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Introduction

Between 2% and 3% of all live-born infants are estimated to have a major developmental defect identified at birth (ICBD 1991; CDC 1995; Holmes 1997; March of Dimes 1999). The percentage increases substantially when all developmental defects—including nonstructural defects such as neurological and behavior problems that often are not detected until childhood or even adulthood—are considered. A developmental defect is defined as a structural or functional anomaly that results from an alteration in normal development.

The causes of most developmental defects are unknown. However, it is known that exposure to chemicals can result in developmental defects. In all, about 3% of developmental defects are attributable to an exposure of the mother to chemicals and physical agents, including environmental agents. A much larger fraction, perhaps 25%, are thought to be due to multifactorial causes resulting from the exposure of genetically predisposed individuals to environmental factors (e.g., infections, nutritional deficiencies and excesses, hyperthermia, ultraviolet radiation, X-rays, and manufactured and natural chemicals). There is concern that greater than 3% of developmental defects may be due to exposures to chemicals and physical agents. One reason for this concern is that only a fraction of the 60,000 to 90,000 chemicals in commercial use have been evaluated for their potential to cause developmental toxicity. Human-health concerns about environmental agents require that scientists and regulators attempt to understand and protect against the potential hazards of those agents on developing embryos, fetuses, and children.

In this committee’s context of addressing the consequences of human prenatal exposure to environmental toxicants, a more inclusive and accurate term is “developmental defect” rather than “birth defect.” The committee will use “de-



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Scientific Frontiers in Developmental Toxicology and Risk Assessment 1 Introduction Between 2% and 3% of all live-born infants are estimated to have a major developmental defect identified at birth (ICBD 1991; CDC 1995; Holmes 1997; March of Dimes 1999). The percentage increases substantially when all developmental defects—including nonstructural defects such as neurological and behavior problems that often are not detected until childhood or even adulthood—are considered. A developmental defect is defined as a structural or functional anomaly that results from an alteration in normal development. The causes of most developmental defects are unknown. However, it is known that exposure to chemicals can result in developmental defects. In all, about 3% of developmental defects are attributable to an exposure of the mother to chemicals and physical agents, including environmental agents. A much larger fraction, perhaps 25%, are thought to be due to multifactorial causes resulting from the exposure of genetically predisposed individuals to environmental factors (e.g., infections, nutritional deficiencies and excesses, hyperthermia, ultraviolet radiation, X-rays, and manufactured and natural chemicals). There is concern that greater than 3% of developmental defects may be due to exposures to chemicals and physical agents. One reason for this concern is that only a fraction of the 60,000 to 90,000 chemicals in commercial use have been evaluated for their potential to cause developmental toxicity. Human-health concerns about environmental agents require that scientists and regulators attempt to understand and protect against the potential hazards of those agents on developing embryos, fetuses, and children. In this committee’s context of addressing the consequences of human prenatal exposure to environmental toxicants, a more inclusive and accurate term is “developmental defect” rather than “birth defect.” The committee will use “de-

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Scientific Frontiers in Developmental Toxicology and Risk Assessment velopmental defect” throughout this report, because it includes the full range of kinds and severity of defects and the full range of times of detection—before, at, and after birth. In recognition of the opportunity to use recent advances in developmental biology and genomics to elucidate further the role of environmental agents in human developmental defects, the NRC approved a project to evaluate the current understanding of the mechanism of action of toxicants that results in developmental defects, and make recommendations for the improvement of toxicant evaluation, ultimately in ways that would improve risk assessment. The specific tasks of the committee were as follows: evaluate the evidence supporting hypothesized mechanisms of developmental toxicity; evaluate the state of the science on testing for mechanisms of developmental effects; evaluate how that information can be used to improve qualitative and quantitative risk assessment for developmental effects; and develop recommendations for future research in developmental toxicology and developmental biology; focusing on those areas most likely to assist in risk assessment for developmental defects. BACKGROUND Awareness of developmental toxicants increased greatly in the early 1960s when the detrimental effect of thalidomide (used at that time as a sedative/hypnotic) primarily on human limb development was recognized (thalidomide causes other developmental defects as well). Before that time, various chemicals had been tested on adult animals but only intermittently on pregnant animals, and it was generally accepted that what was then thought of as the placental barrier protected the fetus from foreign agents. Since the recognition of prenatal vulnerability in the early 1960s, much has been done to detect potential developmental toxicants in the environment and to regulate human exposure to them. Adverse developmental effects of toxicants now are recognized to include not only malformations at birth but also growth retardation, death (including embryonic and fetal loss), and functional defects in the newborn. Over 1,200 specific compounds, pathogens, and conditions have been identified in experimental animals as causing adverse developmental effects, and the impact of human exposure to many of these agents is not understood (Shepard 1998). Since the 1960s, the science of developmental toxicology—that is, the study of the impact of toxicants on critical processes of normal development—has advanced. The science of risk assessment of chemical effects on humans, which depends on the advances in toxicology, has also advanced. To predict risk, assessors rely primarily on two kinds of information: estimates of the level of human exposure to a particular chemical, and estimates of the chemical’s toxicity for humans based on the developmental outcome of offspring from experimental pregnant animals exposed to that chemical. Occasionally, information is avail-

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Scientific Frontiers in Developmental Toxicology and Risk Assessment able from other sources, such as (1) structure-activity relationships relating the toxicity of a chemical to other members of its chemical family; (2) the results of in vitro tests of the chemical; and (3) human epidemiological observations about the effect of the chemical. To make their evaluations of risk, assessors seek accurate mechanism-based empirical data—that is, data based on a solid understanding of the mechanism of a chemical’s toxicity, as determined by developmental toxicologists. Such data are sparse. Several uncertainties limit the estimation of a chemical’s potential for developmental toxicity. Animal bioassays, principally using mammals, currently are considered to provide the most reliable data for extrapolating toxicant effects to humans. Because these bioassays are expensive and time consuming, only a small fraction of the compounds in commerce and in the environment have been fully evaluated for their toxicity potential in animals. The many attempts to devise simpler, less costly test systems involving tissue explants, cell cultures, or purified biological molecules have so far proved to be of only limited value in predicting the actions of compounds on human embryonic and fetal development. Among the reasons for poor predictability are the inherent differences between the simple test systems and humans regarding the uptake, distribution, metabolism, and excretion of chemicals and the lack of understanding about the basic mechanisms of development. In the absence of accurate mechanism-based empirical data, risk assessors often make four kinds of default assumptions when recommending the acceptable levels of exposure of humans to an environmental agent. First, they assume that animal test results are relevant for humans. Unless there is contradictory evidence, humans are assumed to be the most susceptible mammals, and a factor of 10 below the maximum no-effect exposure level in the animal’s development serves as a basis for setting the acceptable human exposure level. Second, a further 10-fold reduction is introduced to take into account the possibility that the animal’s developmental response, which frequently was obtained at subchronic exposure to the chemical, might not reflect human responses at prolonged (chronic) exposures. Third, a 10-fold reduction is introduced to cover the possibility that susceptibility varies among human individuals, some being inherently more sensitive to the chemical. Fourth, a 10-fold reduction is sometimes introduced if the toxicity database for a chemical is incomplete. Because of the susceptibility of developing systems, an additional child specific factor (usually a 10-fold reduction) is sometimes applied. Although many risk assessors would prefer to use mechanism-based empirical data instead of those defaults (up to a 10,000-fold compounded reduction in acceptable human exposure beyond that given by the animal test) to improve their risk assessments for environmental agents, the test data for the assessors’ use often are sparse because of limited resources and are of unknown applicability to humans because of a lack of understanding of basic mechanisms of developmental toxicity and of differences in humans and animals.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment Thus, risk assessors are challenged by the problems of extrapolation, interpretation, cost, and speed. Considering the large number of environmental chemicals (both manufactured and naturally occurring chemicals) that are not adequately tested for potential developmental toxicity, scientists have been asked to develop testing approaches that are based on our rapidly expanding knowledge of normal development to provide more timely information with improved predictions for human developmental outcomes. These issues are ongoing challenges in the effort to assess human risk from environmental toxicants. RECENT ADVANCES IN DEVELOPMENTAL BIOLOGY AND THE PROMISE OF GENOMICS Developmental biology is the study of normal developmental processes. It begins with descriptions of the sequential events of development, from the formation of the oocyte (the egg precursor) and sperm, to fertilization, then to cell division, morphogenesis (the transformation of egg organization into embryonic organization), organogenesis (the formation of organs), cell differentiation, and embryonic and fetal growth. In its full scope, developmental biology covers the development and growth of the infant, child, and adolescent to the time of reproductive maturity. Developmental biology also describes events in the organism’s spatial dimension (the changing number and position of cells, tissues, and organs) in the vast multicellular population of the embryo and fetus (approximately 1 trillion cells in a newborn infant). In the past 15 years, remarkable advances have been made in the knowledge of the components, mechanisms, and processes of normal development, primarily as the result of new insights into the molecular biology of development. Developmental biology has become a study of the mechanisms of development at the molecular level, particularly of the interaction of components of intracellular genetic regulatory circuits with components of intercelluar signaling pathways. To cite a few of those insights, it is now known that the trillions of cells of a large mammal such as a human have the same genetic composition (genetic blueprint). As recently reaffirmed by the cloning of Dolly the lamb (Wilmut et al. 1997), the Cumulina mouse family (Wakayama et al. 1998), and a nonhuman primate (Chan et al. 2000), the genetic content of almost all of the cells in an animal does not change from that of the single-celled fertilized egg from which it developed. Despite having the same genes, the cells in an animal differ widely in their appearance, functions, and responses to environmental impacts. At least 300 cell types are recognized in humans (e.g., red blood cells, Purkinje nerve cells, and smooth or striated muscle cells), and the number of cell subtypes at different stages of development and different parts of the body is perhaps tens of thousands. These cell types differ greatly in their ribonucleic acids (RNA) and proteins, reflecting the different combinations of genes they express from the same genomic repertoire). Development can be viewed as evolution’s foremost ac-

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Scientific Frontiers in Developmental Toxicology and Risk Assessment complishment in gene regulation, entailing a complex orchestration of which cells will express which genes when and where in the embryo and fetus. Two major elements in that regulation are (1) a large variety of specific transcription factors that act in an even larger variety of combinations to control differential gene expression; and (2) the chemical communication between cells during development that allows cells to turn specific genes on and off in response to signals from their neighbors. The following is now realized: Embryonic and fetal development involves repeated signaling among groups of cells, and the expression of particular genes in a cell depends on signaling inputs from other cells in the local environment. The number of signaling pathways used in development is limited. About 17 signaling pathways are now recognized, and probably only a few more remain to be discovered. Each pathway consists of an intercellular chemical signal, a specific receptor on or within the cell, and a set of molecular transducers that transmit each signal to targets, such as to components of the transcription machinery, within the cell. These 17 pathways are used repeatedly at different times and places in the developing embryo and fetus. The roles of these pathways in development are a major focus of current research in developmental biology. Surprisingly, given the morphological diversity of animal embryos and fetuses, the 17 signaling pathways are highly conserved across numerous animal phyla (e.g., nematode worms to arthropods to chordates). The molecular targets and responses within cells also are conserved across phyla, including specific gene expression, cell migration, and cell proliferation. Those signaling and responding aspects of development presumably were already present in the pre-Cambrian common ancestor of animals of modern phyla as diverse as the chordates (including humans), the arthropods (including fruit flies), and the nematodes. The differences in the development of various organisms mostly reflect differences in the particular times, places, and combinations of use of the conserved pathways and responses. Those findings give new validity to the use of model organisms to learn more about basic development in mammals, to provide mechanistic clues about human variability, and to analyze and assess the risks of potential developmental toxicants. With the transformation of developmental biology in the past decade, DNA sequence data from a variety of organisms have accumulated at an explosive rate. The large-scale projects initiated under the Human Genome Project include the complete sequencing of the genomes of several widely used model organisms, such as yeast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, the laboratory mouse, and humans. The sequencing of the yeast, C. elegans, and Drosophilia genomes has already been completed (Goffeau et al.

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Scientific Frontiers in Developmental Toxicology and Risk Assessment 1996; C. elegans Sequencing Consortium 1998; Adams et al. 2000). The C. elegans genome was the first metazoan genome sequenced. The mouse and human genomes should be completed in 3 years, perhaps sooner. Out of these efforts, the field of genomics has emerged. It includes the identification of all genes of an organism, all RNA transcripts of those genes, all the rules for the time, place, and conditions of expression of those genes, and the sequence variants within the population of that organism. Proteomics is the study of all the proteins expressed from all RNA transcripts. The promise of genomics and proteomics is great, because all studies of physiological function, developmental change, and evolutionary diversification will draw upon it. As DNA sequence data from various organisms become available, the need to manage and analyze vast amounts of sequence data will increase. That need has spawned a new field of science called bioinformatics. The ability of scientists to make use of genomic databases will become increasingly important for coordinating developmental biology, developmental toxicology, and genomics. 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 Toxicology (Kimmel et al. 1997) and were used as background information for the second phase of the project in which a multidisciplinary committee with expertise in developmental biology and developmental toxicology was asked to develop a consensus report (this report) that evaluates recent revolutionary advances in the understanding of normal development and gene-environment interactions and in the technology connected to the Human Genome Project and assesses whether these advances provide opportunities for innovation in developmental toxicology and risk assessment. In its report, the committee attempts to make broad-based interdisciplinary proposals by drawing on information from several fields of science—developmental toxicology, developmental biology, molecular biology, epidemiology, and genetics—all of which impinge on the understanding of the action of developmental toxicants. ORGANIZATION OF THE REPORT This report is organized into eight chapters in addition to this Introduction. Chapter 2 describes the type and frequency of developmental defects in more detail, the problems of collecting accurate data on defects, and the general understanding of possible intrinsic and extrinsic causes. Chapter 3 describes the current methods of risk assessment for the evaluation of developmental toxicity and the uncertainties in this assessment that make it

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Scientific Frontiers in Developmental Toxicology and Risk Assessment necessary for assessors to introduce large default corrections in estimating allowable exposure levels. Chapter 4 describes the history and current status of developmental toxicology, summarizing the attempts to identify mechanisms of action of toxicants, and the mechanisms of presentation of active toxicants to the embryo and fetus, detailing a few examples of well-understood developmental toxicants. The committee concludes that although much progress has been made in the analysis of toxicant action, much more remains to be done, probably facilitated by the recent advances in developmental biology and genomics. Chapter 5 describes the fields of human genetics and genomics, including the role of molecular epidemiology in toxicant detection and the difficulties in the detection of complex genotype-environment interactions. The committee concludes that powerful new comprehensive methods from genomics will be of great value in developmental toxicology. Such a method is the newly found capacity to detect human genetic variation. Chapter 6 describes the history of developmental biology and recent advances in that field, stressing the central role of cell-to-cell signaling in development and the repeated use of a small number of signaling pathways at different times and places in development. The committee concludes that the evolutionary conservation of these pathways, of a variety of genetic regulatory circuits and molecular-stress and checkpoint pathways, and of numerous other cellular activities makes it likely that informed use of model organisms to detect and analyze toxicant action will be valuable. Chapter 7 discusses new approaches for using model organisms to test chemicals for developmental toxicity, stressing the value of using those organisms for which development is well understood and for which genetic manipulation can be performed to optimize their usefulness. Chapter 8 outlines a novel multilevel, multidisciplinary approach to improve understanding of the mechanisms of action of toxicants and to improve developmental toxicity risk assessment by applying the recent advances in developmental biology and genomics. The capacity to understand organismal differences in development and toxicant metabolism is now possible, and the importance of that information for extrapolations of animal data to humans is emphasized. The final chapter, Chapter 9, summarizes the committee’s conclusions and recommendations. Here it is emphasized that the recent advances in developmental biology and genomics create an opportunity for improved detection and analysis of toxicants and for a better understanding of the meaning of assay results for risk assessment. Four appendixes are included in the report. Appendix A contains a glossary of definitions of key terms used throughout the report. Appendix B contains descriptions of protein and genomic databases that can be useful to developmental toxicologists. Appendix C contains figures of the 17 known signal transduction pathways. Finally, Appendix D contains bibliographic information on the Committee on Developmental Toxicology.