Executive Summary

Of approximately 4 million births per year in the United States, major developmental defects are identified in approximately 120,000 live-born infants. Major defects are defined as ones that are life threatening, require major surgery, or present a significant disability. The most frequently recognized class of developmental defects are the structural abnormalities (e.g., neural tube and heart defects), which represent the majority of the developmental defects identified at birth. Other manifestations of abnormal development include growth retardation (e.g., low birth weight), functional deficits (e.g., mental retardation), and pre- and postnatal death (including early pregnancy losses). Because of differences in definition, detection, and reporting practices, the actual frequency of developmental defects is not known with certainty.

At present, the causes of the majority of developmental defects are not understood. It is known that prenatal exposure to some chemicals (e.g., mercury, lead, and polychlorinated biphenyls) and physical agents (e.g., radiation) found in the environment can cause developmental defects. Scientists generally agree that approximately 3% of all developmental defects are attributable to exposure to toxic chemicals and physical agents, including environmental factors, and that 25% of all developmental defects may be due to a combination of genetic and environmental factors. These environmental factors include infection, nutritional deficiencies and excesses, life-style factors (e.g., alcohol), hyperthermia, ultraviolet radiation, X-rays, and closer to the concerns of this committee, the myriad of manufactured and natural agents encountered by humans.

THE CHARGE TO THE COMMITTEE

The National Research Council (NRC) undertook the study leading to this report to clarify how environmental agents may be impacting human develop-



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Scientific Frontiers in Developmental Toxicology and Risk Assessment Executive Summary Of approximately 4 million births per year in the United States, major developmental defects are identified in approximately 120,000 live-born infants. Major defects are defined as ones that are life threatening, require major surgery, or present a significant disability. The most frequently recognized class of developmental defects are the structural abnormalities (e.g., neural tube and heart defects), which represent the majority of the developmental defects identified at birth. Other manifestations of abnormal development include growth retardation (e.g., low birth weight), functional deficits (e.g., mental retardation), and pre- and postnatal death (including early pregnancy losses). Because of differences in definition, detection, and reporting practices, the actual frequency of developmental defects is not known with certainty. At present, the causes of the majority of developmental defects are not understood. It is known that prenatal exposure to some chemicals (e.g., mercury, lead, and polychlorinated biphenyls) and physical agents (e.g., radiation) found in the environment can cause developmental defects. Scientists generally agree that approximately 3% of all developmental defects are attributable to exposure to toxic chemicals and physical agents, including environmental factors, and that 25% of all developmental defects may be due to a combination of genetic and environmental factors. These environmental factors include infection, nutritional deficiencies and excesses, life-style factors (e.g., alcohol), hyperthermia, ultraviolet radiation, X-rays, and closer to the concerns of this committee, the myriad of manufactured and natural agents encountered by humans. THE CHARGE TO THE COMMITTEE The National Research Council (NRC) undertook the study leading to this report to clarify how environmental agents may be impacting human develop-

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Scientific Frontiers in Developmental Toxicology and Risk Assessment ment by using the scientific knowledge gained from major advances in developmental and molecular biology over the past 10-15 years. This study was undertaken with a public health goal of understanding mechanisms of developmental defects to improve our preventive actions. The Committee on Developmental Toxicology was formed to evaluate the current understanding of the mechanisms of action of developmental toxicants and to make recommendations for the improvement of developmental toxicity risk assessment. The specific tasks of the committee were as follows: (1) evaluate the evidence supporting hypothesized mechanisms of developmental toxicity; (2) evaluate the state of the science on testing for mechanisms of developmental effects; (3) evaluate how that information can be used to improve qualitative and quantitative risk assessment for developmental effects; and (4) develop recommendations for future research in developmental toxicology and developmental biology; focus on those areas most likely to assist in assessing risk for developmental defects. COMMITTEE’S APPROACH TO ITS CHARGE The project was conducted in two phases. The first phase consisted of a symposium entitled “New Approaches for Assessing the Etiology and Risks of Developmental Abnormalities from Chemical Exposure,” which was held December 11-12, 1995, in Washington, D.C. The proceedings from that symposium were published in Reproductive Toxicology1 and were used as background information for the second phase of the project. In the second phase, a multidisciplinary committee with expertise in developmental biology and developmental toxicology was asked to address the tasks described above. In this report, the committee documents many recent advances in research in the areas of developmental biology and genomics. These extraordinary advances are significant for developmental toxicology and risk assessment because they present opportunities to improve substantially the detection of developmental toxicants and to elucidate the mechanisms by which toxicants induce developmental defects. The committee makes recommendations for incorporating the new scientific information with existing experimental methods to improve the understanding of the role of environmental agents in human developmental disorders. In approaching its charge, the committee evaluated current methods used to assess risk for developmental defects. Specifically, the committee reviewed the types of data commonly used to evaluate chemicals for potential developmental toxicity and explored the limitations of the risk assessment process. The limitations include the lack of information on the mechanisms of action of chemicals 1   Symposium Proceedings. 1997. New Approaches for Assessing the Etiology and Risks of Developmental Abnormalities from Chemical Exposure. Reprod. Toxicol. 11(2/3):261-463.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment and the uncertainties associated with the extrapolation of data among humans due to the variability in their susceptibility to chemicals, and with the extrapolation of data from animals to humans. The committee attempted to determine whether those limitations could be addressed by recent advances in the understanding of normal development, gene-environment interactions, and human susceptibility. In particular, the committee evaluated new developmental biology data from model animals (e.g., fruit fly, roundworm, zebrafish, and mouse), including genetically modified model animals, and from new molecular biology approaches utilizing in vitro and cellular assays. It developed approaches to show how such new information could improve hazard identification and dose-response assessment and clarify the mechanisms of developmental toxicity. The committee also evaluated data on new technologies for assessing human variability in genes involved in developmental processes and the metabolism of chemicals and determined whether the new technologies could improve risk characterization by reducing uncertainty and variability. Finally, the committee evaluated how this information could be integrated into an overall risk-assessment framework. The committee’s major conclusions and recommendations, organized in response to each of the committee’s tasks, are discussed in the remainder of this summary. CONCLUSIONS IN RELATION TO THE CHARGE Charge 1: Evaluate the Evidence Supporting Hypothesized Mechanisms of Developmental Toxicity. There are only a few compounds (e.g., retinoic acid, diethylstilbesterol (DES), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) for which the mechanism of developmental toxicity is partially explained and no compound for which it is fully explained. Reasons for this incomplete understanding include the lack of knowledge about normal developmental processes, the complexity of developmental toxicity, the broad spectrum of agents and chemical mixtures present in the environment, and the variety of potential mechanisms by which they might cause toxicity. Ideally, a full description of the mechanism of action by which a chemical causes developmental toxicity includes the following types of mechanistic information: the chemical’s toxicokinetics (i.e., its absorption, distribution, metabolism, and excretion) within the mother, fetus, and embryo; the chemical’s toxicodynamics (i.e., how the chemical or a metabolite derived from it interacts with specific molecular components of developmental processes in the embryo and fetus or with maternal or extraembryonic components of processes supporting development); the consequences of those interactions on cellular or developmental processes (also part of toxicodynamics); and the consequence of the altered process for a developmental outcome, namely, the generation of a defect.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment Research has been conducted on the toxicokinetics of some toxicants (e.g., retinoic acid, diphenylhydantoin, methotrexate, and methylmercury). For certain toxicants, routes and rates of exposure to the fetus and embryo have been identified, as have the presence of parent compounds and metabolites in the mother, fetus, and embryo. Additionally, the role of drug-metabolizing enzymes (DMEs) in the metabolism of toxicants has been studied extensively. However, knowledge about critical metabolites and their reactivity with specific target tissues is lacking for most environmental agents. Some toxicants (e.g., retinoids, DES, and TCDD) are known to act on molecular components that function as signaling proteins and transcriptional regulators (described below). However, the committee found that little information is available on how most chemicals impact those molecular components. Where information is available, it is generally sparse and does not allow for the association of developmental defects with a toxicant’s action on specific molecular components of developmental processes. Charge 2: Evaluate the State of the Science on Testing for Mechanisms of Developmental Effects. Major discoveries have recently been made about the components, mechanisms, and processes of normal development. Developmental processes have been identified at the molecular level in various model animals, including the fruit fly, the roundworm, the zebrafish, the frog, the chick, and the mouse. Molecular components of these processes are substantially conserved (i.e., the structure and function of the components have not changed throughout evolution) among animal phyla, including mammals; they regulate development by signaling specific cells to activate proteins called transcription regulators, which turn specific genes on and off. Seventeen signaling pathways are currently recognized, and probably only a few more remain to be discovered. These conserved pathways are used repeatedly in various combinations at different times and locations in the developing embryo and fetus. Species differences in development involve different times, locations, and combinations of these pathways. Many of the kinds of cell responses to signals also are conserved, including selective gene expression, secretion, cell proliferation, and cell migration. The sequencing of the human genome and a variety of animal genomes is providing fundamental information about genome organization, genome evolution, and genetic polymorphisms (variations in the deoxyribonucleic acid (DNA) sequence of a particular gene within a population of organisms). Identifying polymorphisms in the human genome might provide opportunities to increase the understanding of genotype-environment interactions and human susceptibility to toxicants. For example, recent insights into the human differences in the activity of various DMEs and the genetic basis for those differences offer a promising direction for research. Components involved in developmental processes such as the signaling pathways that might be important in susceptibility are less well studied.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment Analyses of human and model animal gene sequences will increase the understanding of gene function and gene expression. For example, methods are available that can be used to determine the changes in gene expression in embryos following exposure to toxicants and to allow for assessment of the consequences of such changes for development within a species and among various species. Charge 3: Evaluate How Recent Advances in Developmental Biology and Genomics Can Be Used to Improve Qualitative and Quantitative Risk Assessment for Developmental Effects. The committee concludes that the major recent advances in developmental biology and genomics can be used to improve qualitative and quantitative risk assessments by integrating toxicological and mechanistic data on a variety of model test animals with data on human variability in genes encoding components of developmental processes, genes encoding enzymes involved in the metabolism of chemicals, and genes encoding receptors and transporter proteins that move these chemicals and their metabolites in and out of the cell. For example, as described in Chapter 7 of this report, chemicals could be evaluated for their potential to alter signaling pathways central to normal development by using nonmammalian model animal systems, such as the fruit fly, roundworm, and zebrafish. Those systems are inexpensive, and the assays can be performed rapidly; therefore, large numbers of chemicals, chemical mixtures, and testing conditions could be evaluated for impacts on these key developmental processes. Such mechanistic information from those systems could be used to improve the identification of potential mammalian hazards, because molecular components and processes of development are well understood in those model animals and because the conservation of signaling pathway components is pervasive and extends to humans. The nonmammalian systems, and the laboratory mouse, can be genetically modified to facilitate the identification of vulnerable developmental pathways, target organs, and times of susceptibility during development. In addition, the model animals can be assessed to define their differences from humans in toxicokinetic and toxicodynamic properties. They also can be genetically modified to contain human metabolism genes, which will reduce the toxicokinetic differences between experimental animals and humans. Such information could be used to improve extrapolation of toxicological data from model animals to humans. Individual human susceptibility to toxicants and genotype-environment interactions could be explored using sequence information from genes encoding DMEs and molecular components, such as components of signaling pathways, involved in development. This information would improve the understanding of human variability in metabolism and the identification of genes encoding molecular components that might be particularly susceptible to chemicals during development.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment Charge 4: Develop Recommendations for Research in Developmental Toxicology and Developmental Biology; Focus on Those Areas Most Likely to Assist in Risk Assessment for Developmental Effects. The committee recommends a multilevel, multidisciplinary approach to risk assessment that incorporates information from a range of model systems intended to evaluate chemicals for potential developmental toxicity; provide mechanistic information on toxicants; address several key areas of uncertainty about the relevancy of cross species extrapolation of toxicological information from animals to humans; and further the exploration of the genotype-environment interactions that might underlie a large fraction of developmental defects and could help to explain human variability in response to environmental agents. This novel approach should provide a guide for obtaining the kinds of data that are needed for a comprehensive cross-species model of exposure and development. Specifically, as described in Chapter 9 of this report, the committee recommends that research be conducted in the following areas. Greater use of model systems for developmental toxicity and risk assessment. Model systems should be used to assess and understand chemical effects (or absence of effects). This recommendation is based on the conclusion that model-animal research has been highly informative about mammalian development, especially human development, and therefore, is likely to be informative about mammalian developmental toxicity. The model systems that should be considered include in-vitro and cellular assays, nonmammalian (e.g., fruit fly, roundworm, and zebrafish) developmental assays, mammalian (e.g., the mouse) developmental assays, and in-depth mammalian tests of mechanism and susceptibility. Evaluation of chemicals for developmental toxicity. In-vitro and cellular assays and nonmammalian tests should be used for evaluating chemicals and chemical mixtures so that patterns of toxicity can be more readily recognized. The number of chemicals in commerce is rapidly expanding, and it is a continuing challenge to obtain toxicity data on them. These model systems could be used quickly and are inexpensive, and their use would permit a large number of chemicals and doses to be evaluated for their potential impact on many key developmental processes. Analysis of mechanisms of toxicity. Mechanistic information is essential to our understanding of how chemicals can perturb development and, thus, is an important component of risk evaluation. To improve the understanding of the mechanisms of action of toxicants, critical molecular targets of components of developmental processes should be identified. Potential critical molecular targets that should be further investigated include (1) evolutionarily conserved pathways of development, such as intercellular signaling pathways (including their

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Scientific Frontiers in Developmental Toxicology and Risk Assessment associated transcriptional regulators); (2) conserved molecular-stress and checkpoint pathways; and (3) conserved toxicokinetic components such as those involved in the transport and metabolism of toxicants (e.g., DMEs). It is important to explore how such molecular perturbations can result in altered function and adverse outcomes of development. Model animals such as the fruit fly, roundworm, and zebrafish can be used to study the mechanisms of developmental toxicity. The signaling pathways that operate in the development of the organs of these organisms also operate in the development of mammalian organs; therefore, the effects of chemicals on fundamental processes such as signaling can be detected. Because the same signaling pathways operating in various kinds of organ development in mammals are partially known and will be better known soon, a chemical’s toxicological impact on these pathways can be predicted on the basis of the results in nonmammalian organisms and tested in mammals. Molecular-stress and checkpoint pathways are used by cells to counteract damage to basic cellular functions, including functions involved in development, and investigating these pathways is important to understand the broad responses of cells to environmental stimuli. Multiple pathways are used in the development of organs; however, one pathway at a time can be studied for a specific aspect of development (e.g., the development of a particular organ) by using genetically modified (e.g., sensitized) animals. Human variability of response to developmental toxicants. To define the genetic basis of variability in human response to developmental toxicants, differences in toxicokinetics, signaling pathways, and molecular-stress and checkpoint pathways need to be characterized. Two approaches to studying variability are recommended: (1) a human epidemiological approach making use of genome information, and (2) a model-animal approach making use of molecular biological techniques and insights. Research should be conducted to assess differences in the genes encoding molecular components among various species, including humans, and among human individuals. As human gene polymorphisms are identified, they should be introduced into the mouse, and molecular biological techniques should be used to assess the organisms’ sensitivity or resistance to various chemicals. Assaying across the entire developmental period. All periods of development are susceptible to the actions of toxicants. For example, early fetal loss in human development occurs in 20-30% of initial pregnancies and, although many of these losses are due to chromosomal aberrations, exposure to a toxicant during early times in development can lead to loss of the embryo or fetus as well as specific structural defects and functional deficits. Use of genetically modified model systems could provide mechanistic information to improve the understanding of early fetal loss as well as morphological alterations and later functional deficits by providing sensitized systems for evaluating developmental defects. Extrapolation from animals to humans. Differences in toxicokinetics and toxicodynamics of experimental animals and humans should be better character-

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Scientific Frontiers in Developmental Toxicology and Risk Assessment ized to improve extrapolations from animals to humans. For example, the study of differences in DMEs between humans and experimental animals will improve the ability to extrapolate from animal test results to humans, because it will be known whether the animal embryo or fetus and the human embryo or fetus are exposed to a chemical at corresponding concentrations and times during development. Also, when the differences between animal and human DME activities are understood, mice can be genetically modified to make them more similar to humans in chemical metabolism. Studies on developmental components, such as signaling components and transcriptional regulators, that are similar to those discussed here for DMEs also should be conducted. Sequence information from the human and mouse genomes will facilitate these studies, as will studies on mice bearing targeted gene alterations. Extrapolations from high to low doses. Because exposure to a chemical at high doses might affect a variety of developmental processes, while exposure at low doses might affect only one critically sensitive pathway, studies using model test animals should be conducted to distinguish dose effects and, in particular, to distinguish effects that could potentially occur at exposure levels relevant to humans. Because a large number of chemicals cause apoptosis (cell death) in the embryo and fetus, the molecular-stress and checkpoint pathways should be given particular attention. Studies using sensitized model animals should be especially useful for defining low-dose responses. Improved access to information. To support the growth of knowledge in developmental toxicology and to organize information in a way that is useful for risk assessment, an inclusive national developmental toxicant database should be established, with entries from industry, academia, and government. The developmental toxicant database should include chemical toxicant information as well as information on known molecular targets and associations with developmental defects, both from animal tests and from humans. Steps should be taken to link this database with the databases of developmental biology (e.g., the database of phenotypes of mice with mutations in their signaling components, which are being generated by genetic modification techniques), and genomics. Databases describing metabolic pathways for drugs and environmental agents, and DME and transporter protein polymorphisms should be linked as well. Ideally, a separate relational database in which signaling pathways are grouped should be established and used when chemicals are identified as interacting with an element of the pathway. This relational database could help to suggest potential biological interactions of a chemical with other chemicals that affect components of the same pathway and record the involvement of signaling pathways in all aspects of development from a wide range of organisms. Multidisciplinary outreach. The challenges that investigators face when trying to work across fields, such as developmental biology, developmental toxicology, and risk assessment, are a key issue that the committee identified early in its deliberations. This issue previously impeded the successful application of the

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Scientific Frontiers in Developmental Toxicology and Risk Assessment new scientific information to improve developmental toxicity risk assessment. For the successful application of this report’s findings, the committee believes that multidisciplinary educational and research programs must be conducted. Programs, such as workshops and professional meetings, should be organized so that researchers of developmental toxicology, developmental biology, genomics, medical genetics, epidemiology, and biostatistics can come together to exchange new insights, approaches, and techniques related to the analysis of developmental defects and to risk assessment. By accelerating the necessary research, cooperative research projects would move forward the recommendations of this report.