Chapter 10. Developmental Toxicology

• Developmental toxicology encompasses the study of pharmacokinetics, mechanisms, pathogenesis, and outcome following exposure to agents or conditions leading to abnormal development.
• Developmental toxicology includes teratology, or the study of structural birth defects.
• Gametogenesis is the process of forming the haploid germ cells: the egg and the sperm.
• Organogenesis is the period during which most bodily structures are established. This period of heightened susceptibility to malformations extends from the third to the eighth week of gestation in humans.

Successful pregnancy outcome in the general population occurs at a surprisingly low frequency. Estimates of adverse outcomes include postimplantation pregnancy loss, 31 percent; major birth defects, 2 to 3 percent at birth and increasing to 6 to 7 percent at 1 year as more manifestations are diagnosed; minor birth defects, 14 percent; low birth weight, 7 percent; infant mortality (prior to 1 year of age), 1.4 percent; and abnormal neurologic function, 16 to 17 percent. Thus, less than half of all human conceptions result in the birth of a completely normal, healthy infant. Many hundreds of chemicals are teratogens; most of them produce birth defects by an unknown mechanism. However, Table 10–1 lists chemicals, chemical classes, or conditions known to alter prenatal development in humans.

Thalidomide

In 1960, a large increase in newborns with rare limb malformations of amelia (absence of the limbs) or various degrees of phocomelia (reduction of the long bones of the limbs) was recorded in West Germany. Congenital heart disease; ocular, intestinal, and renal anomalies; and malformations of the external and inner ears were also involved. Thalidomide, identified as the causative agent, was used throughout much of the world as a sleep aid and to ameliorate nausea and vomiting in pregnancy. It had no apparent toxicity or addictive properties in adult humans or animals at therapeutic exposure levels.

As a result of this catastrophe, regulatory agencies developed requirements for evaluating the effects of drugs on pregnancy outcomes.

Diethylstilbestrol

Diethylstilbestrol (DES) is a synthetic nonsteroidal estrogen widely used from the 1940s to the 1970s in the United States to prevent threatened miscarriage. It was soon linked to clear cell adenocarcinoma of the vagina. Maternal use of DES prior to the 18th week of gestation appeared to be necessary for induction of the genital tract anomalies in offspring; the overall incidence of noncancerous alterations in the vagina and cervix was estimated to be as high as 75 percent. In male offspring of exposed pregnancies, a high incidence of reproductive tract anomalies along with low ejaculated semen volume and poor semen quality were observed. The realization of the latent and devastating manifestations of prenatal DES exposure has broadened the magnitude and scope of potential adverse outcomes of intrauterine exposures. A recent study in mice suggests that the increased susceptibility to abnormalities conferred by DES exposure may be passed on to future generations of exposed mothers.

Ethanol

Although the developmental toxicity of ethanol can be traced to biblical times (e.g., Judges 13:3-4), only since the description of the Fetal Alcohol Syndrome (FAS) in 1971 has a clear acceptance of alcohol's developmental toxicity occurred. FAS comprises craniofacial dysmorphism, intrauterine and postnatal growth retardation, retarded psychomotor and intellectual development, and other nonspecific major and minor abnormalities.

In utero exposure to lower levels of ethanol than those that produce full-blown FAS has been associated with a wide range of effects, including isolated components of FAS and milder forms of neurologic and behavioral disorders that have been termed Fetal Alcohol Spectrum Disorder (FASD). Alcohol consumption can affect birth weight in a dose-related fashion.

Tobacco Smoke

Prenatal and early postnatal exposure to tobacco smoke or its constituents may well represent the leading cause of environmentally induced developmental disease and morbidity today. Approximately 25 percent of women in the United States continue to smoke during pregnancy, despite public health programs aimed at curbing this behavior. The consequences of developmental tobacco smoke exposure include spontaneous abortions, perinatal deaths, increased risk of sudden infant death syndrome (SIDS), increased risk of learning, behavioral, and attention disorders, and lower birth weight. One component of tobacco smoke, nicotine, is a known neuroteratogen in experimental animals and can by itself produce many of the adverse developmental outcomes associated with tobacco smoke. Perinatal exposure to tobacco smoke can also affect branching morphogenesis and maturation of the lung, leading to altered physiologic function. Environmental (passive) tobacco smoke also represents a significant risk to the pregnant nonsmoker.

Cocaine

Cocaine is a local anesthetic with vasoconstrictor properties. Effects on the fetus are complicated and controversial and demonstrate the difficulty of monitoring the human population for adverse reproductive outcomes. Accurate exposure ascertainment is difficult, as many confounding factors including socioeconomic status and concurrent use of cigarettes, alcohol, and other drugs of abuse may be involved. In addition, reported effects on the fetus and infant (neurologic and behavioral changes) are difficult to identify and quantify. Nevertheless, adverse effects reliably associated with cocaine exposure in humans include abruptio placentae, premature labor and delivery, microcephaly, altered prosencephalic development, decreased birth weight, SIDS, and a neonatal neurologic syndrome of abnormal sleep, tremor, poor feeding, irritability, and occasional seizures.

Retinoids

Vitamin A (retinol) exposure can cause malformations of the face, limbs, heart, central nervous system, and skeleton. Spontaneous abortion, live-born infants having at least one major malformation, and numerous exposed children having full-scale IQ scores below 85 at age 5 years have been documented.

Angiotensin Converting Enzyme (ACE) Inhibitors and Angiotensin Receptor Antagonists

ACE inhibitors and angiotensin receptor blockers are widely prescribed and, when used in the second half of pregnancy, are known to cause oligohydramnios, fetal growth retardation, pulmonary hypoplasia, joint contractures, hypocalvaria, neonatal renal failure, hypotension, and death. Some studies suggest that exposure in the first trimester should be avoided.

Some basic principles of teratology put forth by Jim Wilson in 1959 and listed in Table 10–2 are still valid today.

Critical Periods of Susceptibility and Endpoints of Toxicity

Development is characterized by various changes that are orchestrated by a cascade of factors regulating gene transcription throughout development. Intercellular and intracellular signaling pathways essential for normal development rely on transcriptional, translational, and posttranslational controls. The rapid changes occurring during development alter the nature of the embryo/fetus as a target for toxicity. Timing of some key developmental events in humans and experimental animal species is presented in Table 10–3.

Gametogenesis is the process of forming the haploid germ cells: the egg and the sperm. These gametes fuse in the process of fertilization to form the diploid zygote, or one-celled embryo. Gametogenesis and fertilization are vulnerable to toxicants.

Following fertilization, the embryo moves down the fallopian tube and implants in the wall of the uterus. The preimplantation period comprises mainly an increase in cell number through a rapid series of cell divisions with little growth in size (cleavage of the zygote) and cavitation of the embryo to form a fluid-filled blastocoele. This stage, termed the blastocyst, contains cells destined to give rise to the embryo proper and other cells that give rise to extraembryonic membranes and support structures.

Toxicity during preimplantation is generally thought to result in no or slight effect on growth (because of regulative growth) or in death (through overwhelming damage or failure to implant). Patterning of the limbs and lower body may begin at this time. Because of the rapid mitoses occurring during the preimplantation period, chemicals affecting DNA synthesis/integrity or those affecting microtubule assembly would be expected to be particularly toxic if given access to the embryo.

Following implantation the embryo undergoes gastrulation, the process of formation of the three primary germ layers—the ectoderm, mesoderm, and endoderm. As a prelude to organogenesis, the period of gastrulation is quite susceptible to teratogenesis. A number of toxicants administered during gastrulation produce malformations of the eye, brain, and face. These malformations are indicative of damage to the anterior neural plate, one of the regions defined by the cellular movements of gastrulation.

The formation of the neural plate in the ectoderm marks the onset of organogenesis, during which the rudiments of most bodily structures are established. This period of heightened susceptibility to malformations extends from approximately the third to the eighth week of gestation in humans. The rapid changes of organogenesis require cell proliferation, cell migration, cell–cell interactions, and morphogenetic tissue remodeling. Within organogenesis, there are periods of peak susceptibility for each forming structure. The peak incidence of each malformation coincides with the timing of key developmental events in the affected structure.

The end of organogenesis marks the beginning of the fetal period, which is characterized primarily by tissue differentiation, growth, and physiologic maturation. All organs are present and grossly recognizable, although not yet completely developed.

Exposure during the fetal period is most likely to result in effects on growth and functional maturation. Functional anomalies of the central nervous system and reproductive organs—including behavioral, mental, and motor deficits as well as decreases in fertility—are among the possible adverse outcomes.

Dose–Response Patterns and the Threshold Concept

The major effects of prenatal exposure, observed at the time of birth in developmental toxicity studies, are embryo lethality, malformations, and growth retardation. For some agents, these endpoints may represent a continuum of increasing toxicity, with low dosages producing growth retardation and increasing dosages producing malformations and then lethality.

Another key element of the dose–response relationship is the shape of the dose–response curve at low exposure levels. Because of the high restorative growth potential of the mammalian embryo, cellular homeostatic mechanisms, and maternal metabolic defenses, mammalian developmental toxicity has generally been considered a threshold phenomenon. Assumption of a threshold means that there is a maternal dosage below which an adverse response is not elicited because some repair or defense system is able to combat the exposure.

The term mechanisms refers to cellular-level events that initiate the process leading to abnormal development. Pathogenesis comprises the cell-, tissue-, and organ-level sequelae that are ultimately manifest in abnormality. Mechanisms of teratogenesis include mutations, chromosomal breaks, altered mitosis, altered nucleic acid integrity or function, diminished supplies of precursors or substrates, decreased energy supplies, altered membrane characteristics, osmolar imbalance, and enzyme inhibition. Although these cellular insults are not unique to development, they may trigger unique pathogenetic responses in the embryo, such as reduced cell proliferation, cell death, altered cell–cell interactions, reduced biosynthesis, inhibition of morphogenetic movements, or mechanical disruption of developing structures.

Cell death plays a critical role in normal morphogenesis. The term programmed cell death refers specifically to apoptosis, which is under genetic control in the embryo. Apoptosis is necessary for sculpting the digits from the hand plate and for assuring appropriate functional connectivity between the central nervous system and distal structures. Cell proliferation rates change both spatially and temporally during ontogenesis. There is a delicate balance between cell proliferation, cell differentiation, and apoptosis in the embryo. DNA damage might lead to cell cycle perturbations and cell death.

Damage to DNA can inhibit cell cycle progression at the G1–S transition, through the S phase, and at the G2–M transition. If DNA damage is repaired, the cell cycle can return to normal, but if damage is too extensive or cell cycle arrest too long, apoptosis may be triggered. The relationship between DNA damage and repair, cell cycle progression, and apoptosis is depicted in Figure 10–1. From the multiple checkpoints and factors present to regulate the cell cycle and apoptosis, it is clear that different cell populations may respond differently to a similar stimulus, in part because cellular predisposition to apoptosis can vary.

In addition to affecting proliferation and cell viability, molecular and cellular insults can affect essential processes such as cell migration, cell–cell interactions, differentiation, morphogenesis, and energy metabolism. Although the embryo has compensatory mechanisms to offset such effects, production of a normal or malformed offspring will depend on the balance between damage and repair at each step in the pathogenetic pathway.

Advances in the Molecular Basis of Dysmorphogenesis

Advances in gene targeting and transgenic strategies now allow modification of gene expression at specific points in development and in specific cell types. Conditional knockouts or knockins, inducible gene expression, and other techniques are being used to study the effects of specific gene products on development in great detail. The use of synthetic antisense oligonucleotides allows temporal and spatial restriction of gene ablation by hybridizing to mRNA in the cell, thereby inactivating it. In this way, gene function can be turned off at specific times.

Gain of gene function can also be studied by engineering genetic constructs with an inducible promoter attached to the gene of interest. Ectopic gene expression can be made ubiquitous or site-specific depending on the choice of promoter to drive expression. Transient overexpression of specific genes can be accomplished by adding extra copies using adenoviral transduction.

The extent and the form in which chemicals reach the conceptus are important determinants of whether the agent can impact development. The maternal, placental, and embryonic compartments comprise independent yet interacting systems that undergo profound changes throughout the course of pregnancy. Alterations in placental physiology can have significant impact on the uptake, distribution, metabolism, and elimination of xenobiotics. For example, decreases in intestinal motility and increases in gastric emptying time result in longer retention of ingested chemicals in the upper gastrointestinal tract in the mother. Cardiac output increases by 50 percent during the first trimester in humans and remains elevated throughout pregnancy, whereas blood volume increases and plasma proteins and peripheral vascular resistance decrease. The relative increase in blood volume over red cell volume leads to borderline anemia and a generalized edema with a 70 percent elevation of extracellular space. Thus, the volume of distribution of a chemical and the amount bound by plasma proteins may change considerably during pregnancy. Other changes occur in the renal, hepatic, and pulmonary systems as well. Clearly, maternal handling of a chemical influences the extent of embryotoxicity.

The placenta also influences embryonic exposure by helping to regulate blood flow, offering a transport barrier, and metabolizing chemicals. The placenta permits bidirectional transfer of substances between maternal and fetal compartments. It is important to note that virtually any substance present in the maternal plasma will be transported to some extent by the placenta. The passage of most drugs across the placenta seems to occur by simple passive diffusion. Important modifying factors to the rate and extent of transfer include lipid solubility, molecular weight, protein binding, the type of transfer (passive diffusion, and facilitated or active transport), the degree of ionization, and placental metabolism. Blood flow probably constitutes the major rate-limiting step for more lipid-soluble compounds.

Maternal metabolism of xenobiotics is an important and variable determinant of developmental toxicity. As for other health endpoints, the field of pharmacogenomics offers hope for increasing our ability to predict susceptible subpopulations based on empirical relationships between maternal genotype and fetal phenotype.

Although all developmental toxicity must ultimately result from an insult to the conceptus at the cellular level, the insult may occur through a direct effect on the embryo/fetus, indirectly through toxicity of the agent to the mother and/or the placenta, or a combination of direct and indirect effects. Some conditions that may adversely affect the fetus are depicted in Figure 10–2.

The distinction between direct and indirect developmental toxicity is important for interpreting safety assessment results in pregnant animals, as the highest dosage level in these experiments is chosen based on its ability to produce some maternal toxicity (e.g., decreased food or water intake, weight loss, and clinical signs). However, maternal toxicity defined only by such crude manifestations gives little insight to the toxic actions of a xenobiotic. When developmental toxicity is observed only in the presence of maternal toxicity, the developmental effects may be indirect (i.e., caused by an inappropriate growing condition because of an altered maternal environment rather than by a direct interaction of the fetus with the toxin). Greater understanding of the physiologic changes underlying the observed maternal toxicity and elucidation of the association with developmental effects is needed before one can begin to address the relevance of the observations to human safety assessment.

Maternal Factors Affecting Development

Genetics

The genetic makeup of the pregnant female has been well documented as a determinant of developmental outcome. The incidence of cleft lip and/or palate [CL(P)], which occurs more frequently in whites than in blacks, has been investigated in offspring of interracial couples in the United States. Offspring of white mothers had a higher incidence of CL(P) than offspring of black mothers after correcting for paternal race, whereas offspring of white fathers did not have a higher incidence of CL(P) than offspring of black fathers after correcting for maternal race.

Disease

Chronic hypertension in the mother, uncontrolled maternal diabetes mellitus, and certain infections in the mother (i.e., cytomegalovirus and Toxoplasma gondii) are leading causes of several types of defects in the fetus. Exposure to hyperthermia (such as febrile illness in the mother) is also implicated in neural defects in the fetus.

Nutrition

A wide spectrum of dietary insufficiencies ranging from protein-calorie malnutrition to deficiencies of vitamins, trace elements, and/or enzyme cofactors is known to adversely affect pregnancy. In fact, folate supplementation by pregnant women can reduce neural tube defect recurrence by over 70 percent.

Stress

Diverse forms of maternal toxicity may have in common the induction of a physiologic stress response. Various forms of physical stress have been applied to pregnant animals in attempts to isolate the developmental effects of stress. Noise stress of pregnant rats or mice throughout gestation can produce developmental toxicity. Restraint stress produces increased fetal death in rats, and malformations of cleft palate, fused and supernumerary ribs, and encephaloceles in mice. There is a positive correlation in humans between stress and adverse developmental effects, including low birth weight and congenital malformations.

Placental Toxicity

The placenta is the interface between the mother and the conceptus, providing attachment, nutrition, gas exchange, and waste removal. The placenta also produces hormones critical to the maintenance of pregnancy, and it can metabolize and/or store xenobiotics. Placental toxicity may compromise these functions. Known placental toxicants include cadmium, arsenic or mercury, cigarette smoke, ethanol, cocaine, endotoxin, and sodium salicylate.

Maternal Toxicity

A retrospective analysis of relationships between maternal toxicity and specific types of prenatal effects found species-specific associations between maternal toxicity and specific adverse developmental effects. Various adverse developmental outcomes include increased intrauterine death, decreased fetal weight, supernumerary ribs, and enlarged renal pelvises.

A number of studies directly relate specific forms of maternal toxicity to developmental toxicity, including those in which the test chemical causes maternal effects that exacerbate the agent's developmental toxicity. However, clear delineation of the relative role(s) of indirect maternal and direct embryo/fetal toxicity is difficult.

Diflunisal, an analgesic and anti-inflammatory drug, causes axial skeletal defects in rabbits. Developmentally toxic dosages resulted in severe maternal anemia and depletion of erythrocyte ATP levels. Teratogenicity, anemia, and ATP depletion were unique to the rabbit. The teratogenicity of diflunisal in the rabbit was probably due to hypoxia resulting from maternal anemia.

Phenytoin, an anticonvulsant, can affect maternal folate metabolism in experimental animals, and these alterations may play a role in the teratogenicity of this drug. A mechanism of teratogenesis was proposed relating depressed maternal heart rate and embryonic hypoxia. Supporting studies have demonstrated that hyperoxia reduces the teratogenicity of phenytoin in mice.

There is the growing concern that exposure to chemicals that can interact with the endocrine system may pose a serious health hazard. An “endocrine disruptor” has been broadly defined as an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones responsible for the maintenance of homeostasis and the regulation of developmental processes. Due to the critical role of hormones in directing differentiation in many tissues, the developing organism is particularly vulnerable to fluctuations in the timing or intensity of exposure to chemicals with hormonal or antihormonal activity. Chemicals from a wide variety of chemical classes induce developmental toxicity via at least three modes of action involving the endocrine system: (1) by serving as ligands of steroid receptors; (2) by modifying steroid hormone metabolizing enzymes; and (3) by perturbing hypothalamic-pituitary release of trophic hormones.

Laboratory Animal Evidence

Estrogenic or antiestrogenic developmental toxicants include DES, estradiol, antiestrogenic drugs such as tamoxifen and clomiphene citrate, and some pesticides and industrial chemicals. Female offspring are generally more sensitive to these toxicants than males and altered pubertal development, reduced fertility, and reproductive tract anomalies are common findings.

Antiandrogens represent another major class of endocrine-disrupting chemicals. Principal manifestations of developmental exposure to an antiandrogen are generally restricted to males, and include hypospadias, retained nipples, reduced testes and accessory sex gland weights, and decreased sperm production. Polychlorinated biphenyls (PCBs) may act at several sites to lower thyroid hormone levels during development, and cause body weight and auditory deficits. PCBs also cause learning deficits and alter locomotor activity patterns in rodents and monkeys.

Human Evidence

Whether human health is being adversely impacted from exposures to endocrine disruptors present in the environment is equivocal. Reports in humans are of two types:

1. Observations of adverse effects on reproductive system development and function following exposure to chemicals with known endocrine activities that are present in medicines, contaminated food, or the workplace. These have tended to involve relatively higher exposure to chemicals with known endocrine effects.

2. Epidemiologic evidence of increasing trends in reproductive and developmental adverse outcomes that have an endocrine basis. For example, secular trends have been reported for cryptorchidism, hypospadias, semen quality, and testicular cancer, but due to the lack of exposure assessment, such studies provide limited evidence of a cause and effect relationship.

Impact on Screening and Testing Programs

The findings of altered reproductive development following early life-stage exposures to endocrine-disrupting chemicals helped prompt revision of traditional safety evaluation tests. These now include assessments of female estrous cyclicity, sperm motility and sperm morphology in both parental and F1 generations, the age at puberty in the F1s, histopathology of target organs, anogenital distance in the F2s, and primordial follicular counts in the parental and F1 generations. For the new prenatal developmental toxicity test guidelines, one important modification aimed at improved detection of endocrine disruptors was the expansion of the period of dosing from the end of organogenesis (i.e., palatal closure) to the end of pregnancy in order to include the developmental period of urogenital differentiation.

Experience with chemicals that have the potential to induce developmental toxicity indicates that both laboratory animal testing and surveillance of the human population (i.e., epidemiologic studies) are necessary to provide adequate public health protection.

Regulatory Guidelines for In Vivo Testing

New and internationally accepted testing protocols rely on the investigator to meet the primary goal of detecting and bringing to light any indication of toxicity to reproduction. Key elements of various tests are provided in Table 10–4. The general goal of these studies is to identify the NOAEL, which is the highest dosage level that does not produce a significant increase in adverse effects in the offspring.

Multigeneration Tests

Information pertaining to developmental toxicity can also be obtained from studies in which animals are exposed to the test substance continuously over one or more generations. For additional information on this approach, see Chapter 20.

Children's Health

Infants and children differ both qualitatively and quantitatively from adults in their exposure to pesticide residues in food because of different dietary composition, intake patterns, and different activities, such as crawling on the floor or ground, putting their hands and foreign objects in their mouths, and raising dust and dirt during play. Even the level of their activity (i.e., closer to the ground) can affect their exposure to some toxicants. In addition to exposure differences, children are growing and developing, which makes them more susceptible to some types of insults. Effects of early childhood exposure, including neurobehavioral effects and cancer, may not be apparent until later in life. Debate continues over the approach to be used in risk assessment in consideration of infants and children.

Alternative Testing Strategies

Various alternative test systems have been proposed to refine, reduce, or replace reliance on the standard regulatory mammalian tests for assessing prenatal toxicity (Table 10–5). These can be grouped into assays based on cell cultures, cultures of embryos in vitro (including submammalian species), and short-term in vivo tests. It was initially hoped that the alternative approaches would become generally applicable to all chemicals, and help prioritize full-scale testing. Indeed, given the complexity of embryogenesis and the multiple mechanisms and target site of potential teratogens, it was perhaps unrealistic to have expected a single test, or even a small battery, to accurately prescreen the activity of chemicals in general.

An exception to the poor acceptance of alternate tests for prescreening for developmental toxicity is the Chernoff/Kavlock in vivo test. In this test, pregnant females are exposed during the period of major organogenesis to a limited number of dosage levels near those inducing maternal toxicity, and offspring are evaluated over a brief neonatal period for external malformations, growth, and viability. It has proven reliable over a large number of chemical agents and classes.

Epidemiology

Reproductive epidemiology studies the associations between specific exposures of the father or pregnant woman and her conceptus and the outcome of pregnancy. The likelihood of linking a particular exposure with a series of case reports increases with the rarity of the defect, the rarity of the exposure in the population, a small source population, a short time span for study, and biological plausibility for the association. In other situations, such as occurred with ethanol and valproic acid, associations are sought through either a case–control or a cohort approach. Both approaches require accurate ascertainment of abnormal outcomes and exposures, and a large enough effect and study population to detect an elevated risk. Another challenge to epidemiologists is the high percentage of human pregnancy failures related to a particular exposure that may go undetected in the general population. Furthermore, with the availability of prenatal diagnostic procedures, additional pregnancies of malformed embryos (particularly neural tube defects) are electively aborted. Thus, the incidence of abnormal outcomes at birth may not reflect the true rate of abnormalities, and the term prevalence, rather than incidence, is preferred when the denominator is the number of live births rather than total pregnancies.

Other issues particularly relevant to reproductive epidemiology include homogeneity, recording proficiency, and confounding. Homogeneity refers to the fact that a particular outcome may be described differently by various recording units and that there can be multiple pathogenetic origins for a given specific outcome. Recording difficulties relate to inconsistencies of definitions and nomenclature, and to difficulties in ascertaining or recalling outcomes as well as exposures. For example, birth weights are usually accurately determined and recalled, but spontaneous abortions and certain malformations may not be. Last, confounding by factors such as maternal age and parity, dietary factors, diseases and drug usage, and social characteristics must be considered in order to control for variables that affect both exposure and outcome.

Epidemiologic studies of abnormal reproductive outcomes are usually undertaken with three objectives in mind: the first is scientific research into the causes of abnormal birth outcomes and usually involves analysis of case reports or clusters; the second aim is prevention and is targeted at broader surveillance of trends by birth defect registries around the world; and the last objective is informing the public and providing assurance. Cohort studies, with their prospective exposure assessment and ability to monitor both adverse and beneficial outcomes, may be the most methodologically robust approach to identifying human developmental toxicants.

Information on differential genetic susceptibility to birth defects continues to accrue. This new knowledge promises to elucidate links between genetics and disease susceptibility. Understanding the genetic basis of vulnerability to environmentally induced birth defects will allow more inclusive risk assessments and a better appreciation of the mechanisms of action of developmental toxicants.

Concordance of Data

Studies of the similarity of responses of laboratory animals and humans for developmental toxicants support the assumption that results from laboratory tests are predictive of potential human effects. Concordance is strongest when there are positive data from more than one test species. Humans tend to be more sensitive to developmental toxicants than is the most sensitive test species.

Elements of Risk Assessment

Extrapolation of animal test data for developmental toxicity follows two basic directions, one for drugs where exposure is voluntary and usually to high dosages and the other for environmental agents where exposure is generally involuntary and to low levels. For drugs, a use-in-pregnancy rating is utilized, wherein the letters A, B, C, D, and X are used to classify the evidence that a chemical poses a risk to the human conceptus. For example, drugs are placed in category A if adequate, well-controlled studies in pregnant humans have failed to demonstrate a risk, and in category X (contraindicated for pregnancy) if studies in animals or humans, or investigational or postmarketing reports, have shown fetal risk that clearly outweighs any possible benefit to the patient. The default category C (risks cannot be ruled out) is assigned when there is a lack of human studies and animal studies are either lacking or are positive for fetal risk, but the benefits may justify the potential risk. Categories B and D represent areas of relatively lesser or greater concern for risk, respectively.

For environmental agents, the purpose of the risk assessment process for developmental toxicity is generally to define the dose, route, timing, and duration of exposure that induces effects at the lowest level in the most relevant laboratory animal model. The exposure associated with this “critical effect” is then subjected to a variety of safety or uncertainty factors in order to derive an exposure level for humans that is presumed to be relatively safe. In the absence of definitive animal test data, certain default assumptions are generally made:

1. An agent that produces an adverse developmental effect in experimental animals will potentially pose a hazard to humans following sufficient exposure during development.

2. All four manifestations of developmental toxicity (death, structural abnormalities, growth alterations, and functional deficits) are of concern.

3. The specific types of developmental effects seen in animal studies are not necessarily the same as those that may be produced in humans.

4. The most appropriate species is used to estimate human risk when data are available (in the absence of such data, the most sensitive species is appropriate).

5. In general, a threshold is assumed for the dose–response curve for agents that produce developmental toxicity.

One troubling and subjective aspect of risk assessment for developmental toxicants is to distinguish between adverse effects that are detrimental to health and lesser effects that are considered not significant to human health. The interpretation of reduced fetal growth in developmental toxicity studies illustrates most of the issues. Whereas we have accepted definitions of low birth weight in humans and understand how intrauterine growth retardation translates to an elevated risk of infant mortality and mental retardation, similar knowledge in rodents is lacking. Further concerns arise from recent epidemiologic evidence suggesting that birth weight in humans is a predictor of adult-onset diseases including hypertension, cardiovascular disease, and diabetes.

There are several mechanisms of normal development that are conserved in diverse animals, including the fruit fly, roundworm, zebrafish, frog, chick, and mouse. Seventeen conserved intercell-ular signaling pathways are described that are used repeatedly at different times and locations during development of these and other animal species, as well as in humans (Table 10–6). The conserved nature of these key pathways provides a strong scientific rationale for using these animal models to advantage for developmental toxicology. These organisms have well-known genetics, embryology, and rapid generation times, and they are also amenable to genetic manipulation to enhance the sensitivity of specific developmental pathways or to incorporate human genes to answer questions of interspecies extrapolation.

Increased understanding of human genetic polymorphisms and their contribution to susceptibility to birth defects, use of sensitized animal models for high- to low-dose extrapolation, use of stress/checkpoint pathways as indicators of developmental toxicity, implementation of bioinformatic systems to improve data archival and retrieval, and increased multidisciplinary education and research on the causes of birth defects will aid assessment of the developmental risk of toxicants.