fetal processing/examination compared with at the end of gestation. Despite encountering higher than anticipated embryo-fetal death, morphological defects clearly associated with a known teratogen, pyrimethamine, were detected. Although the gonads are poorly differentiated macroscopically at mid-term, a histological examination confirmed that external sexing of the fetuses was accurate. Double staining of the bone and cartilage of the mid-term fetal skeleton allowed a more refined examination.

Ethical approval for multicenter cohort studies on drug exposure during pregnancy: A survey among members of the European Network of Teratology Information Services (ENTIS).

The European Network of Teratology Information Services (ENTIS) is in a privileged position to perform independent post-marketing surveillance of drugs in pregnancy. The aim of this survey was to describe the legal requirements and procedures involved in obtaining ethical approval for collaborative cohort studies. We sent a survey questionnaire to all 28 Teratology Information Services (TIS), of which 25 (89%) in 18 countries completed our questionnaire. For 15 TIS, specific research ethical approval was mandatory. The review process was estimated to last from 2 up to 16 weeks. Procedures for patients' information and consent were oral (12), written (5) or both (3). Five TIS had no requirement to inform patients and seek consent. Since data on drug exposure during pregnancy are scarce, ENTIS research efforts should be further encouraged, and procedures optimized so that legitimate ethical and legal requirements do not translate into deterrent administrative constraints and costs.

Use of Human Embryoid Bodies for Teratology.

Human birth defects are relatively common and can be caused by exposure to environmental teratogens or to pharmaceuticals with teratogenic activities. Human embryonic stem cells (hESCs), by virtue of their pluripotent nature, provide an excellent cellular platform for teratogen detection and risk assessment. This unit describes detailed protocols for the preparation and validation of highly pluripotent hESCs, the production of large quantities of aggregated multicellular spheroids composed of hESCs, and these spheroids' differentiation into embryoid bodies (EBs). EBs contain a variety of cells of endodermal, ectodermal, and mesodermal origin and can be subjected to compound exposure in vitro. Hence, they are useful for the detection of chemicals with teratogenic activities. Beyond describing protocols to assemble and culture EBs, this unit details methods to exploit the EB system for teratological assessment. In addition, strategies to distinguish compounds with bona fide teratogenic activity versus simple toxicity are discussed. © 2018 by John Wiley & Sons, Inc.

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).

Comparison of a modified mid-coronal sectioning technique and Wilson's technique when conducting eye and brain examinations in rabbit teratology studies.

BACKGROUND: There are two methods used when examining fetal rabbit eyes and brain in teratology studies. One method employs prior fixation before serial sectioning (Wilson's technique) and the other uses fresh tissue (mid-coronal sectioning). METHODS: We modified the mid-coronal sectioning technique to include removal of eyes and brain for closer examination and to increase the number of structures that can be evaluated and compared it to the Wilson's technique. We found that external examination of the head, in conjunction with either sectioning method, is equally sensitive in identifying developmental defects. We evaluated 40,401 New Zealand White (NZW) and Dutch-Belted (DB) rabbit fetuses for external head alterations, of which 28,538 fetuses were further examined for eye and brain alterations using the modified mid-coronal sectioning method (16,675 fetuses) or Wilson's technique (11,863 fetuses). The fetuses were from vehicle control or drug-treated pregnant rabbits in embryo-fetal development studies conducted to meet international regulatory requirements for the development of new drugs. RESULTS: Both methods detected the more common alterations (microphthalmia and dilated lateral cerebral ventricles) and other less common findings (changes in size and/or shape of eye and brain structures). CONCLUSIONS: While both methods are equally sensitive at detecting common and rare developmental defects, the modified mid-coronal sectioning technique eliminates the use of chemicals and concomitant fixation artifacts that occur with the Wilson's technique and allows for examination of 100% intact fetuses thereby increasing potential for detecting eye and brain alterations as these findings occur infrequently in rabbits.

Imaging and morphology in reproductive toxicology - progress to date and future directions.

This review looks at the recent development and application of imaging techniques for the morphological examination of fetuses from preclinical regulatory reproductive toxicology studies. Full replacement of the examination methods currently used in routine studies (microdissection, Bouin's fluid fixation/sectioning and alizarin red S/alcian blue preparations) by imaging techniques has yet to be achieved. Progress, especially in the application of micro-CT for skeletal examination, has been made but challenges, particularly the financial investment required, remain. Despite this apparent lack of progress the application of imaging techniques to "non-routine" preclinical reproductive toxicology studies has been used to good effect. The ability to acquire multiple images over a time course i.e. longitudinally has enabled the fate, particularly of skeletal features, to be determined. The additional evidence gained from such studies can be used to better inform the prenatal developmental hazard assessment of test compounds.

Zebrafish models for assessing developmental and reproductive toxicity.

The zebrafish is increasingly used as a vertebrate animal model for in vivo drug discovery and for assessing chemical toxicity and safety. Numerous studies have confirmed that zebrafish and mammals are similar in their physiology, development, metabolism and pathways, and that zebrafish responses to toxic substances are highly predictive of mammalian responses. Developmental and reproductive toxicity assessments are an important part of new drug safety profiling. A significant number of drug candidates have failed in preclinical tests due to their adverse effect on development and reproductivity. Compared to conventional mammal testing, zebrafish testing for assessing developmental and reproductive toxicity offers several compelling experimental advantages, including transparency of embryo and larva, higher throughput, shorter test period, lower cost, smaller amount of compound required, easier manipulation and direct compound delivery. Toxicity and safety assessments using zebrafish have also been accepted by the FDA and EMEA for investigative new drug (IND) approval.

Zebrafish model systems for developmental neurobehavioral toxicology.

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models.

The developmental toxicity testing of biologics.

The characteristics of biologic drugs, as compared with small molecules, confer significant advantages for both the drug developer and the prospective patients. The necessity for, and the timing of, developmental toxicity testing in the preclinical program must be considered. Choice of an appropriate test system is of particular importance, one that shows pharmacodynamic activity comparable to man. Where the conventional rodent/non-rodent species show such functional cross-reactivity, those species can be used in developmental testing, but often the only relevant species will be a nonhuman primate, in which case an extended study design (the ePPND) should be the default. Such an approach provides appropriate toxicity screening while reducing animal usage.

Teratology testing under REACH.

REACH guidelines may require teratology testing for new and existing chemicals. This chapter discusses procedures to assess the need for teratology testing and the conduct and interpretation of teratology tests where required.

The teratology testing of food additives.

The developmental and reproductive toxicity testing (including teratogenicity) of new foods and food additives is performed worldwide according to the guidelines given in the FDA Redbook. These studies are not required for substances that are generally recognized as safe, according to the FDA inventory. The anticipated cumulated human exposure level above which developmental or reproduction studies are required depends on the structure-alert category. For food additives of concern, both developmental (prenatal) and reproduction (multigeneration) studies are required. The developmental studies are performed in two species, usually the rat and the rabbit. The reproduction study is generally performed in the rat. The two rat studies are preferably combined into a single experimental design, if possible. The test methods described in the FDA Redbook are similar to those specified by the OECD for the reproductive toxicity testing of chemicals.

Developmental toxicity testing of vaccines.

Preventative and therapeutic vaccines are increasingly used during pregnancy and present special considerations for developmental toxicity testing. The various components of the vaccine formulation (i.e., protein or polysaccharide antigen, adjuvants, and excipients) need to be assessed for direct effects on the developing conceptus. In addition, possible adverse influences of the induced antibodies on fetal and/or postnatal development need to be evaluated. A guidance document on the preclinical testing of preventative and therapeutic vaccines for developmental toxicity was issued by the FDA in 2006. Preclinical studies are designed to assess possible influences of vaccines on pre- and postnatal development. The choice of model animal for these experiments is influenced by species differences in the timing and extent of the transfer of the induced maternal antibodies to the fetus. The cross-placental transport of maternal immunoglobulins generally only occurs in late gestation and tends to be greater in humans and monkeys than in non-primate species. For many vaccines, the rabbit shows a greater rate of prenatal transfer of the induced antibodies than rodents. For biotechnology-derived vaccines that are not immunogenic in lower species, nonhuman primates may be the only appropriate models. It may be advisable to test new adjuvants using the ICH study designs for conventional pharmaceuticals in addition to the developmental toxicity study with the final vaccine formulation.

The teratology testing of cosmetics.

In Europe, the developmental toxicity testing (including teratogenicity) of new cosmetic ingredients is performed according to the Cosmetics Directive 76/768/EEC: only alternatives leading to full replacement of animal experiments should be used. This chapter presents the three scientifically validated animal alternative methods for the assessment of embryotoxicity: the embryonic stem cell test (EST), the micromass (MM) assay, and the whole embryo culture (WEC) assay.

Teratology studies in the mouse.

The rat is the routine species of choice as the rodent model for regulatory safety testing of xenobiotics such as medicinal products, food additives, and other chemicals. However, the rat is not always suitable for pharmacological, toxicological, immunogenic, pharmacokinetic, or even practical reasons. Under such circumstances, the mouse offers an alternative for finding a suitable rodent model acceptable to the regulatory authorities. Since all essential routes of administration are possible, the short reproductive cycle and large litter size of the mouse make it a species well adapted for use in teratology studies. Given that good quality animals, including virgin mated females, can be acquired relatively easily and inexpensively, the mouse has been used in reproductive toxicity studies for decades and study protocols are well established.

Teratology studies in the rat.

The rat is the rodent species of choice for the regulatory safety testing of xenobiotics, such as medicinal products, food additives, and other chemicals. Many decades of experience and extensive data have accumulated for both general and developmental toxicology investigations in this species. The high fertility and large litter size of the rat are advantages for teratogenicity testing. The study designs are well defined in the regulatory guidelines and are relatively standardized between testing laboratories across the world. Teratology studies address maternal- and embryo-toxicity following exposure during the period of organogenesis. This chapter describes the design and conduct of a teratology study in the rat in compliance with the regulatory guidelines. The procedures for the handling and housing of the pregnant animals, the caesarean examinations and the sampling of fetuses for morphological examinations are described. The utility and design of preliminary studies and the inclusion of satellite animals in the main study for toxicokinetic sampling are discussed.