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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., (Copyright © 2013 Wiley Periodicals, Inc.)

Under normal circumstances, fertility and embryotoxicity studies are run separately according to the ICH S5(R2) guideline for the detection of toxicity to reproduction of medicinal products (1). However, the flexible approach of the S5(R2) guideline also allows the reproduction stages covered in the fertility and embryo-fetal development studies (stages A to D) to be combined into a single study design. The administration period covers the pre-mating and gestation phases through to closure of the hard palate. The principal advantages of the combined study include reductions in the number of animals required and cost. Although the rat is the routine species of choice, the mouse may also be used.

The enhanced pre- and postnatal (ePPND) study design has been developed in response to new scientific knowledge and subsequent guideline changes [ICH M3(R2) and ICH S6(R1)]. The changes in study design were basically driven by the experiences obtained during preclinical development of biopharmaceuticals. The standard ePPND concept does not apply to conventional small molecule pharmaceuticals. In essence, the ePPND design is a pre- and postnatal development (PPND) study in which key elements of an embryo-fetal development study are investigated in newborns and infants rather than in the fetus. The cynomolgus monkey is the current relevant nonhuman primate model. The ICH S6(R1) guideline reached step 5 in June 2011 and provides detailed recommendations on various parameters and the conduct of an ePPND study. This chapter provides working guidance for monitoring menstrual cycles to generate pregnant animals, ultrasound monitoring of pregnancy, morphometric measurements of fetuses and newborns, in vivo skeletal examination, various protocols for evaluation of infants (e.g., neurobehavioral assessment, learning and memory test, grip strength, immune system evaluation) and a comprehensive list of additional infant evaluation parameters for the cynomolgus monkey.

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.

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.

Human health risk assessment for pesticides is based mainly on animal studies submitted by the applicant and aims to determine safe exposure levels for operators (farmers and agricultural workers) and consumers of all age groups. Critical effects, including those resulting from reproductive toxicity, are identified during hazard assessment from an evaluation of all studies in the toxicity package. Reproductive or developmental effects are considered critical if they are more severe or occur at lower doses than other toxicities. Reference values for human exposure are then derived from No Adverse Effect Levels for the relevant critical effects by applying safety factors. This paper describes methods and caveats applicable to the evaluation of prenatal toxicity and two-generation studies from the view of a regulator, stressing the importance of individual litter data and the relationship between different endpoints.

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.

As a model for teratogenicity research, zebrafish are gaining popularity and creditability. Zebrafish embryos have been proven to be a highly valuable tool in genetics and developmental biology research and have advanced our understanding of a number of known developmental toxicants. It has yet to be determined conclusively how reliably a zebrafish embryo screening assay predicts what will happen in mammalian models, but results from initial assessments have been encouraging. Here we have presented procedures for the basic care of a zebrafish colony to support embryo production, embryo collection and culturing, and teratogenicity experiments.

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.

Although demonstration of some degree of maternal toxicity is required in regulatory developmental toxicology studies, marked maternal toxicity may be a confounding factor in data interpretation. Reduction in maternal body weight gain is the far most frequently used endpoint of toxicity, but alternative endpoints, like organ toxicity or exaggerated pharmacological response, can also be taken into consideration. The following conclusions are based on literature data and discussions at maternal toxicity workshops attended by representatives from regulatory agencies, academia, and industry: (1) Available results do not support that maternal toxicity (defined as clinical signs, decreased body weight gain or absolute body weight loss of up to 15% in rats or 7% in rabbits) can be used to explain the occurrence of major malformations. (2) There is clear evidence that substantial reductions in maternal weight gain (or absolute weight loss) are linked with other manifestations of developmental toxicity. Among these can be mentioned decreased fetal weight, and skeletal anomalies (e.g., wavy ribs) in rats and decreased fetal weights, post implantation loss, abortions, and some skeletal anomalies in rabbits. (3) There are several examples of misinterpretation among companies, where it was incorrectly expected that regulatory authorities would not label chemicals/drugs as "teratogens/developmental toxicants" because embryo fetal adverse effects were only observed at doses also causing signs of maternal toxicity. (4) Similarly, even if mechanistic studies indicate that a substance causes developmental toxicity via exaggerated pharmacological effects in the mother, such a mechanism does not automatically negate the observed fetal adverse effects.From a regulatory perspective, an observed developmental toxic finding is considered to be of potential human relevance (even if it is mediated via maternal pharmacological effects or occur at doses causing signs of maternal toxicity) unless the company can provide appropriate mechanistic and/or other convincing evidence to the contrary.

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 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.

The rabbit is generally the non-rodent species or second species after the rat recommended by the regulatory authorities and is part of the package of regulatory reproductive studies for the detection of potential embryotoxic and/or teratogenic effects of pharmaceuticals, chemicals, food additives, and other compounds, including vaccines (see Chapters 1-7).Its availability, practicality in housing and in mating as well as its large size makes the rabbit the preferred choice as a non-rodent species. The study protocols are essentially similar to those established for the rat (Chapter 9), with some particularities. The study designs are well defined in guidelines and are relatively standardized between testing laboratories across the world.As for the rat, large litter sizes and extensive background data in the rabbit are valuable criteria for an optimal assessment of in utero development of the embryo or fetus and for the detection of potential external or internal fetal malformations.

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.

The emergence of toxicogenomic applications provides new tools to characterize, classify, and potentially predict teratogens. However, due to the vast number of experimental and statistical procedural steps, toxicogenomic studies are challenging. Here, we guide researchers through the basic framework of conducting toxicogenomic investigations in the field of developmental toxicology, providing examples of biological and technical factors that may influence response and interpretation. Furthermore, we review current, diverse applications of toxicogenomic-based approaches in teratology testing, including exposure-response characterization (dose and duration), chemical classification studies, and cross-model comparisons study designs. This review is intended to guide scientists through the challenging and complex structure of conducting toxicogenomic analyses, while considering the many applications of using toxicogenomics in study designs and the future of these types of "omics" approaches in developmental toxicology.

There is a long history for the use of nonhuman primates in toxicological research. This unit reviews applications in reproductive toxicology and teratology, neural toxicology and neurobehavioral toxicology, immunotoxicology, respiratory (lung) toxicology, and chemical carcinogenesis.

Cryopreservation of semen from Japanese White rabbits was examined to reduce the number of their males for use in teratological studies. Semen was frozen with liquid nitrogen, preserved, thawed, and tested for motility according to the method of Chen et al. Even after cryopreservation an average of 52% of the thawed sperms were motile. In a previous study, frozen- thawed sperms with a motility of 40% resulted in a high conception rate (approximately 88%) on artificial insemination when New Zealand White rabbits were used [5]. These results indicate the possibility that cryopreservation of semen from Japanese White rabbits may be used in teratological studies to reduce the number of males.