Concern has been raised in recent years regarding potential adverse effects of various environmental contaminants often called "endocrine disruptors" and referred to in this report as "hormonally active agents" (HAAs). In part, this concern originated from the finding that some synthetic chemicals in the environment that are associated with adverse reproductive and developmental effects in wildlife mimic the actions of the female sex hormone estradiol. In addition, the effects of in utero exposure to the potent synthetic estrogen diethylstilbestrol (DES) in the offspring of treated women and the replication of these effects in mice have focused attention on embryonic development as a target for the potential disruptive effects of environmental agents with hormonal activity. Although it is clear that exposures to HAAs at high concentrations can affect wildlife and human health, the extent of harm caused by exposure to these compounds in concentrations that are common in the environment is debated.
Driven by considerable public interest, the U.S. Environmental Protection Agency (EPA), the U.S. Department of the Interior (DOI), the U.S. Centers for Disease Control and Prevention (CDC), and the U.S. Congress requested that the National Research Council (NRC) conduct an independent study of this topic. In response to that request, the NRC convened a multidisciplinary expert committee, the Committee on Hormonally Active Agents in the Environment, and charged it with the following tasks: (1) review critically the literature on HAAs in the environment; identify the known and suspected toxicologic mechanisms and impacts on fish, wildlife, and humans; identify significant uncertainties, limitations of knowledge, and weaknesses in the available evidence; develop a science-based conceptual framework for assessing observed phenomena: and recommend research, monitoring, and testing priorities; (2) to the extent practi-soft
cable with available information and study resources, identify particular chemical substances, geographic areas, contaminant sources, human subpopulations, and fish and wildlife populations of special concern with respect to HAAs; and (3) if possible and warranted, suggest general approaches for identifying and mitigating toxicologic problems.
The charge to the committee did not include, nor did the committee attempt to evaluate, risk-management policy options. Due to constraints of time and resources, the committee also did not consider approaches for mitigating toxicologic problems.
The Committee's Approach and Difficulties Encountered
To evaluate the endocrine-disruptor hypothesis, the committee's approach involved ( ) identification of chemicals with hormonal or antihormonal activity: (2) evaluation of scientific literature on the effects in both the adult and the developing organism associated with those chemicals in vertebrateshumans. laboratory animals, and wildlife; and (3) consideration of whether the effects can be attributed to the hormonal properties of the chemicals and environmental exposure to them.
The committee focused its attention on compounds that have been reported to induce reproductive changes, developmental defects, neurobehavioral abnormalities, immunologic deficits, carcinogenesis, and ecologic effects.
It became clear as the work of the committee progressed that limitations and uncertainties in the data could lead to different judgments among committee members with regard to interpreting the general hypothesis, determining appropriate sources of information, evaluating the evidence, defining the agents of concern, and evaluating environmental and biologic variables.
Some of the differences reflect areas where additional research would help; others reflect differing judgments about the significance of the existing information. The differences are not confined to this committee but are reflected in the scientific community at large. Some differences appear to stem from different views of the value of different kinds of evidence obtained by experiments, observations, weight-of-evidence approaches, and extrapolation of results from one compound or organism to others, as well as allowable sources of information and criteria for arriving at meaningful conclusions and recommendations.
Other difficulties arise from questions about the observed effects: for example, is human sperm concentration really in decline? In other cases, the effect is clear (e.g., developmental abnormalities in some wildlife species), but the cause is in question. Often, organisms are exposed to many environmental chemicals as well as other environmental and ecologic perturbations. In addition, one must recognize that under multiple exposures, there is the potential for interaction among agents. These factors make assigning cause to specific chemicalscontinue
extremely difficult. Understanding the mechanism of action of various compounds is inseparable from defining HAAs and is one reason for the difficulty in defining HAAs. As an example, many compounds that have hormone-like activity might also affect organisms through pathways unrelated to any hormonal activity. Despite these differences, however, the many areas of consensus among the committee members are described below.
The Committee's Evaluation
Mechanism of Action
For most associations reported between HAAs and various biologic outcomes, the specific mechanism of action is not well understood. However, lack of knowledge about a mechanism does not mean that a reported effect is unconfirmed or unimportant, nor does demonstration of a mechanism document that the resulting effects are unique to that mechanism or are pervasive in natural systems. It does suggest that additional studies are warranted. In general, the committee does not believe that HAAs can function only through receptor binding and recommends research to distinguish between direct and indirect effects and between primary and secondary effects of HAAs. Underlying mechanisms of action should be investigated with both in vitro and in vivo systems that can detect diverse responses.
Because of the difficulties associated with establishing cause and effect in humans and wildlife populations, for which exposure to HAAs is often the result of unintended releases of chemicals into the environment and involves exposures to multiple chemicals, the committee evaluated laboratory studies on individual HAAs in conjunction with available data from human studies and from field observations of wildlife. The following sections summarize the committee's consensus with respect to developmental, reproductive, neurologic, immunologic, carcinogenic, and ecologic effects of HAAs.
Developmental Effects and Reproduction
Adverse reproductive and developmental effects have been observed in human populations, wildlife, and laboratory animals as a consequence of exposures to HAAs. In humans, the effects of prenatal exposure to polychlorinated biphenyl (PCBs), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and other contaminants from maternal consumption of contaminated fish or other food products have been studied in several populations in the United States and abroad. Collectively, these studies indicate that prenatal exposure to PCBs can causecontinue
lower birth weight and shorter gestation and have been correlated with deficits in IQ and memory as well as delayed neuromuscular development. Prenatal and postnatal exposure to PCBs and polychlorinated dibenzofuran (PCDFs) from accidental contamination of rice oil in Yusho, Japan, and Yu-Cheng, Taiwan, have resulted in various developmental defects. Reported increases in the incidence of male reproductive disorders such as hypospadias (urethra opening found at the bottom rather than the tip of the penis), cryptorchidism (undescended testes), and testicular cancer cannot be linked to exposures to environmental HAAs at this time. With respect to the end point most closely studied, sperm concentration, retrospective analyses of trends over the past half-century remain controversial. When the data from large regions are combined and analyzed. some data sets indicate a statistically significant trend consistent with declining sperm concentrations. However, aggregation of data over larger geographic regions might not be an appropriate spatial scale for this analysis, given the significant geographic heterogeneity. The current data are inadequate to assess the possibility of trends within more appropriately defined small regions. Acquiring data at smaller regional scales is critical to assessing the significant geographic variation in sperm concentration.
Laboratory studies using male and female rats, mice, and guinea pigs and female rhesus monkeys have shown that exposure of these animals during development to a variety of concentrations of certain HAAs (e.g., DDT, methoxychlor, PCBs, dioxin, bisphenol A, octylphenol, butyl benzyl phthalate (BBP), dibutyl phthalate (DBP), chlordecone, and vinclozolin) can produce structural and functional abnormalities of the reproductive tract.
Many wildlife studies show associations between reproductive and developmental anomalies and exposure to environmental contaminants, some of which are HAAs. Reproductive and developmental abnormalities have also been observed in several populations of fish exposed to effluents from sewage treatment plants and paper mills and polluted waters of the Great Lakes. Effects observed include intersexes in trout exposed to sewage-treatment-plant effluent (STPE); increased egg and fry mortality in Great Lakes trout and salmon; thyroid enlargement in Great Lakes salmon; and changes in plasma sex-steroid concentrations, decreased egg and gonad size, and delayed sexual maturity in white suckers exposed to effluents from paper mills along Lake Superior.
Laboratory experiments with specific HAAs found in those effluents and polluted waters have produced effects consistent with those wildlife observations. For example, certain HAAs found in STPEs induce estrogenic responses in male trout. Specifically, ethinylestradiol and alkylphenol ethoxylates have been shown to induce vitellogenin synthesis, a hallmark of estrogen exposure, and to decrease the rate of testicular growth in male fish in tests that duplicate concentrations found in some effluents. Dioxin and structurally related compounds have been shown to induce blue-sac disease in developing trout and growth and sur-soft
vival reduction in salmon. Thyroid enlargement in salmon of the Great Lakes is hypothesized to be caused by exposure to PCBs, which also have been shown to induce goiter formation in laboratory rodents fed PCB-contaminated salmon from the Great Lakes. Finally, ß-sitosterol found in paper-mill effluent has been shown to alter the reproductive physiology of goldfish under experimental conditions, in which goldfish were injected with 10-100 µg/g.
Laboratory studies are also consistent with some reproductive and developmental abnormalities (e.g., skewed sex ratios, behavioral modifications, and morphologic abnormalities of the gonads) observed among North American gull populations. Specifically, gull eggs injected with DDT at concentrations found in wild gull eggs induce gonadal abnormalities that are similar to those observed in contaminated gulls. Also, doves fed mixtures containing DDE and PCBs exhibit abnormal breeding behavior.
Similarly, defects seen in alligators from Lake Apopka (the site of a chemical spill containing dicofol and DDT), including small penis size and abnormal testes in males and abnormal ovaries in females, are consistent with structural and functional reproductive abnormalities that occur following perinatal exposure of laboratory rodents to estrogenic and antiandrogenic chemicals.
Wildlife and human populations should continue to be monitored for abnormal development and reproduction. Studies of wildlife species that exhibit population declines, abnormal sociosexual behavior, or deformities should be designed to investigate those phenomena with regard to chemical contamination. In human populations suspected of being affected by HAAs, prospective and cross-sectional studies using cohorts tracked from conception through adulthood are particularly needed on female and male reproductive end points, such as sperm concentration, cryptorchidism, and hypospadias. Regional differences in those end points should be studied prospectively to determine whether the differences can be associated with genetic and environmental factors.
In humans, results of cognitive and neurobehavioral studies of mother-infant cohorts accidently exposed to high concentrations of PCBs and PCDFs and of mother-infant cohorts eating contaminated fish and other food products containing mixtures of PCBs, dioxin, and pesticides (such as DDE, dieldrin, and lindane) provide evidence that prenatal exposure to these HAAs can affect the developing nervous system. Similarly, monkeys exposed to PCBs in utero and during lactation have deficits in cognitive function when assessed at 14 months post-exposure and rats and mice exposed prenatally to PCBs suffer impaired locomotor ability and learning.break
In human populations suspected of being affected by HAAs, longitudinal tests should be conducted on developmental milestones from conception through adulthood. A standardized set of criteria should be established to study neurobiologic and social development.
Several HAAs affect diverse elements of the immune system in laboratory animals. There is evidence of suppression of the immune system by exposure to organochlorines (predominantly PCBs) in birds in the Great Lakes region. There is also evidence of suppression of innate and acquired immune responses in seals fed fish from the PCB-contaminated Baltic Sea. Such immunosuppression is believed to be the reason for the increased incidences of bacterial and viral infections in seals in similarly contaminated waters. In humans, data on the immunologic effects of HAAs are inadequate to support any definitive conclusions.
Studies are needed on the prevalence of autoimmune problems in cohorts suspected of being affected by HAAs, especially offspring whose mothers were exposed during pregnancy. Cohort studies that include various life phases should use clinically relevant immunologic assays to clarify the relationship between HAA exposure and human health. The immunologic activity of HAAs currently in use (e.g., endosulfan and lindane) should also be investigated in laboratory studies.
With the exception of several bioassays in which mice were exposed to DDT during fetal life, during lactation, and after weaning without apparent adverse effects, perinatal exposure to environmental HAAs has not been assessed with respect to carcinogenesis in laboratory animals or humans, nor have transgenerational effects been investigated. Although some HAAs (e.g., toxaphene, DDE, DDT, TCDD, and endrin) have been associated with tumors of the thyroid, pituitary, or renal glands in particular species and strains of laboratory animals, HAAs in general have not been shown to induce tumors in reproductive or other endocrine organs after postnatal exposure, but the suitability of animal models might be in question. An evaluation of the available studies conducted to date does not support an association between adult exposure to DDT, DDE, TCDD, and PCBs and cancer of the breast. Although the current literature does not support associations between those HAAs and other hormonally sensitive can-soft
cers (testicular, prostate, or endometrial cancer), few studies have examined measured concentrations of those compounds in adults in relation to cancer risk. Moreover, no studies have been conducted to examine associations between risk of any cancer and exposure to any HAAs during development, particularly during fetal life. However, a recent study reported an association between dieldrin and breast cancer; additional epidemiologic and laboratory studies are needed to help confirm or refute this possible relationship.
Appropriately designed and conducted case-control and retrospective cohort studies are needed to document the presence or absence of associations between HAAs and various cancers in humans. If such associations are found, the possibility of causality must be investigated. Such studies should take into account the latency period between exposure and disease, the timing of likely exposure windows with respect to cancer, indicators of susceptibility, and the role of potential confounders. In addition, markers of total xenoestrogen exposure and chemical concentrations in blood or adipose tissue should be measured to provide an accurate assessment of internal dose and, therefore, to identify groups experiencing different exposures during the recent decade.
Research in appropriate animal models is needed on the role of prenatal exposure to suspected chemicals in inducing cancers later in life or in subsequent generations. Initial studies should focus on HAAs that have been shown to induce cancers of the thyroid, pituitary, and adrenal glands in some laboratory animals.
Environmental HAAs probably have contributed to declines in some wildlife populations, including fish and birds of the Great Lakes and juvenile alligators of Lake Apopka, and possibly to diseases and deformities in mink in the United States, river otters in Europe, and marine mammals in European waters. Such contaminants, along with inbreeding, might have contributed to the poor reproductive success of the endangered Florida panther and the increased embryonic mortality of the snapping turtle in the Great Lakes.
Biologic communities in all the Great Lakes, including species from plankton to top predators, have undergone very large changes in the past 100 years. The changes have resulted from introduction of exotic species, pollution, fishing, development of the shorelines, changes in the lakes' hydrology and that of their tributaries, and other factors. To the degree that HAAs have affected population sizes of individual species, they might also have contributed to those changes in community structure, but it is difficult to account quantitatively for the various causal factors. There is epidemiologic and experimental evidence that somecontinue
persistent, bioaccumulative HAAs (also referred to as persistent organic pollutants) produce adverse effects on wildlife populations, but whether the primary effect is mediated by hormonal activity remains to be determined.
Long-term studies of populations subjected to HAA exposures are needed to assess the effects of these chemicals in altering population size, age structure, and dynamics. Observational and experimental studies of the linkages between chemical exposures and alterations of key aspects of life histories should be undertaken to understand how chemical exposures affect long-term ecologic attributes of natural systems. Ultimately, the physiologic and biochemical basis of these linkages, once established, should be determined.
Exposure, Dosimetry, and Screening and Monitoring
Determining the risk of environmental HAAs to humans and wildlife is difficult because exposure to these agents has not been routinely monitored, and effects that might be attributed to background concentrations could be complicated by endogenous hormones, pharmacologic estrogens (e.g., hormonal contraceptives), and naturally occurring HAAs (e.g., phytoestrogens) that are ubiquitous in the environment. Synthetic HAAs have been detected in all environmental media, although concentrations of some compounds, such as PCBs and DDT, have declined in some regions, because their use has been discontinued in those countries. However, those HAAs and others can persist in some media, such as sediments, for years and can contaminate areas far removed from the original site of contamination (e.g., via atmospheric transport). Some human populations have been found to have relatively high exposures to HAAs, primarily from diets that include frequent consumption of contaminated foods, especially fish, or foods containing phytoestrogens.
Understanding the relationships between exposure, absorption, disposition, metabolism, excretion, and response is important for predicting whether exposure to an agent will be harmful. Once inside an organism, the transport of an HAA to potential target organs is influenced by its binding to plasma proteins. Lipophilic HAAs will cross the placenta and can have effects on the developing organism. Adipose and other tissues can accumulate HAAs and serve as reservoirs or depots.
There are important differences among species and between adult and developing organisms in their responses to HAAs. These differences could have important implications when assessing toxicity studies or extrapolating data from one species or subpopulation to another. In addition, biologic responses to some HAAs might be greater at low doses than at high doses.
There are no generally accepted, adequately validated methods for routinecontinue
identification or monitoring of exposures to HAAs. In addition, most in vitro and in vivo screening assays do not address the full range of putative actions of HAAs.
Better monitoring of contaminated media is required to determine environmental concentrations of HAAs and to assess the persistence and recycling of HAAs in and between the various environmental media. Background concentrations of HAAs in humans, particularly in adipose tissue and blood, and other biota need to be established. In particular, routes of exposure and the effects of diet need to be assessed to provide a framework for examining the effects of these compounds in the general population and in highly exposed subpopulations.
Differences in response to HAAs among species and between adult and developing organisms need to be investigated further, especially at concentrations encountered in the environment. Dose-response relationships of recognized actions of various HAAs also need to be investigated at those concentrations in vitro and in vivo studies.
The committee's recommendations for screening and monitoring are consistent, in principle, with those of EPA's Endocrine Disruptor Screening and Testing Advisory Committee. Those recommendations include (1 ) a battery of short-term assays for rapid and inexpensive screening of putative HAAs that need to be validated, replicated, and deployed appropriately; (2) the development and validation of additional biomarkers that screen for embryonic and fetal events that predict long-term, delayed effects; (3) the monitoring of wildlife as environmental sentinels; (4) further investigation of species- and tissue-specific effects resulting from exposure to HAAs; and (5) better characterization of dose-response relationships of various HAAs through in vitro and in vivo assays at concentrations encountered in the environment.break