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WHITE PAPER

ENVIRONMENTAL ENDOCRINE DISRTUPTORS AND ITS POTENTIAL IMPACTS ON PUBLIC HEALTH AND THE ENVIRONMENT IN NEW JERSEY

May 2003

Prepared By:

Thomas Ledoux, Ph.D.,

DEPARTMENT OF ENVIRONMENTAL PROTECTION DIVISION OF SCIENCE, RESEARCH AND TECHNOLOGY

Acknowledgements

This paper would not have been possible without the unceasing assistance of Mary Kearns-Kaplan, Dorothy Alibrando and Maria Baratta, technical librarians at the New Jersey DEP, Information Resource Center. Their help in acquiring numerous reprints, volumes, and other technical information is gratefully acknowledged and appreciated. Also acknowledged is the technical assistance of Terri Tucker, DSRT. Also gratefully acknowledged is the assistance of Dr. Alan Stern, Chief, Bureau of Risk Analysis, DSRT; Dr. Eileen Murphy, Assistant Director, DSRT; Dr. Keith R. Cooper, Dean of Research & Graduate Programs, Cook College; Dr. Mark G. Robson, Chairman, Department of Environmental & Occupational Health, UMDNJ-School of Public Health; Dr. Michael A. Gallo, Professor of Environmental Medicine, UMDNJ-Robert Wood Johnson Medical School; Dr. Perry Cohn, New Jersey Department of Health & Senior Services; and Drs. Michael Aucott and Branden Johnson, Division of Science Research & Technology for critical comments in the review and editing of the manuscript.

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TABLE OF CONTENTS I. EXECUTIVE SUMMARY ...... 1

II. PURPOSE ...... 3

III. BACKGROUND ...... 4

IV. ENDOCRINE DISRUPTORS...... 6

V. BASIC BIOLOGY AND MAJOR CONCEPTS ...... 7 DEVELOPMENTAL BIOLOGY AND ...... 7 VI. ...... 10 A. HOW HORMONES WORK / MECHANISM OF ACTION ...... 10 B. FEMALE HORMONES...... 10 1. ...... 10 2. ...... 11 3. FOLLICLE-STIMULATING (FSH), (LH), AND THYROID-STIMULATING HORMONE (TSH) ...... 11 C. MALE HORMONES ...... 11 1. ...... 11 2. THYROID HORMONE ...... 11 3. ...... 12 VII. SPECIFIC ENDOCRINE DISRUPTORS ...... 13 A. PHARMACEUTICAL ESTROGEN MIMICS & ... 13 B. ...... 13 C. CLASSES OF COMPOUNDS OF POTENTIAL CONCERN...... 14 D. SPECIFIC ENDOCRINE-MEDIATING CHEMICALS OF POTENTIAL ENVIRONMENTAL CONCERN...... 15 1. (DES)...... 15 2. POLYETHOXYLATES ...... 17 3. A...... 18 4. ...... 20 5. POLYCHLORINATED BIPHENYLS (PCBs)...... 21 6. DIOXINS ...... 24 7. POLYBROMINATED DIPHENYL ETHERS ...... 25 8. ...... 26 9. ...... 28 10. GROWTH PROMOTERS...... 31 11. ORGANOTIN COMPOUNDS...... 31 12. PHARMACEUTICAL COMPOUNDS...... 32 13. DIESEL GASES ...... 32 E. IMPACTED ANIMALS...... 33 1. SNAILS...... 33 2. MOLLUSKS...... 33

ii 3. FROGS...... 33 4. FISH...... 36 5. ALLIGATORS ...... 38 6. TURTLES...... 38 7. HUMANS ...... 39 VII. ENVIRONMENTAL TRANSPORT OF ENDOCRINE DISRUPTIVE CHEMICALS ...... 42

IX. ALTERATIONS IN THE POPULATION DISTRIBUTIONS OF VARIOUS ENDOCRINE-RELATED PARAMETERS ...... 42

X. RELATIONSHIP OF TO CANCER...... 43 A. ...... 43 B. PROSTATE CANCER...... 44 XI. OTHER MALE EFFECTS...... 45

XII. ROLE OF GOVERNMENT - FEDERAL, STATE, AND INTERNATIONAL ORGANIZATIONS...... 46 A. FEDERAL GOVERNMENT ...... 46 B. STATE GOVERNMENT...... 49 C. INTERNATIONAL...... 50 XIII. POSITIONS TAKEN BY NATIONAL SOCIETIES ...... 51 A. NEHA POSITION ON ENDOCRINE DISRUPTERS (1997)...... 51 B. TERATOLOGY SOCIETY POSITION PAPER: THE DEVELOPMENTAL OF ENDOCRINE DISRUPTORS TO HUMANS (1999) ...... 51 C. NATIONAL ACADEMY OF SCIENCES REPORT: HORMONALLY ACTIVE AGENTS IN THE ENVIRONMENT (1999) ...... 52 XV. DISCUSSION ...... 55

XV. NEW JERSEY-SPECIFIC RECOMMENDATIONS ...... 56 A. GENERAL ...... 56 B. INDICATOR DEVELOPMENT/IMPLEMENTATION...... 56 C. ENVIRONMENTAL SURVEILLANCE ...... 57 D. RESEARCH...... 58 D. OTHER ACTIVITIES...... 59 XVI. EXAMPLES OF RECENT AND ONGOING ENDOCRINE DISRUPTOR RELATED NEW JERSEY RESEARCH PROJECTS...... 59 A. NJDEP, DIVISION OF SCIENCE, RESEARCH & TECHNOLOGY ...... 59 1. New York/New Jersey Harbor Estuary Project ...... 60 2. Development of a Method to Access In Utero Exposure of Infants to Environmental Contaminants ...... 60 3. Investigation of Endocrine Disruption Effects of on New Jersey Frogs...... 60 B. , ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES INSTITUTE (EOHSI) ...... 60

iii C. PRINCETON UNIVERSITY ...... 61 XVII. REFERENCES AND BIBLIOGRAPHY...... 62

XVIII. APPENDIX I - GLOSSARY ...... 90

XVIII. APPENDIX II - SUMMARY OF THE ENDOCRINE DISRUPTOR SCREENING AND TESTING ADVISORY COMMITTEE FINAL REPORT AND RECOMMENDATIONS...... 94

iv Acronyms and abbreviations used in this report:

AGD: Anogenital distance Ah: Aryl hydrocarbon receptor APEOs: Alkylphenol polyethoxylates BPA: CERHR: Center for the Evaluation of Risks to Human Reproduction DBP: Dibutyl DDE: 1,1-Dichloro-2, 2-bis (p-cholorphenyl) ethylene or dichlorodiphenyldichloro-ethylene DDT: 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane or dichloro-diphenyl trichloroethane DEP/M3: Diesel exhaust particles per cubic meter DEHP: Di(2-ethylhexyl) phthalate DES: Diethylstilbestrol DHT: DINP: Diisononyl phthalate DSRT: Division of Science, Research, and Technology E2: 17 Beta- ED: Endocrine disruption EDMVS: Endocrine Disruptor Methods Validation Subcommittee EDSTAC: Endocrine Disruptor Screening and Testing Advisory Committee EDTA: Endocrine Disrupter Testing and Assessment EE: ELISA: Enzyme-Linked Immunosorbent Assay FSH: Follicle-Stimulating Hormone GR: Receptors HAA: Hormonally Active Agents HPLC: High Performance Liquid Chromatography IC50: Concentration that inhibits 50% of activity IgG: Immunoglobulin G IUPAC: International Union of Pure and Applied Chemistry 11-KT: 11-ketotestosterone LH: Luteinizing hormone mg/kg/day: milligram per kilogram per day MOA: Mode of Action MCLs: Maximum Contaminant Levels NEHA: The National Environmental Health Association NOAEL: No-Observed-Adverse-Effect Level NTP: National Toxicology Program ng/g: nanogram per gram ng/L: nanogram per liter ng/M3: nanograms per cubic meter OECD: Organization for Economic Co-operation and Development OPPTS: EPA's Office of Prevention, Pesticides and Toxic Substances ORD: EPA's Office of Research and Development OTC: over the counter PBDE: Polybrominated diphenyl ethers PCBs: Polychlorinated biphenyls PCDDs: Polychlorinated dibenzo-p-dioxins PCDFs: Polychlorinated dibenzofuran POPs: Persistent Organic Pollutants ppb: parts per billion ppm: parts per million ppt: parts per trillion PVC: SWHS: Seveso Women's Health Study TBT: Tributytin TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin 2,3,7,8-TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin, commonly known as dioxin TPT: triphenyltin TSH: Thyroid-Stimulating Hormone ug/L: micrograms per liter YES: Yeast Estrogen Screening bioassay

v I. EXECUTIVE SUMMARY

ABSTRACT The purpose of this White Paper on endocrine disruption (ED) is threefold: 1. provide a brief review of the current state of the science regarding the potential environmental health impacts of ED; 2. consider how ED effects may be impacting environmental health in New Jersey now and in the future; and 3. suggest practical approaches for investigating the existence and extent of such ED effects in New Jersey for the purpose of designing strategies to mitigate ED impacts.

BACKGROUND In the decades since the publication of Rachel Carson's Silent Spring, there has been a gradual increase in public awareness of the potential for abnormal growth and development in various wildlife and human populations associated with the vast increase in the use of synthetic organic chemicals. Reports in the scientific literature have noted effects including: abnormal genitalia in alligators; gender changes in fish; poor reproductive outcomes in certain fish, birds, frogs, and humans; and abnormal sexual development in fish, mollusks, and avian species. The term endocrine disruption has been coined for such phenomena and applies most properly when the effect is related to the endocrine system. The scientific literature from the past several decades contains reports of persistent and bioaccumulative chemicals in the environment that may act via an endocrine mechanism to cause adverse health impacts in a number of species, including humans. It is critical to understand the effects of these compounds because they potentially have long-term adverse consequences, including adverse developmental effects.

SPECIFIC ENDOCRINE DISRUPTORS OF POTENTIAL ENVIRONMENTAL CONCERN Many thousands of chemicals remain to be investigated for their potential endocrine disrupting properties. Among those currently recognized or suspected are: , a common breakdown product of and anti-foaming agents; bisphenol A, a common constituent of commercial plastics; phthalates, PVC additives that are now pervasive in the environment; PCBs (polychlorinated biphenyls); polybrominated diphenyl ethers (PBDE), high volume flame retardants whose concentrations in the chain and human milk are rising dramatically; DDT and its main metabolite DDE, still prevalent in the environment despite its ban in 1972; pyrethroid pesticides, the most used indoor pest control agents; atrazine, the most commonly applied herbicide in the U.S.; and various pharmaceuticals, including synthetic estrogens in birth control pills and metabolites of synthetic estrogens discharged with sewage treatment effluent.

ANIMAL POPULATIONS IMPACTED BY ENDOCRINE DISRUPTORS Frogs - Recent evidence suggests that concentrations of atrazine common in New Jersey surface waters can cause hermaphroditism in male frogs. Causes of gross frog deformities are still unresolved, but environmental contaminants may play a role. Fish - Male fish in receiving waters of sewage treatment plants have been found with female characteristics including egg-containing testes. The discharge of female hormones including birth control metabolites appears to be involved.

1 Alligators - A 1980 spill of DDT and dicofol, organochlorine pesticides, in Florida's Lake Apopka resulted in a range of hormonally-mediated effects including altered reproductive tracts and abnormal genitalia in male alligators.

ENDOCRINE DISRUPTION EFFECTS IN HUMANS While more speculative than effects in animals, potential effects of endocrine-disrupting contaminants on humans are plausible and of considerable concern. These include: Premature Female (thelarche) - Puerto Rico has the highest reported rate of thelarche. A recent study found significantly elevated serum levels of several phthalates in thelarche cases with little or no elevation in those without thelarche. While not conclusive, the ubiquity of phthalates in the environment raises concerns for the issue of various secondary sexual processes in girls. Sex Ratio - A decrease of about 0.4% in the proportion of males to female births has been noted in birth data since the 1950s in western Europe and since the 1970s in the U.S. and Canada. The reasons for this are not clear. However, the dramatic drop in male births in Seveso, Italy following the well-known dioxin release suggests a plausible connection of decreasing male births to environmental contaminants. Male Fertility - Recent studies suggest a general decline in sperm in men in developed countries since about 1940. In the U.S., the decline appears to be about 1.5% per year and about 3% per year in Europe. Although no clear cause has been suggested, the ability of several common contaminants (including nonylphenols and dioxins) to interfere with sperm production raises concerns for environmental exposure. Breast Cancer - Several studies of varying quality have reported higher blood levels of DDE and PCBs in patients with breast cancer than in controls. Such a relationship, however, remains controversial.

THE ROLE OF THE FEDERAL GOVERNMENT IN ADDRESSING ENDOCRINE DISRUPTION The Protection Act and the Safe Drinking Water Act amendments of 1996 required the EPA to develop screening and testing strategies to determine whether certain substances may have hormonal effects in humans. In response, the EPA's Office of Prevention, Pesticides and Toxic Substances (OPPTS) established an Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). While the EDSTAC proposed framework has been finalized, much developmental, validation, and implementation work remains. The work is significantly behind the schedule set by the legislation and systematic testing has not yet begun.

THE ROLE OF STATE GOVERNMENTS States are in the best position to predict likely areas and populations at risk from endocrine disruption. They should also be able to identify those potential endocrine disruptor substances produced, used, and discharged in their jurisdictions with potential for local impact. Such substances may escape scrutiny at the national level. Existing state programs can be utilized to identify potential problem areas in the state. Cross-program efforts will be necessary to fully address the issue.

2 NEW JERSEY-SPECIFIC RECOMMENDATIONS General The NJDEP should formulate an endocrine disruptor-specific policy that addresses the development of endocrine disruptor indicators, surveillance, and research. The policy should also provide mechanisms for staying current with ongoing scientific developments. A useful mechanism for development of this policy and for interchange of information would be a NJ Endocrine Working Group consisting of scientists from State government, academia, chemical and pharmaceutical companies, and environmental groups. Indicator development Establish a New Jersey monitoring program to identify and track maternal and infant exposures to persistent endocrine disrupting chemicals. Such a program would identify specific chemicals, potential hot spots of exposure, and population groups with elevated exposures. This approach would also establish a baseline against which trends in exposures could be detected and tracked over time. Environmental surveillance Institute surveillance that focuses both on the environmental occurrence of known and suspected endocrine-disrupting chemicals and on the identification of effects in populations of sentinel biota potentially mediated by endocrine disruptors. Priorities for surveillance should include waterways impacted by current or historical industrial activity or high population density (e.g., Newark Bay); waterways which support both sewage treatment plant discharges and drinking water intakes in close proximity (e.g., the Passaic River basin); and waterways receiving significant runoff from cultivated land (i.e., golf courses and farmland). Likely chemical candidates for surveillance include pesticides (atrazine), personal care and cleaning products (e.g., nonylphenol polyethoxylates), pharmaceuticals and their metabolites (including synthetic estrogens), and flame retardants (PBDEs). Research 1. Support the development in NJ academic institutions of specific analytical capabilities for detection of low concentrations of endocrine disruptors in various environmental matrices. Such capability is essential for environmental surveillance. 2. Continue and expand studies of organochlorine effects on reproduction in endangered and sentinel NJ wildlife species including ospreys and eagles in the Delaware Bay region. Efforts should be made to identify other species and populations similarly affected. 3. Characterize the environmental chemistry and fate of known and potential endocrine- disrupting chemicals and their metabolites in New Jersey. This should specifically include the chemistry relevant to the passage of such chemicals through wastewater treatment plants. 4. Characterize fish and other aquatic populations in heavily utilized waters including receiving waters of sewage treatment plants. Such characterization should include gender shifts, sexual development, and reproductive competency.

II. PURPOSE

The purpose of this White Paper on endocrine disruption (ED) is threefold: 1. To provide a brief (and by no means comprehensive) review of the current state of the science regarding the potential environmental health impacts of ED; 2. To consider how ED effects may be impacting

3 environmental health in New Jersey now and in the future; and 3. To suggest practical approaches for investigating the existence and extent of such ED effects in New Jersey for the purpose of developing indicators and designing strategies to mitigate ED impacts.

As ED is extremely complex and technical in nature, this paper will not be an exhaustive and comprehensive review on all aspects of this topic; rather it will provide sufficient background and summary information to demonstrate the type and range of impacts that may result from endocrine disruption in New Jersey. This limitation is necessary as endocrine disruption encompasses a wide spectrum of biological and medical sciences, including endocrinology, toxicology, , physiology, immunology, reproductive toxicology, neonatology, biochemistry, and environmental sciences. To assist with the broad technical range of the subject, the document includes a brief section on pertinent biology and the major concepts involved. A glossary of terms used is included in the appendix.

III. BACKGROUND

Many people can trace their earliest knowledge of the potential disruption of wildlife from use to the publication of Silent Spring by Rachel Carson in 1962. She was one of the first to popularly describe the hazards of pesticides, such as DDT, that can accumulate in the fatty tissues of many species of animals, killing not only targeted insects but many others besides, and which can remain active in the environment for long periods of time, eventually accumulating in wildlife and human food sources.

In the decades since then, there has been a gradual increasing public awareness of reports in the scientific and lay press concerning abnormal growth and development in various wildlife species associated primarily with the vast increase in the use of synthetic organic chemicals. These reports have included: abnormal penises in alligators, gender changes in fish such as salmon and trout, poor reproduction in certain fish, birds, frogs, and possibly man, and abnormal sexual development in fish, mollusks, and avian species. The term endocrine disruption has been coined for such phenomena and applies most properly when the effect is related to the endocrine system.

The term endocrine refers to secretion of hormones from specialized organs and tissues in animals into blood or lymph in the body. These hormones have specialized regulatory effects on sensitive organs or tissues in animals at some distance from their origin. Hormones are biochemical substances produced in endocrine glands in the body that serve to coordinate and regulate rates of specific biochemical reactions either by augmentation or restraint of specific enzyme systems. To accomplish their intended purpose, hormones must be released at precisely the proper time and in the correct amounts. Adverse or less than optimal physiological action may occur if the hormone release occurs too early, too late, not at all, or not in the proper amount. Hormones are without effect unless they are available at the proper target tissues and those tissues are capable of responding properly. The competence of any particular hormone may also be altered by other pharmacologically active substances present in the body (Turner and Bagnara, 1976). Endocrinology, the science of hormones and their actions, is very young and was only born in the late years of the 19th century.

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Scientific literature from the past several decades contains reports of chemicals that may act via an endocrine mechanism to cause adverse health impacts in a number of species. Chemicals possessing this activity are popularly termed endocrine disruptors or endocrine mimics. It is critical to understand the effects of these compounds because they can persist in the body for prolonged periods of time, have long-term adverse consequences, and transfer to the offspring where they can again cause adverse developmental consequences. In the last decade, there has been intense interest in whether endocrine-active chemicals in the environment may cause endocrine related effects similar to (or perhaps qualitatively different from) those which have been observed in wildlife populations and/or in laboratory studies.

In 1991, a group of scientists met at the Wingspread Conference Center in Racine, Wisconsin to assess what was known about the endocrine disruptor issue. The proceedings of this conference later became published as "Chemically Induced Alterations in Sexual Development: The Wildlife/Human Connection" (Colborn and Clement, 1992). While scientists at this conference were from many different biological fields, they shared a common interest in the adverse health impacts observed in wildlife in the Great Lakes' region and in Europe and in the marine mammals in the Northern Hemisphere (Colborn, et al, 1992). A consensus statement they issued at the end of the conference, stated that, with "certainty" a large number of man-made chemicals that had been released into the environment, as well as a few natural ones, have the potential to disrupt the endocrine system of animals and man. Further, some of these chemicals are persistent, bioaccumulative, organohalogen compounds that include pesticides, fungicides, herbicides, insecticides, and industrial chemicals. Certain wildlife populations are known to be affected by these compounds, showing effects such as: thyroid dysfunction in birds and fish; decreased fertility in birds, fish, shellfish, and mammals; decreased hatching success in birds, fish, and turtles; gross birth deformities in birds, fish, and turtles; metabolic abnormalities in birds and fish; of male fish, birds, and mammals; defeminization and masculinization of female fish and birds; and compromised immune systems in birds and mammals. While most of these conclusions were based on results from field studies, some laboratory studies corroborated the effects observed in the field (Colborn, et al, 1992).

The Conference attendees noted that some of the developmental impairments reported in humans are seen in adult offspring of parents exposed to synthetic hormone disruptors (antagonists and antagonists) released in the environment. They noted that concentrations of some synthetic sex hormones measured in the human population now are within the range at which effects are seen in wildlife populations. Further, experimental results are seen at the low end of current concentrations found in the environment (Colborn, et al, 1992).

Scientists at the Wingspread Conference thought that, unless the environmental loading of these synthetic hormone disruptors is lessened and controlled, large-scale dysfunction at the population level is possible. As more research is conducted on this problem more parallels in wildlife, laboratory, and human research are expected to be revealed (Colborn, et al, 1992).

In a speech at the Rio + 5 Forum March 14, 1997 Dr. J. P. Myers stated the issue of endocrine disruption this way:

5 Let me challenge you with two simple facts. 1. Every one sitting here today is carrying at least 500 measurable chemicals in your bodies that were not part of human chemistry before the 1920s. We are walking experiments, differing from all previous generations of human ancestry. 2. There is now incontrovertible scientific proof that a mother shares some of these man-made chemicals with her baby while it is in her womb. No baby has been born on the planet for the last two decades without some exposure to novel chemicals in the womb. Some with little exposure. Some with a lot. But none with none. In all likelihood, some, perhaps many of these compounds will turn out to be benign, with no impact. But some we know already cause problems (Jost, 1998).

IV. ENDOCRINE DISRUPTORS

Endocrine disruptors are defined not so much by what they are, but rather by what they do, that is, how they function. EDs for the U.S. EPA, are chemicals that are thought to mimic or inhibit the action of natural hormones, or alter the normal regulatory function of the immune, nervous, and endocrine systems (U.S. EPA, 1997). The National Research Council expert workgroup, known as the Committee on Hormonally Active Agents in the Environment, had trouble accepting the frequently used term, endocrine disruption, and so instead used the term hormonally active agents (HAA) to refer to the adverse effects of environmental contaminants, which most other groups refer to as endocrine disruptors. Committee members differed on some basic epistemologic issues, which led to different interpretations and conclusions on the issues of HAAs in the environment (NRC, 2000). Unfortunately, after half a decade of intensified investigation, there is no consensus definition for endocrine disruption between some of the most prestigious governmental groups in the United States.

In Europe, a well accepted working definition of an endocrine-disrupting chemical is an exogenous substance that causes adverse health effects in an intact organism or its progeny consequent to changes in endocrine function. A potential endocrine-disrupting chemical is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism (OECD, 1996). Such an ED chemical need not be remotely associated in chemical structure with the hormone it mimics. The structure of the organochlorine pesticide DDT and the structure of diethylstilbestrol (DES) bear no resemblance to the structure of estrogen. However, due to the estrogenic activity that both of these chemicals possess, it is reasonable to believe that some part of their structure binds to estrogenic receptor sites in target organs of estrogen and thus elicits estrogenic hormone-type activities.

Chemicals most often associated with endocrine disruption usually possess estrogen-like, anti- estrogen-like, androgenic-like, anti-androgenic-like, and/or thyroid hormone-like or anti-thyroid hormone-like activities, or in some way inhibit or modify those activities. This list is not necessarily exhaustive, and various lists of endocrine disruptors contain broader categories. Some lists of ED chemicals include chemicals that affect hormone-releasing or hormone-receiving tissues only indirectly (e.g., secondarily through developmental effects on the growth or organization of these tissues) and alterations in hormone that occur as a result of enzyme induction that can influence clearance rates.

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Thus, endocrine disruptors are considered as a group because of endocrine activity or ability to influence endocrine function in either in vivo or in vitro tests, rather than because they share some similarity of chemistry or structure or origin. Furthermore, the term endocrine disruptor is a bit of a misnomer, as it conveys the concept that something is stopped abruptly from occurring. The terms endocrine modulators or endocrine mimics probably more closely convey the true meaning of what is occurring. In this paper, the terms endocrine disruptors, endocrine mimics, endocrine modulation, and hormonally active agents will be used synonymously.

V. BASIC BIOLOGY AND MAJOR CONCEPTS

As stated earlier, endocrine disruption is a very complicated multidisciplinary issue. Prior to reviewing the current state of knowledge regarding endocrine disruptors, a brief overview of developmental biology, toxicology, and hormones will be beneficial for a proper understanding of this topic. A glossary of terms used is provided in the appendix.

DEVELOPMENTAL BIOLOGY AND TOXICOLOGY

Developmental biology is concerned with the complex series of events involved in the growth and development of a fertilized egg, through birth, growth, sexual maturation, and adulthood. The aspects of these events in different species can be quite varied. Disruption and/or alterations of these basic processes during development may lead to alterations in normal development ranging from slight variations in structure and function through severe effects that can be life threatening and fatal.

Developmental toxicology is concerned with adverse effects of substances that may be manifest as prenatal or early postnatal death, structural abnormalities, altered growth, and/or functional deficits. Failure of fertilization is considered under the aegis of reproductive toxicology; however, if an adverse effect occurs after fertilization it is then considered to be in the realm of developmental toxicology.

In humans, it is estimated that over one third of early human embryos die, and about 15 % of recognized abort spontaneously (Dixon and Hall, 1982). Further, birth defects are the leading cause of infant mortality and a major cause of disability in the young in the United States (March of Dimes, 1998). It is thought that approximately 20 percent of birth defects are associated with gene mutations and about 5 percent with chromosomal aberrations. Of the remaining birth defects, less than 10 percent are known to be related to teratogenic agents (Shepard, 1986). Over 60 percent of birth defects have unknown causes (March of Dimes, 1998). Approximately 3 percent of newborns have significant congenital malformations at birth and an additional 3% are recognized by one year of age (Dixon and Hall, 1982 and Shepard, 1986). There is concern that exposure to endocrine disruptors and/or environmental contaminants may be contributing to the incidence of several adverse effects including embryonic mortality, increased incidence of certain birth defects (e.g., hypospadias), and increases in certain disease states (e.g., testicular cancer).

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The periods during which exposure to endocrine disrupting chemicals may result in developmental effects include times prior to conception (i.e., exposure of germ cells of either parent), during , or, postnatally, up to the time of sexual maturation. Developmental effects may occur in systems that are not functional until puberty or adulthood and, therefore, may not become evident until some time during adulthood. Consequently, all stages of development are potentially susceptible to disruption, through either excess or deficiencies of endogenous or exogenous agents.

Gametogenesis is the process wherein haploid sex cells are formed from somatic diploid cells. This process is called oogenesis and spermatogenesis in the female and male, respectively. Female mammals and birds have, at birth, all the primary oocytes they will ever possess. Therefore, anything that adversely impacts oocyte development or oocyte number is irreversible. This is unlike the situation in the male where sperm cells are made throughout a substantial portion of adulthood.

Preimplantation is the time period between fertilization and implantation of the egg in the . The duration of the preimplantation period is approximately the same in most mammals, 5 - 7 days, regardless of differences in gestational length. Previously it was thought that the preimplantation period was resistant to outside adverse influences. This notion is no longer viewed as true. Recently, it has been learned that as little as one hour of inhalation exposure to certain chemicals during the preimplantation period can increase the malformation and decrease viability rates of laboratory animals.

The embryonic or organogenic period is the time interval between implantation and the fetal period. During the embryonic period, derivatives of the fertilized ovum undergo very rapid development to produce the groundwork for all the essential organs and tissues of the adult. Developing embryos during organogenesis are often spectacularly sensitive to developmental perturbations that later become manifest as malformations and/or functional deficits. The organogenic period is variable among mammals, lasting from week 3 to week 8 of in humans

Formation of organ systems occurs with cell proliferation, cell migration, cell differentiation, induction, and programmed cell death or . Cell proliferation occurs by a process of cellular division called mitosis. Cell migration occurs by cells following certain chemical or physical trophic influences. Cell differentiation and induction, whereby multi-potential embryonic cells become fixed as a specific tissue type, occurs under both genetic and chemical influences. Apoptosis is an important mode of programmed cell death in which cells are deleted from tissues. It is an equal and opposite force to mitosis (cell division) and plays any essential role in the maintenance of renewable tissues. Apoptosis accounts for most of the tissue remodeling in vertebrate development. For instance, without apoptosis, fingers and toes would be webbed.

The fetal period is the time between the embryonic period and birth when in utero development occurs. Structural abnormalities from toxicants are less likely to occur in the fetal period because the fetus is more resistant to lethal effects than the embryos. Effects resulting from fetal exposure to toxicants include cell depletion, growth alterations, functional disorders, and carcinogenesis.

8 The postnatal period of life is the time period after birth until puberty. Depending on the species, some significant development of tissues and organs occurs during this period. Exposures to the organism during this period can occur through inhalation, dermal absorption, and ingestion routes, including maternal breast milk and regular food. Alterations occurring during the postnatal period may not be expressed until after puberty.

Effects detected during the postnatal period resulting from prenatal exposure can be illustrated by the example of diethylstilbestrol (DES), which was administered to pregnant women from the 1940s through the end of 1972 to prevent miscarriages. DES was used to stabilize pregnancies at risk of spontaneous abortion. One advertisement in an obstetrics/gynecology medical journal read, "Recommended for routine prophylaxis of all pregnancies... 96 percent live delivery with desPLEX in one series of 1200 patients - bigger and stronger babies, too. No gastric or other side effects with duplex - in either high or low dosage" (AJOBGYN, 1957). DES treatment was found to result in increased rates of otherwise very rare genital tract cancer in female offspring and high rates of reproductive tract abnormalities in male and female offspring (Kimmel and Seed, 1997). In addition, daughters of women given DES are less likely to have full-term live births during their first pregnancy (64 percent versus 84 percent for unexposed women) (Kaufman, et al, 2000). It is estimated that 5 to 10 million women were directly exposed to DES during pregnancy (NCI, 1998). In 1971, the FDA banned DES use in humans; however, its use as a feed additive in cattle continued for another decade, until it was finally banned in 1980. While USDA stopped testing beef for DES residues in 1991, Switzerland reported detecting DES-contaminated American beef exported from our fourth largest meat packer in July 1999 (DES Action, 2000).

Well-established health effects associated with exposure of women to DES in utero (DES daughters) are: clear cell adenocarcinoma of the vagina in young women, vaginal epithelial changes, reproductive tract abnormalities (e.g., gross anatomical changes of the cervix, T-shaped and hypoplastic uteri), ectopic pregnancies, miscarriages, and premature births. These effects in the total population are low, but the risk for reproductive problems has been reported to be as high as 90% in the exposed population. An increased breast cancer risk has also been noted in DES mothers. Probable effects of DES exposure are in DES daughters and reproductive tract abnormalities (e.g., epididymal cysts, hypoplastic testis, and cryptorchidism) in DES sons. Other possible effects include cervical dysplasia and carcinoma in situ and autoimmune disorders (DES daughters), and infertility and testicular effects (DES sons). Suspected effects include breast cancer (DES daughters), psychosocial effects (DES daughters and sons), prostatic hyperplasia and cancer (DES sons), and third-generation effects (DES grandchildren) (NCI, 1999).

While the endocrine disrupting effects of DES are primarily a pharmaceutical problem rather than an environmental problem, DES provides a strong example of the types and extent of endocrine disruptor effects on development that can occur in populations exposed to endocrine disrupting chemicals. It is also an excellent example of an in utero exposure resulting in effects passed on to the child.

9 VI. HORMONES

A. HOW HORMONES WORK / MECHANISM OF ACTION

There are three classes of steroid hormones: adrenal (e.g., , ), sex (e.g., progesterone, estrogen, and ,), and vitamin D3 (recently reclassified as a hormone) which have chemical structures similar to and in most cases are derived from cholesterol (Bashirelahi, et al, 1997). Generally, steroid hormones strongly bind to receptors in target cells, and the hormone-receptor complex is ultimately transported to the cell nucleus where it can interact with DNA and affect the production of specific proteins. Under certain circumstances, however, steroid hormones may act by binding to receptors on the outside of cell membranes. (Turner and Bagnara, 1976; Strobl and Thomas, 1990; Baulieu E.E., 1978, Sanchez-Bueno, et al, 1991, and Majewska and Schwartz, 1987, as cited by Bashirelahi, et al, 1997).

Low concentrations of a hormone can stimulate a tissue, while higher concentrations can have the opposite effect. For example, in mice, the prostate becomes enlarged as a result of fetal exposure to low doses of estradiol or DES, while high doses result in decreased prostate (vom Saal, et al, 1997). At 0.02, 0.2 and 2.0 ng DES per gram of body weight per day fed to pregnant female mice from gestational days 11 to 17 increased adult prostate weight, whereas 200 ng per gram body weight resulted in decreased adult prostate weight in male offspring. Thus there was a 100-fold difference in concentration, or two orders of magnitude, between stimulation and inhibition of prostatic weights.

B. FEMALE HORMONES

1. ESTROGENS

Estrogens are steroid endocrine hormones produced during maturation of the female that are responsible for growth and development of the vagina, uterus, oviducts, and other structures required in reproduction. Estrogen is also involved in the promotion of closure of bone junctures during development and in the production of secondary sexual characteristics that occur in females during puberty (Smith, et al, 1983, as cited by Bashirelahi et al, 1997). Estrogens are found in female tissues such as the , adrenals, and and in male tissues such as the testes, adrenals, and small amounts in spermatozoa (Turner, C. D. and J. T. Bagnara, 1976). While the mammalian differentiates properly in the absence of estrogen, estrogen is required for proper ovarian development in birds, reptiles, and lower vertebrates (Jeyasuria, P. and A. R. Place 1998). In the human female, three estrogens are present in the plasma: 17 B-estradiol, , and . Binding of estrogens to cellular receptors is initiated and maintained at the aromatic A ring structure common to estrogens. Agonists and antagonists of estrogen usually have similar A ring type structures (Smith et al, 1983, and Sluyser, 1985, as cited by Bashirelahi et al, 1997). 17 B- Estradiol is the most potent of the estrogens. Other estrogens activity is often referenced back to 17 B-estradiol potency which is approximately 10 times more potent (ability to elicit an effect) than

10 estrone, and 80 times as potent as estriol (Guyton, 1986, and Smith et al, 1983, as cited by Bashirelahi et al, 1997). The plasma concentration of these hormones is extremely low (Chrousos, 1989). Absorption of estrogens may occur by oral, percutaneous and respiratory routes of exposure.

2. PROGESTERONE

Progesterone is an endocrine hormone produced in the ovary. In pregnancy, progesterone is responsible for the preparation of the reproductive tract for zygote implantation and maintenance of pregnancy (Hadley, 1996). Progesterone has been identified as providing a rapid chemotaxic signal in follicular fluid that facilitates sperm migration.

3. FOLLICLE-STIMULATING HORMONE (FSH), LUTEINIZING HORMONE (LH), AND THYROID-STIMULATING HORMONE (TSH)

FSH, LH, and TSH are glycoprotein chemicals originating from the adenohypophysial (anterior) portion of the pituitary. Each of these hormones is composed of a similar alpha subunit and a unique beta subunit, which provides receptor specificity. That is, TSH is composed of an alpha subunit bound to a TSH beta subunit. Together, TSH only associates with its own receptor. Individually, separate alpha and beta subunits have essentially no biological activity. FSH and LH are both required for successful development and maturation of the reproductive organs and successful completion of reproductive function in both male and female animals, however, they play different roles in both male and female reproduction.

C. MALE HORMONES

1. ANDROGENS

Androgens, endocrine hormones responsible for masculinization, mediate their action by interacting with cytoplasmic and nuclear receptors in the cell. Testosterone and are the main circulation androgens of testicular origin and are responsible for regulation of gonadotropic secretion, spermatogenesis, and wolffian duct differentiation to form the epididymis, vas deferens, and seminal vesicle. Androgens are also produced, although usually to a lesser extent, by the adrenal glands in both males and females. FSH and LH are also required for successful maturation and function of the male reproductive system.

2. THYROID HORMONE

Thyroid deficiency in man, during the second and third trimesters of gestation and during the first months after delivery, results in mental retardation and other neurologic deficits. During mid and late gestation thyroid hormone is supplied by both fetus and mother, but mostly by the mother.

11 Recently it was discovered that the neonatal thyroid status is a crucial determinant of adult testis size. Hypothyroidism (deficiency of thyroid activity) during the neonatal period can produce unexpected overgrowth of the male reproductive system, whereas hyperthyroidism (excessive functional activity and excess secretion of thyroid hormones marked by goiter, etc.) can lead to dramatically smaller testes. Untreated rat testes reach a maximal size around days 90-100, whereas, animals made hypothyroid in utero have testes 80% larger than controls at 160 days of age. Neonatal hypothyroidism causes an extension of this proliferative stage of the Sertoli cells of the testis and thus allows an increase in the population of Sertoli cells with the concomitant increase in total number of germ cells, creating a larger testis that produces more sperm than normal.

3. ESTROGEN

Estrogen, while not usually considered a male hormone, is found in male tissues such as the testes, adrenals, and small amounts in spermatozoa (Turner, C. D. and J. T. Bagnara, 1976). Estrogen is also necessary in males, as in females, for the closure of the epiphyses, which stops the growth of long bones on late puberty and thus determines final height (Carani et al, 1997; Smith et al, 1994; and Morishima et al, 1995, as cited by Sharpe, 1997).

Exposure of males to estrogen at levels above the low levels normally found in male tissues causes (the excessive development and function of the male mammary glands). Studies using high doses of estrogen have demonstrated that exposure to estrogen during fetal development can inhibit prostate development. However, studies in mice, using extremely low doses (in the part-per-trillion range of the artificial estrogens estradiol and DES during fetal development, have shown a permanent increase in the size of the prostate. The possibilities of causation of such effects at extremely low doses raises concerns that exposure of the fetus to low doses of estrogenic chemicals present in pharmaceuticals (birth control pills, etc.) or to environmental concentrations of estrogenic chemicals present in food, water, or air (from pesticides, components of plastics, detergents, hand creams, and other products) (vom Saal et al, 1997) can result in significant effects in later life.

Recently, estrogen in males was discovered to regulate fluid reabsorption in the efferent ductules, the tubules carrying sperm from the testis to epididymis. Disrupting this essential reabsorption function causes the sperm to enter the epididymis diluted, resulting in potential infertility. This is so because dilution or rather lack of concentration of spermatozoa can limit the number and concentration of sperm available for delivered to the site of fertilization and affect their ability to penetrate the outer layer of the egg, the zona pellucida. Therefore, a lack of or impairment in fluid reabsorption in efferent ductules raises concern over the potential direct effects environmental estrogens may have on male reproduction and reported declines in human sperm counts (Hess, et al, 1997).

12 VII. SPECIFIC ENDOCRINE DISRUPTORS

A. PHARMACEUTICAL MIMICS & ANTIESTROGENS

Some nonsteroid compounds possess estrogenic activity, the most potent being diethylstilbestrol (DES). Antiestrogens, such as the clomiphene and , through competition with estrogen receptors, prevent actions of endogenous estrogens from expressing their full effect on target tissues. By this action, they antagonize a variety of estrogen-dependent processes like uterine growth. These compounds have a pharmaceutical use in preventing the growth of estrogen- dependent breast tumors.

B. ANTIANDROGENS

The most potent antiandrogens are derivatives of progesterone, a hormone most associated with female reproductive activity. Although possessing weak androgenic activity, progestens inhibit the actions of testosterone by occupying receptors and thus preventing androgen binding. Treatment of the mature male with causes atrophy of seminal vesicles, prostate, and other androgen-responsive target organs (Hadley, 1996).

Some environmental chemicals having adverse reproductive and developmental effects are mediated through inhibition of the . is a systemic dicarboximide fungicide used widely in Europe and the United States on grapes, fruits, vegetables, hops, ornamental plants, and turf. It was reported by the registrant to possess antiandrogenic effects. Oral exposure of pregnant rats to 100 or 200 mg/kg/day on Gestational Day 14 through Postnatal Day 3 resulted in male offspring exhibiting nipple development, reduced anogenital distance (more indicative of female development), cleft phallus with hypospadias, suprainguinal ectopic testes, a vaginal pouch, epididymal and testicular granulomas, and atrophic seminal vesicles and ventral prostate gland (Gray et al, 1994, as cited by Kelce et al, 1996). Studies suggest vinclozolin metabolites act as antiandrogens either by inhibiting androgen receptor activity or by inhibiting the enzymatic activity of 5 alpha-reductase (the enzyme that converts testosterone to the more active androgen 5 alpha-dihydrotestosterone) (Gray et al, 1994, as cited by Kelce et al, 1996). Vincozolin has been detected in ground water in , in surface water samples from a water supply catchment area in the central Mountain Lofty ranges of South Australia, in Dutch surface water resources, the Rhine river in Dusseldorf, and in France (Toxline, 2000).

Several environmental antiandrogens disrupt development in rats and/or rabbits at fetal concentrations at, or near, exposure levels seen in some segments of the human population. In rats, fetal tissue concentrations of 10-20 ppm of p,p'-DDE, a DDT metabolite, are correlated with reproductive abnormalities observed in male offspring. These concentrations are similar to those found in first-trimester human fetal tissues in the late 1960s. In addition to vinclozolin, , and DDT are androgen receptor antagonists. They reduce the anogenital distance in rats and induce areolas at relatively low dosage. Hypospadias, agenesis of sex

13 accessory tissue and retained nipples are seen at medium dosage, while undescended testes and epididymal agenesis are seen at higher doses (Gray, Jr., et al., 2001).

The ability to extrapolate from effects seen in rodents and other experimental animals to those potentially either expected or anticipated to be occurring in humans would be enhanced if in future studies, determination of tissue concentrations of the active metabolites was incorporated into the research protocols.

C. CLASSES OF COMPOUNDS OF POTENTIAL CONCERN

The following are some examples of chemicals either known or suspected of possessing adverse ED properties. The level of evidence of ED activity associated with these chemicals is not equal in all respects, and some qualitative and quantitative evidentiary differences exist. Source: WWF Canada Eagle's Eye, Special Issue on Toxics that Tamper with Hormones, Summer 1995, and Wingspread I Statement, recorded in Chemically Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, eds. T. Colborn and C. Clement, Princeton Scientific Publishing, Princeton, NJ, 1992. Website database of Our Stolen Future: Book Basics: Chemicals Implicated, http://www.ourstolenfuture. org/basics/chemlist.htm (2002). While not everyone will agree on any one listing of endocrine disruptor chemicals, this list should provide an ample idea of the kinds of chemicals various organizations consider to have adverse impact on development of the reproductive, immune, nervous, and endocrine systems of humans and wildlife by mimicking hormones, blocking hormonal action or by other interference with the endocrine system.

Pesticides (commercial and/or domestic) 2,4,5,T, 2,4,D, alachlor, , aldrin, amitrol, atrazine, benomyl, , carbaryl, chlordane, cypermethrin, DBCP, DDT, dicofol, , , esfenvalerate, ethyl parathion, fenvalerate, , hexachlorobenzene, , mancozeb, maneb, methomyl, , metiram, metribuzin, mirex, nitrofen, oxychlordane, permethrin and other synthetic pyrethroids, toxaphene, transnonachlor, tributyltin oxide, trifluralin, vinclozolin, zineb, and ziram are examples of synthetic pesticides. Some of these pesticides are associated with affecting estrogen, androgen, or thyroid hormone levels, by binding to estrogen receptors (aldrin), inhibiting androgen binding to androgen receptors (DDT metabolite, p, p'-DDE), and/or interfering with thyroid hormone synthesis and/or secretion (amitrol). The mechanism of action by which some pesticides and other environmental chemicals interfere with the proper functioning of the endocrine systems still remains unclear.

Heavy Metals Examples of heavy metals of concern include (used in nickel/cadmium batteries, coatings, pigments, stabilizers in plastics and synthetic products and alloys, fossil fuels); lead (used in lead batteries, paints, pipes, leaded gasoline); and mercury (formerly used in alkaline batteries, fluorescent lighting tubes, chlorine production, dental amalgams, and found in fossil fuels and fish). Heavy metals are known to affect glucocorticoid, estrogenic, and reproductive/thyroid hormones. Arsenic and cadmium are believed to act by inhibition of DNA transcription normally stimulated

14 by the glucocorticoid-GR complex and by activating the through an interaction with the hormone-binding domain of the receptor, respectively.

Organochlorines Dioxin (2,3,7,8-TCDD) is a by-product of other organochlorine production, use, and disposal (not intentionally produced). Other examples include incinerator emissions; metal smelting; polyvinylcloride (PVC) plastic production; chlorine-bleached pulping; polybrominated biphenyls (PBBs); and polychlorinated biphenyls (PCBs) (Production is banned but PCBs are still found in electrical transformers, landfills, toxic waste dumps, and sediments), (wood used in textile industry). These persistent organochlorines are believed to interfere with the proper functioning of the thyroid and estrogen hormonal systems by acting as antiestrogens through binding with the Ah receptor which inhibits estrogen receptor binding to the estrogen response elements, thereby inhibiting proper estrogen action (dioxins and furans), or by binding to thyroid hormone possibly through a direct effect on the thyroid gland (pentachlorophenol).

Plasticizers & Examples of plasticizers and surfactants are phthalates, polycarbonates, styrenes (used to make plastics soft), and alkyl and nonyl ethoxylates (used in detergents, pulp and paper and textile industry, some plastic products, paints, and pesticides). Phthalate compounds are known to affect estrogen and/or androgen hormone systems by inhibition of binding to the estrogen receptor (butyl benzyl phthalate, di-n-butyl phthalate, and di-ethylhexyl phthalate) and, in addition, some also act as an anti-androgen (di-n-butyl phthalate and di-ethylhexyl phthalate).

D. SPECIFIC ENDOCRINE-MEDIATING CHEMICALS OF POTENTIAL ENVIRONMENTAL CONCERN

Exposure to exogenous estrogens (those occurring from outside the body) and estrogen mimics can occur from natural estrogenic substances found in certain food plants; synthetic estrogens intentionally used in the production of food (growth promotants); indirect unintentional food additives resulting from leaching from packaging material; medicine intentionally taken, as in birth control pills, estrogen supplementation for menopausal women to promote retention for bone strength; certain plants; fungal products; pesticides; plasticizers; and other agricultural and industrial chemicals, etc. The potential for exposure through most of these routes still exists in the United States and Canada.

1. DIETHYLSTILBESTROL (DES) DES has been used as a growth promoter in chickens, cattle and sheep; a veterinary to treat estrogen deficiency; and in human medicine for menstrual disorders, post-partum breast engorgement, chemotherapy for breast and prostate cancer, a postcoital contraceptive, and to lessen the risk of spontaneous abortion (Spectrum Laboratories, 1999). DES bears marked structural similarities to DDT and other DDT-related chlorinated pesticides. DES was utilized as a growth promoter in livestock because it caused animals to gain weight and muscle mass in less time and with less feed, thus achieving a marketable weight more efficiently and providing an economic

15 advantage to those who used it. In 1958, the Food Additive Amendment contained a Delaney clause that stated, "That no additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal..." (Kilgore and Li, 1980). In 1979, DES was banned by the FDA due to the Delany clause for use in food products. Ten years later in 1989, the U.S. Department of Agriculture stopped testing for DES residues in U.S food animals. Although banned over 20 years ago, DES is still reported in U.S. beef exports, which were rejected by Switzerland in 1999 and 2002 (Hawkins, 2002 and Lawless, 1977).

As far back as the 1940s, 1950s, and 1960s, DES was known to cause feminization of male fetuses and partial retention of wolffian bodies in female fetuses, clitoromegaly in female newborns whose mothers were treated with DES during the first trimester, and adenocarcinoma of the vagina of young women whose mothers had received DES during the first trimester of pregnancy, respectively (Greene et al, 1940, Bongiovanni et al, 1959, and Herbst et al, 1971). In addition, DES led to anatomical and functional abnormalities of the reproductive tract in both male and female offspring of mothers who were threatening miscarriages and treated during pregnancy. The daily dosage of DES recommended to prevent accidents of pregnancy gradually increased from 5 milligram (mg) per day during the 7th and 8th week, to 20 mg per day in the 13th and 14th week, to 40 mg. per day in the 18th week, and increased gradually to 125 mg. per day in the 35th week of pregnancy (Levine, et al, 2001).

There is evidence that these adverse transplacental effects of DES endure and persist across generations, in both animals and man, that is, they have been passed on to the children of treated mothers and to the children of their children. Mice were exposed to DES subcutaneously at three different developmental periods: 1) mothers dosed on days 9-16 of pregnancy (2.5, 5, or 10 ug/kg maternal body weight)- the period of major organogenesis; 2) mothers dosed only on day 18 of pregnancy (1000 ug/kg maternal body weight), just prior to birth; and 3) pups dosed on days 1-5 (0.002 ug/pup/day), just after birth. The offspring of these treated groups of mice were allowed to breed when mature. The fertility of the three treated groups was reduced, time required to produce first litters was increased, and a decreased percentage of females was produced. When female granddaughter mice from these litters were allowed to mature and mate, the DES-treated offspring had an increased incidence over control group values of uterine adenocarcinoma in all three treatment groups. The highest cancer rate was 16 %, consisting of a very rare vaginal cancer found in adult descendants of Group 1, whose grandmothers were exposed prenatally to DES. The number of cysts, lesions, tumors, and cancer of the ovaries, cervix, vagina, uterus, and other reproductive tissue abnormalities increased with age in treated groups compared with control animals. The researchers concluded that all the developmental periods examined were susceptible to adverse effects of DES. In humans, while reduced fertility observed in daughters of DES-treated mothers is not transmitted to granddaughters, an increased susceptibility to reproductive tumors is passed on to subsequent generations. The mechanism by which DES can affect multiple generations is not understood; however, other studies support a genetic connection (Newbold et al, 1998). DES-associated reproductive tract anomalies and cancer are temporally related to DES exposures occurring in the first trimester of pregnancy. Mice lacking the Wnt7a gene have malformed female reproductive tracts that closely resemble the reproductive tract morphologies observed in female mice prenatally exposed to DES, i.e., shallow vaginal fornices, malformed oviducts lacking coils, wolffian duct remnants, vaginal concretions and adenotic lesions in the vagina. Recently, it was learned that DES suppresses the activity of the gene in mice, which is

16 required for the normal development of the male and female reproductive tracts (Miller et al, 1998, as cited by Travis, J., 1999).

2. NONYLPHENOL POLYETHOXYLATES

While nonylphenol (NP) is only weakly estrogenic compared to estradiol, its widespread occurrence and ubiquitousness in aqueous environments has raised concerns both for wildlife and humans. The minimum level for responses related to estrogenic activity of NP in reproductive and mammary tissues is approximately 40 mg/kg/day based on a multi-generation dietary study (NTP, 1997) and uterotrophic and mammary gland assays where NP was dosed orally or with subcutaneous implants (Ashby et al, 1997; Odum et al, 1997, 1999a,b). To assess the potential effects over multiple generations, 4-nonylphenol was administered in the diet at concentrations of 0, 200, 650, and 2000 ppm to groups of Sprague-Dawley rats for three generations by the National Toxicology Program. This study demonstrated that NP is a male and female reproductive toxicant at concentrations equal or greater than 650 ppm based on: decreased epididymal sperm density and testicular spermatid head counts in males; and increased estrous cycle length and decreased ovarian weights in females.

Administration of estradiol or nonylphenol to immature female Sprague-Dawley rat pups (20-21 days of age) in graded intraperitoneal injections leads to dose-dependent increases in uterine protein, DNA contents, and the marker enzyme, uterine peroxidase. The dose-response relationship comparison between estradiol and NP was quite different however, with only doses of NP >1 mg/animal being effective, whereas doses of estradiol at 2.5 ug/animal were effective. The results demonstrate that while NP is estrogenic it is only 1/1000 to 1/2000 as potent as estradiol (Lee and Lee, 1996).

Nonylphenols are widely used in commercial and personal care products. Nonylphenol (NP) is also a breakdown product of alkylphenol polyethoxylates (APEOs) which are widely used domestic and commercial detergents (Dachs, et al, 1999). Nonionic surfactants are used in a wide variety of products to decrease foaming, to increase rates of chemical reactions, and are integral to most pesticide formulations to increase leaf retention of sprayed solutions, and to generally improve the effectiveness of active ingredients in products. These surfactants bind to proteins and phospholipids influencing enzyme activity by stimulating or inhibition and altering membrane permeability (Cserhati, 1995). Estimates are that 60% of APEOs end up in aquatic environments as nonylphenol and octaphenol (OP)(Lee and Lee, 1996). These chemicals are either not detected in current water testing protocols or are under-reported using the standard analytical protocols (Clark, et al., 1992). APEOs have been detected in untreated wastewater and septage samples at concentrations ranging from 1350 to 11,000 ug/L in Cape Cod, Massachusetts. Nonylphenol was detected in all septage samples at concentrations above 1000 ug/L. In groundwater downgradient of an infiltration bed for secondary treatment of effluent, nonyl/octylphenol and ethoxylates were found at concentrations up to 30 ug/L. Bisphenol A was detected in six of 28 private drinking water well samples at concentrations greater than that detected in any analytical blank. Additionally, two of these samples were higher that the quantation limit, (0.044 and 0.020 ug/L). Nonyl/octylphenol tetraethoxylate were detected by High Performance Liquid Chromatography (HPLC) in one drinking water well at 32.9 ug/L (Rudel, et al, 1998).

17

Nonylphenols (NP) had originally been detected only in aquatic environments. Recently, however, NPs have been documented in air samples of urban-industrial New Jersey (Liberty Science Center and Sandy Hook) in the vicinity of the Lower Hudson River Estuary (HRE). Concentrations of NPs in the water from the HRE ranged from 11.6 to 94.5 ng/L in the dissolved phase and 2.6 to 21.6 ng/L in total suspended matter. NP were detected in all atmospheric samples analyzed and the concentrations ranged from 0.2 to 68.6 ng/M3 for the gas phase and from 0.1 to 51.4 ng/M3 for the aerosol phase. Since equilibrium conditions favor net volatilization from the estuarine waters, this is the likely source of NPs to the regional atmosphere. These atmospheric NPs concentrations are higher than those of polycyclic aromatic hydrocarbons and 2 orders of magnitude higher than PCB concentrations in urban and industrial areas. The atmospheric occurrence of NPs in highly populated areas raises concern since this is a pervasive route of exposure (Dachs et al, 1999).

Additional work is necessary to determine the estrogenic activity of NPs and related products before assessing the actual risk posed by these compounds on ecosystems and humans. Based on present data from this country, it appears unlikely that acute exposures to current environmental concentrations of NP are sufficient to elicit estrogenic effects. While the possibility for bioaccumulation of NP to concentrations disruptive of the endocrine system is low, little is known concerning the transfer into eggs and the resulting chronic exposures. In addition, the developmental stage at the time of exposure must be considered. If exposure occurs during critical periods when hormones play critical roles in development, acute exposures could trigger developmental effects which can be permanent and/or result in latent changes in reproductive capability that will not become manifested until later in life (Nimrod and Benson, 1996).

3. BISPHENOL A

Bisphenol A (BPA) is a constituent of plastics widely used on consumer products, such as durable plastic materials, , dental sealants, and epoxy coatings on the inside of food and drink cans. Practically speaking, everyone in the developed world is exposed. Annual production exceeds 1.6 billion pounds in the U.S. alone. Bisphenol A can potentially leach out of these products (Hileman, 1997).

In a study described by von Saal et al. (1998), BPA fed to mice at 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb) from day 11-17 of gestation permanently increased the size of the preputial glands and reduced the size of the epididymides in the testis. These organs develop from different embryonic tissues. At 20 ng/g-body wt. BPA, sperm production was decreased by 20% relative to control males. Since the 2 ppb dose of BPA is lower than the amount reported to be swallowed after application of a plastic dental sealant (up to 931 micrograms: 13.3 ppb in a 70 kg person) it is of interest whether humans ingest sufficient bisphenol A to cause adverse health effects since saliva is capable of softening the surface of dimethacrylate used in dental fillings (Olea et al., 1996; Larsen and Munksgaard, 1991; Atkinson, et al., 2002; Manabe et al., 2000; and Fung, et al., 2000).

The effects of prenatal exposure of BPA on the female rat reproductive system were examined by Talsness and Chahoud (2000). Oral 0.02 mg/kg/day BPA or 0.02 mg/kg-body wt./day 17-alpha

18 ethinylestradiol (EE) were administered to pregnant Sprague Dawley rats during gestation days 6 through 21. On postnatal day 35, female offspring were examined for the occurrence of vaginal openings. Vaginal opening was delayed by 11.6 days in the BPA animals and 9.5 days in the EE offspring compared to controls. Vaginal opening is considered an important reproductive developmental milestone that occurs approximately 5 days before the first ovulation. In an earlier study, a delay in vaginal opening occurred with 0.1 mg/kg/day BPA while precocious vaginal opening occurred at a 50 mg/kg/day BPA dose. Postnatal exposure of estrogens is known to cause precocious vaginal opening. Thus, the effects of in utero exposure to low doses of BPA or EE are consistent with an anti-estrogenic action, while BPA at high dose levels leads to estrogen-like effects on vaginal opening. Results of FDA screening of BPA in the uterotrophic assay suggested that while bisphenol A does act as an estrogen, it does so much less effectively than the physiological estrogen, estradiol, which is 10,000 times more potent than BPA (FDA, 1997).

Measurable effects of BPA were demonstrated on the offspring of Sprague-Dawley female rats that were exposed, via their drinking water, to either 0.1 or 1.2 mg/kg BPA body weight from day 6 of pregnancy through the period of lactation by Ruben et al., (2001). Offspring exposed to BPA exhibited an increase in body weight that was apparent soon after birth and continued into adulthood. In addition high dose BPA female offspring exhibited altered patterns of estrous cyclicity and decreased levels of plasma luteinizing hormone in adulthood. Neither of the doses of BPA that caused effects during perinatal exposure nor a 10-fold higher dose of BPA was able to evoke an uterotropic response in ovariectomized postpubertal females indicating a decreased sensitivity to BPA during the perinatal period and the need for careful evaluation of the current levels of exposure to this compound.

BPA stimulates secretion in Fischer 344 rats, however, not in Sprague-Dawley rats. In F344 rats, BPA increased DNA synthesis in vaginal epithelium at a median effective dose of 37.5 mg/kg body weight. DNA synthesis was not stimulated in Sprague-Dawley rats at any dose tested. Clearance of tritiated labeled BPA from blood followed the same time course in both strains of rats. These findings point to the need for caution in choosing suitable end points and animal models when seeking to test estrogenic effects of chemicals foreign to biological systems (xenobiotics) (Long et al, 2000).

When data from eight independent immature mouse uterotrophic assays, using the subcutaneous and oral routes of administration, were pooled and assessed, the data indicated no cumulative uterotrophic activity for BPA. In a subset of these studies, however, where control uterine weights were relatively low there was a significant, but weak uterotrophic activity observed over a range of dose levels but without the presence of a good dose response relationship. It was concluded that BPA might be weakly uterotrophic under specific conditions in the mouse (Tinwell, et al, 2000). These inconsistent results may reflect the previously discussed variability in BPA activity among rodent strains.

Hormones are active when they occupy only a small proportion of available receptors. When too much hormone is in a system, the system begins to turn off or down-regulate. This does not mean high doses are benign, but rather that the effects of high doses are qualitatively different from those at low doses. Therefore, extrapolation from high dose effects to ascertain effects at low doses may be misleading. Beer and soda cans, which are normally kept cool, lined with epoxy resins made

19 from bisphenol A and epichlorohydrin were examined by a trade association using an analytical technique (HPLC) sensitive to 5 ppb and did not detect any BPA. It is known that bisphenol A does leach from cans into food that requires high-temperature sterilization, such as vegetables, meats, infant formula, and tomato products. Tests by the Society of Plastics Industry showed that extracts from cans had BPA levels ranging from 7 - 77 ppb, with an average 37 ppb (Hileman, 1997).

BPA in biologically significant amounts was detected in human amniotic fluid during the first trimester of pregnancy, indicating early prenatal exposure (Ikezuki, et al, 2002). Investigators detected BPA using a novel enzyme-linked immunosorbent assay (ELISA) in blood samples of healthy nonpregnant women, women in early pregnancy, women in late pregnancy, umbilical cords at delivery, ovarian follicular fluids, and amniotic fluid at full-term Caesarean sections. The range of BPA concentrations varied from near zero to the high teens ppb. It was detected in all the maternal/fetal stages tested, while the highest amount in amniotic fluid was noted in early pregnancy. The authors interpreted this to indicate BPA is entering amniotic fluid from maternal serum and enzymes capable of metabolizing BPA are not fully activated in the fetus. As the fetus matures those enzymes become more effective which leads to a reduction of BPA in amniotic fluid as pregnancy progresses and amniotic fluid is diluted by fetal .

BPA concentrations was determined in blood samples from 37 mothers between weeks 32 and 41 of gestation, and in placental tissue and umbilical cord blood from the same patients by Schonfelder et al., (2002). Concentrations of BPA ranged from 0.3 to 18.9 ng/mL (median 3.1 ng/mL) in maternal plasma, from 0.2 to 9.2 ng/mL (median 2.3 ng/mL) in fetal plasma, and from 1.0 to 104.9 ng/g (median 12.7 ng/g) in placental tissue. BPA blood concentrations were higher in male than female fetuses. Exposure levels of parent BPA were found within a range typical of those used in animal studies and were shown to be toxic to reproductive organs of male and female offspring.

4. PHTHALATES

Phthalates have been found in plants, fungi, air, food, milk, soil, blood, lipoid solutions, oil, body tissue, and water. Although they are used to create flexibility in polyvinyl chloride plastics, they do not bind chemically in the plastic matrix, and are capable of leaching from these plastics. While some phthalates may be present as the result of natural processes, such as microbial action, the major share of the present environmental burden is due to contamination from various polymers. Synthetic organic chemicals have been detected in water that flows through tubing made of various plastic materials. The individual chemical detected at the highest concentration (5000 ppb), butylglycolybutylphthalate, was from water passing through Food-Beverage Grade PVC tubing (Junk, et al, 1974).

One of the most widely used and studied phthalates is di (2-ethylhexyl) phthalate (DEHP). DEHP is widely used as a softening agent in plastics like polyvinyl chloride, which is used in a variety of medical devices like intravenous tubing and blood bags. DEHP leaches from these medical devices into various body fluids. Male infants in intensive care units are known to receive the highest exposures to DEHP, which has adversely affected testicle and sertoli cell development and caused deformities in male rats. The National Toxicology Program's CERHR panel after reviewing the

20 data and considering these risks concluded the available research and testing data are cause for "serious concern" as to the reproductive and developmental effects of DEHP in critically ill male infants receiving medical treatment in intensive care units (NTP, 2000).

National Toxicology Program (NTP) studies showed DEHP induced a strong liver tumor response in adult rats and mice. In response to this and to additional findings suggesting that DEHP may be toxic to the reproductive system, U.S. toy manufacturers agreed in 1986 to limit the concentration of the chemical to 3% in teethers, pacifiers, and squeeze toys. Many toy makers began substituting diisononyl phthalate (DINP) for DEHP in these products. DINP in industry-sponsored studies was linked with liver tumors and damage to the liver, , and other organs in mice and rats. The U.S. Consumer Product Safety Commission examined different products containing DINP and concluded the amount of DINP ingested by children was well below harmful levels and did not recommend a ban on products containing the chemical. The newly formed NTP Center for the Evaluation of Risks to Human Reproduction (NTPCERHR) examined as its first review the reproductive risks to plastic workers and consumers from seven phthalates including DEHP and DINP (EHP, 1999).

Phthalate esters have been associated with the premature breast development (thelarche) in girls younger than 8 years of age without other manifestations of puberty. Puerto Rico has the highest known incidence of thelarche. Since this public health problem was observed two decades ago, no explanation for this phenomenon has been found. As various organic pollutants, including pesticides and some plasticizers can disrupt normal sexual development in wildlife, an investigation was launched to identify pollutants in the serum of Puerto Rican girls with premature thelarche. Serum samples of 41 thelarche patients and 35 control samples were analyzed for the presence of phthalates. However, significantly elevated levels of dimethyl, diethyl, dibutyl, and di- (ethylhexyl) phthalate and a metabolite, mono- (1-ethylhexyl) phthalate, were identified in 68 % of the samples from thelarche patients. Only one control sample showed a significant level of phthalate. No pesticides or pesticide metabolites were detected in the serum of the study or control subjects. As the phthalates identified have been classified as endocrine disruptors, this research suggests a possible association between plasticizers with estrogenic and potentially antiandrogenic activity, and the cause of premature breast development in a human female population. Phthalates such as dibutyl phthalate, besides having estrogenic activity, have antiandrogenic activity. Further research needs to address if these properties of the phthalate esters play a role in premature breast development (Colon, et al, 2000).

5. POLYCHLORINATED BIPHENYLS (PCBs)

Polychlorinated biphenyls (PCBs) are a large class of 209 chlorinated isomers (congeners) that includes the dibenzofurans and dioxins. These congeners do not all result in the same impacts, rather, their effects are dependent on their structure and degree of chlorination. Starting in the 1930s, PCBs, because of their nonflammability and heat-resistant properties, became widely used in products like hydraulics/, transformers, capacitors, plasticizer applications, and petroleum additives. In addition, they were widely used in industry as sealants and as additives to paints, plastics, rubber adhesives, printing ink, and pesticides (Peakall and Lincer, 1970 and Pichirallo, 1971, as cited by Gellert, 1978). Domestic sales of PCB mixtures known as Aroclors,

21 ranged from 32 million pounds in 1957 to almost 80 million pounds in 1970 when voluntary restriction of usage began (Cairns et al, 1986, and Stout, 1986). PCBs are in vivo notable among synthetic organic chemicals because of their extreme stability, low solubility in water, high octanol/water partition coefficients, high bioaccumulation coefficients and resistance to degradation. As a consequence, PCBs exhibit very high bioaccumulative tendencies in fish, and other aquatic organisms, as well as terrestrial animals (National Research Council, 1979; Sawhney, 1986).

Although while PCBs have been banned, and no new PCBs are being intentionally added to products, they continue to enter the environment and are routinely found in animal and human tissue as micro contaminants. Historically, the true amount of these compounds entering waterways was underestimated due to the use of analytical methods that were not as sensitive as those available now. The large inputs of PCBs into our waterways are frequently associated with storm events when high flow rates in streams and rivers appear to cause more leaching from surface runoff, and stir up more sediment, resulting in increased transport of PCBs (Delaware River Basin Commission, 1998).

Determining the biological activity of PCBs is complicated because they normally occur as mixtures of many individual isomers and homologs, which may differ in their biological activity. PCBs exhibit various degrees of toxicity in organisms such as yeast, fungi, phytoplankton, aquatic invertebrates, and fish. Due to the high lipid solubilities of PCBs and their enormous tendency to bioaccumulate, their concentration in aquatic organisms can reach 85,000 times the concentration in the surrounding water (Lowe et al, 1972, as cited by Hamdy and Gooch, 1986).

There is strong evidence that gestational exposure to PCBs can cause adverse neurologic developmental effects in humans (Jacobson and Jacobson, 1996; Jacobson and Jacobson, 1997; Rogan et al, 1986; Huisman et al, 1995a). However, these effects are not clearly linked to endocrine disruption.

PCBs have no known teratogenic effects in the male reproductive tract of mammals nor do they exhibit direct effects on male fertility (Fuller and Hobson, 1986). However, the reproductive effects noted in female terrestrial mammals are many, varied, and appear to be dependent on species, percent chlorination of the PCB isomer, mode of administration, target endpoint, and age of the animal. PCB-associated alterations noted under a variety of experimental conditions include changes in uterine weight, early onset of puberty, changes in estrous or menstrual cycles, and fertility. Younger animals are more sensitive than older animals and females are more sensitive than males (Gellart, 1978, as cited by Fuller and Hobson, 1986). Lower chlorinated PCBs appear to diminish reproductive competency. However, it is not known whether the effect is directly on the reproductive system, indirectly through hepatic enzyme induction, or on nonreproductive endocrine organs (Gellart, 1978, as cited by Fuller and Hobson, 1986).

Oral administration of the PCB Clophen7 A 60 (containing 60 percent chlorine) to mice for 10 weeks caused female estrous cycles to significantly lengthen, and the number of implanted ova to be significantly reduced (Orberg and Kihlstrom, 1973, as cited by Fuller and Hobson, 1986). Aroclors consist of specific mixtures of PCBs with specific characteristics. The last two digits in their identifying number refers to the average degree of chlorination by weight of that specific

22 Aroclor, i.e., Aroclor 1254, is 54 percent chlorine by weight. Aroclor 1221, but not Aroclors 1254 and 1260, administered to female rat pups on post-natal days two and three induced precocious puberty, persistent vaginal estrus, and by 6 months of age. It was estimated that Aroclor 1221 has roughly one millionth the estrogenic activity of 17-B-estradiol. Nonetheless, these results provide evidence that there is an association between the estrogenic activity of PCB mixtures and their effectiveness in altering neuroendocrine differentiation and inducing premature reproductive aging. Additionally, they indicate that not all PCBs isomers exhibit the same activity (Gellart, 1978, as cited by Fuller and Hobson, 1986). To date, much of the focus on endocrine disruption has concentrated on the "reproductive hormones" estrogen and testosterone and insufficient attention has been paid to examining the ability of pollutants and/or mixtures of pollutants to interact or interfere with other receptors such as glucocorticoid receptors (GR). Recently it was demonstrated that metabolites of persistent polychlorinated biphenyls interact with human glucocorticoid receptors. In the Baltic Sea, harbor and gray seal populations are decreasing due to an unknown disease syndrome characterized by deformations of the claw, skin abnormalities, adrenocortical hyperplasia, , uterine lesions, and an impaired immune system. In this area the two most abundant pollutants are PCBs and dichlorodiphenyldichloro-ethylene (DDE). Studies were initiated which showed methylsulfonyl PCBs (metabolites of PCBs) compete with 3H- for binding to the with an IC50 (concentration that inhibits 50% of activity) of 1 uM. While it is still unclear how these findings impact human health, it is known that PCB methyl sulfones are ubiquitous pollutants present in human mother's milk (Johansson et al, 1998).

Polar bears in the Svalbard archipelago region in the Norwegian arctic contain body burdens of PCBs about 10 times higher than any other place in the world. Studies indicate these polar bears with elevated levels of PCBs also produce lower amounts of immunoglobulin G (IgG) and therefore are possibly more susceptible to infection than polar bears with lower PCB body burdens. The IgC concentration increased with polar bear age and was higher in males than females. IgG was negatively correlated with total PCB level and with three PCB congers (IUPAC numbers 99, 194, and 206). Hexachlorobenzene was also negatively correlated with IgG. Significant negative organochlorine contaminant correlation (PCBs, chlordanes, DDE, hexachlorobenzene, and hexachlorocyclohexanes) with IgG levels in the blood plasma from these polar bears may indicate an immunotoxic effect (Bernhoft, et al, 2000; Skaare, et al, 2000). A decade ago, hermaphroditic polar bears were never observed in this Svalbard area. Now however, the rate of female pseudohermaphroditism is 1.5 percent. Examination of two yearling polar bears revealed normal vaginal openings and a 20 mm penis containing a baculum (a bone forming the skeleton of the penis in all insectivores, bats, rodents, carnivores, pinnipeds, and in primates except man; also known as os penis). The penis was caudal to the location in a normal male and was concealed with the vaginal opening by a single pair of labia. The urethral opening was laterally situated about 5 mm from the distal end of the penis. These animals showed no signs of Y chromosomes (needed for a male genotype in mammals), so these animals are regarded as female pseudohermaphrodites. Pseudohermaphroditism in this polar bear population could result from excessive androgen excretion by a mother with a certain tumor, or it could result from endocrine disruption from environmental pollutants (Wiig et al, 1998).

Xenobiotics suspected of possessing estrogenic activities, such as, PCB Aroclor 1242, the pesticides toxaphene, dieldrin, p, p'-DDD, cis-Nonachlor, trans-Nonachlor, p, p'-DDE, and

23 chlordane were tested for their ability to override male-producing incubation temperatures and result in female hatchlings in the red-eared slider turtle, a species with temperature-dependent sex determination. Compounds were tested at environmentally relevant concentrations previously detected in alligator eggs from Lake Apopka, Florida, singly, in combinations, and with estradiol. Single compounds resulting in significant sex reversal were trans-Nonachlor, cis-Nonachlor, Aroclor 1242, p, p'-DDE, and chlordane. When tested with estradiol, only chlordane caused sex reversal at significant levels. However, when applied together, the eight compounds resulted in significant sex reversal (Willingham and Crews, 1999). Red-eared slider male turtles exposed to Aroclor 1242 or chlordane exhibited significantly lower testosterone concentrations than controls, whereas chlordane-treated females had significantly lower progesterone, testosterone and 5[8,1][alpha]-dihydortestosterone concentrations than controls. This demonstrates the effects of these endocrine disruptors extends beyond embryonic development, altering sex-steroid physiology in exposed animals (Willingham et al, 2000).

6. DIOXINS

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly known as dioxin, is the most toxic member of a class of related chlorinated chemicals including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), co-planar polychlorinated biphenyls (PCBs), polychlorinated naphthalenes, and polychlorinated azo- and azoxy-benzenes whose can be expressed as fractional equivalencies of TCDD. Dioxin is practically insoluble in water, and due to its physical and chemical properties bioaccumulates to high levels in the food chain by distributing into fat. TCDD and TCDD-like compounds are formed as unwanted byproducts associated with many widely different processes and activities, such as, chemical synthesis, bleached chemical pulp and paper mills, cement kilns burning hazardous waste, crematoria, drum and barrel reclamation facilities, hazardous waste incinerators, motor vehicles powered with unleaded gasoline, coal and oil-fired electric generating plants, primary non-ferrous metal smelting, petroleum refining catalyst regeneration, residential oil combustion, sewage sludge incineration, diesel-powered vehicles, municipal solid waste incinerators, scrap electric wire recovery, tire combustion, medical waste incinerators, and industrial wood combustion. A comprehensive database of congener-specific chlorinated dibenzo-p-dioxin/dibenzofuran emissions data is maintained by the EPA (U.S. EPA, 2001a).

One of the largest identified sources of newly made dioxin and dioxin-like compounds is the incineration of medical waste (25% of all medical products contain vinyl as a primary component, e.g., disposable or single-use products such as IV containers and components, blood bags, drip chambers, device packages, basins, and surgical gloves etc.) (Birnbaum, 1993). Polyvinyl chloride plastic, the main source of organically bound chlorine in medical waste streams, forms dioxin during combustion at 300 – 400 oC. The dioxin formed is subsequently released into the atmosphere where it binds with dust particles which are subject to long-range atmospheric transport (Costner, 1995; PSR, 1998; and HCWH, 2001). Another principle source of dioxin may be its presence as a contaminant in technical-grade pentachlorophenol, widely used in the past as an outdoor wood preservative. As a consequence of the plethora of sources and activities that produce dioxin, there is no place globally, regardless of how remote, that is free of dioxin today. While trace amounts of dioxin existed before the heavy use of chlorinated organics began in industry in

24 the 1930s, analysis of dated sediments show the concentrations were low, frequently below the detection limit (Birnbaum, 1993 and Thornton et al, 1996).

Binding to the aryl hydrocarbon (Ah) receptor, a specific internal cellular protein, appears to be necessary for dioxin mediated endocrine effects. Immunotoxicity and reproductive effects appear to be among the most sensitive responses. The Ah receptor transports dioxin molecules to the cell nucleus, and functions in initiation of the transcription of mRNA for a specific suite of protein. Interaction with specific base sequences in DNA appear to be modulated by the presence of growth factors, hormones and their receptors, as well as other regulatory proteins. Consequently, dioxin appears to function as a hormone, initiating a series of events that is dependent upon the environment of each cell and tissue. Prenatal exposure to TCDD was determined to interfere with fetal development, in smaller doses than those necessary to cause overt toxicity in adult animals. Pregnant Long Evans rats receiving a single oral dose of 0.20 microgram of TCDD/kg on gestational day 15 delivered pups that displayed delayed puberty, decreased epididymal sperm counts in male rat pups, and malformations of external genitalia in female pups (Hurst, et al, 2000). In utero and lactational exposure to TCDD causes a decrease in size of the accessory glands of the male reproductive tact, including the seminal vesicles as determined by total weight of the tissue. Administration of 1.0 ug/kg TCDD during gestation caused a significant decrease in seminal vesicle weights of Long Evans rat offspring 8-11 months of age. This is well past the time of puberty.

Food appears to be the major source of dioxin exposure in the U.S., deriving principally from meat, fish and dairy products. Nursing infants can be exposed to elevated levels of dioxin-like chemicals due to the mobilization of dioxin from maternal lipid stores during lactation. It has been estimated that 5 to 16 percent of the lifetime dose of these compounds may occur in the infant as the result of breast feeding (U.S. EPA, 2000a).

Since dioxins are highly bioaccumulative in aquatic food chains, fishers, particularly those who frequently fish in contaminated waterways, may be highly exposed to dioxins (U.S. EPA, 2000). This is particularly true in certain areas of New Jersey, such as the Newark Bay Complex. This complex consists of the Hackensack River downstream of Oradell Dam, the Passaic River downstream of the Dundee Dam, Newark Bay, the Arthur Kill, the Kill Van Kull, and tidal portions of all rivers and streams that feed into these water bodies. In the Newark Bay Complex, concentrations of 2,3,7,8-TCDD in the blue crab have been detected in excess of 1 ppb in certain tissues. The do-not-eat advisory level for dioxin in fish is 50 ppt (FDA, 1993). TCDD sediments concentrations adjacent to known contaminated waste sites on the Passaic River have been recorded as high as 6.9 ppb (NJDEP, DSR, 1985, NJDEP, DSR, 1993, and NJDEP, 1995). Despite prohibitions and advisories for the Newark Bay Complex, consumption of blue crabs from this area remains popular, and many consumers frequently eat large numbers of crabs per meal (Pflugh et al, 1999 and Burger et al, 1999, Pflugh et al, 2002).

7. POLYBROMINATED DIPHENYL ETHERS

Polybrominated diphenyl ethers (PBDEs) are a class of chemicals used as flame retardant additives in high-impact plastics, foams, and textiles comprising 5-30 percent of these products by weight

25 (U.S. EPA, 1991). Swedish data indicate that PBDE concentrations in human breast milk have been doubling every 5 years for the last 25 years. PBDEs are transferred by breast milk from the mother to the offspring. Young pilot whales have 2-3 fold higher levels of 19 PBDE congers than adults (Lindstrom, et al, 1999). PBDEs are found as residues in sediments, wildlife (marine mammals, fish, and bird eggs) and humans (milk, serum, and adipose tissue). PBDEs are lipophilic and metabolically resistant, and thus similar to environmentally persistent compounds such as DDT, PCBs and halogenated dibenzo-p-dioxins and dibenzofurans. PBDE mixtures are not strongly bound in the products to which they are added, and are selectively released over the products' lifetimes. Their toxicology is not well characterized, but appears to resemble that of PCBs. Little is known of PBDE breast milk levels or body burdens in the United States (Hooper and McDonald, 2000). Environmental and wildlife concentrations indicate that PBDEs increase in concentration as the trophic level increases. Persistent Organic Pollutants (POPs) have three characteristics that make them intrinsically hazardous: stability (persistent), lipophilicity (fat seeking), and the potential to be endocrine disruptors (Hooper and McDonald, 2000).

PBDEs disrupt thyroid hormone balance as evidenced by studies in several species. In rats, penta- BDE reduced thyroid hormone levels and increased incidences of thyroid hyperplasia at doses as low as 2 mg/kg/day, the lowest level tested (U.S. EPA, 1999a). In mice, penta-BDE significantly reduced T4 levels a week after a single exposure at the lowest dose tested (0.8 mg/kg) (Fowles, et al, 1994). Effects of tetra-BDE were additive with coadministered PCBs (Aroclor 1254) or chlorinated paraffins in reducing levels of thyroid hormones (Hallgren and Darnerud, 1998, as cited by Hooper and McDonald, 2000).

8. PHYTOESTROGENS

Phytoestrogens are naturally occurring compounds in some 300 species of plants that have estrogen-like effects in animals. The biological relevance and potency of many of these have not been well characterized. These chemicals of plant origin have 2-phenylnaphthalene-type chemical structures similar to estrogens, which bind to estrogen receptors, and have estrogenic and antiestrogenic effects. Their estrogenic activity is dependent on factors such as their concentration, the concentrations of endogenous estrogens, and individual characteristics such as gender and menopausal status (Adlercreutz and Mazur, 1997; and Knight and Eden, 1996, as cited by Tham et al, 1998). Phytoestrogens are known as and related metabolically-generated compounds including , , and mammalian . Plants that have relatively high content of phytoestrogens are barley, bluegrass, oats, orchard grass, rye, ryegrass, rice, wheat, sage, alfalfa, clovers, soybeans, and garlic. In terms of human dietary consumption, the richest sources of phytoestrogens are legumes and grains. The content within a given species is influenced both by cultivar and location where the crop is grown (Tham et al, 1998 and Hughes, 1996). In young children and infants, consumption of soy-based formulas and soy milk can lead to high levels of exposure to phytoestrogens with only limited data available concerning any potential benefits or adverse effects (Stark and Madar, 2002). Some phytoestrogens have been isolated from cow's milk and have been detected in the blood of children and adults who drank milk (Farnsworth et al, 1975, as cited by Molteni et al, 1995).

26 While phytoestrogens are known to have anti-oxidant properties, it is unlikely that this accounts for the full range of their effects (Hughes, 1996). While phytoestrogens exhibit weak estrogenic activity, on the order of 10-2 to 10-3 that of 17-B-estradiol (Adlercreutz et al, 1993 and Adlercreutz et al, 1986, as cited by Tham et al, 1998), they may be present in the body at concentrations 100- fold those of endogenous estrogens (Martinex-Campos et al, 1986; Martin et al, 1978, as cited by Tham et al, 1998) and may compete for estrogen receptor sites when administered concomitantly with estradiol (Molteni et al, 1995). Much of the phytoestrogen hormone-modulating activity may be due to the chemical structural similarity to estrogens, 17-B-estradiol and diethylstilbestrol. Several nonclassical mechanisms of estrogen action are being examined which may account for the estrogenic activity of phytoestrogens despite their relatively low binding capacity to estrogen receptors compared to endogenous estrogens. These involve membrane receptors for estrogens, binding sites, Type II estrogen receptors, and orphan receptors (Hughes, 1996). Phytoestrogens are also structurally related to the weak estrogen/antiestrogen tamoxifen, which is used in the treatment of breast cancer (Tham et al, 1998).

Rodent tests suggest that, depending on the timing of exposure during gestation, phytoestrogens can exert developmental effects including advancing the age of neuroendocrine senescence in females; enlarging the sexually dimorphic nucleus of the medial preoptic area in the brain during the postpubertal interval; reducing the anogenital distances in progeny and delay vaginal openings; affecting female genital tract development; and protecting against chemically-induced mammary cancer (Hughes, 1996).

Estrogenic actions of , in vitro at levels (0.009 - 0.07 %) found in normal rat and human diets were investigated. Coumestrol is one of the few phytoestrogens that do not compete for binding to the progestin receptor (Whitten and Naftolin, 1991). Studies showed coumestrol exhibited a relative binding affinity of about 19% that of estradiol. Further study indicated that coumestrol inhibits binding of estradiol in a competitive manner. In commercial laboratory chow diets, rats ingest daily doses of approximately 84 - 8,900 ug coumestrol. Studies demonstrated oral doses of 50 ppm coumestrol were inactive when dosed for a 4-day period, but showed positive uterotrophic responses when dosed at the same concentration for an 8-day interval. This response parallels the uterotrophic action of steroidal estrogens.

Coumestrol, fed in a semipurified diet at concentrations found naturally in human and animal feeds (100 ppm) to immature rats, stimulated the onset of estrous cycles when provided prepubertally but inhibited ovarian cycles in adult cycling females. Female rats perinatally exposed to exogenous estrogens develop a premature anovulatory syndrome characterized by persistent vaginal cornification and inability to elicit a luteinizing hormone surge. Pregnant rats fed a low-dose coumestrol diet (100 ppm) either for the first 10 postnatal days, or throughout 21 days of lactation, showed no significant alteration in adult estrous cyclicity in the 10-day treatment group. However, the 21-day treatment resulted in a persistent estrous state by 132 days of age. Perinatal exposure of male rats to exogenous estrogen prevents masculinization of sexual behavior. Exposure to coumestrol in the diet increased the latency to mount and ejaculate and reduced mount and ejaculation rates. These actions demonstrate that a low-dose coumestrol diet (such as one which may normally be consumed by humans) can alter neuroendocrine development and can act as a developmental toxicant under certain time dependent circumstances (Whitten et al, 1995; Whitten et al, 1991).

27

Effects of phytoestrogens on fertility or on patterns of secretion of reproductive hormones are more distinct in females than males. The adverse effects of phytoestrogens may include premature sexual differentiation and development of the brain, ovulation and puberty, and the female genital tract, however, they are possibly beneficial in acting to limit proliferation of tumors in mammary tissues. In rodent studies, phytoestrogens show uterotrophic effects that are estrogenic or antiestrogenic depending on experimental design. The patterns of effect are not simply those of a weak estrogen. Considering all the evidence, phytoestrogen effects must be considered predominantly adverse with regard to fertility of female mammals (Hughes, 1996).

The dietary intake of these chemicals can be quite high depending on the diet Oriental people, in particular the Japanese, consume, on average, markedly more soybean-derived food than the average American. This is thought to be responsible for the comparatively low incidence of breast cancer in Japanese women. Japanese women who immigrate to U.S. show a higher incidence of breast cancer after a number of years, suggesting that diet can be an important risk factor in disease incidence.

Beneficial effects of dietary soy phytoestrogens in the postreproductive interval include reduction of risk for several endocrine-dependent cancers, osteoporosis, and coronary artery atherosclerosis. While not known yet with certainty, inclusion of soy phytoestrogens in children and young adult diets could reduce the risk of these diseases later in life (Hughes, 1996).

9. PESTICIDES

In the 1960s and 1970s, DDT, methoxychlor, and other chlorinated pesticides involved in eradication of malaria-carrying mosquitoes, control of farm pests, etc. were implicated in declining bird populations through a mechanism of eggshell thinning. This occurred because technical grade DDT (containing both the p, p' and o, p-isomers) interfered with the activity of carbonic anhydrase, an enzyme involved in the incorporation of calcium in eggshells. The o, p-DDT isomer, which is estrogenic and makes up about 10-20% of the technical mixture, is thought to be responsible for this phenomenon. The p, p'-DDT isomer does not exhibit estrogenic activity. Laboratory studies in Japanese quail on a low calcium diet, receiving 100 ppm of DDT or DDE (a DDT breakdown product) in the diet showed a reduction in the thickness and calcium content of eggshell. Later investigators showed that when the diet contained normal calcium DDT had little or no effect on eggshell thickness (Bitman et al, 1969; Cecil et al, 1971, as cited by Chang and Stokstad, 1975).

Technical DDT, comprised of 70% p,p'-DDT, 21% o,p'-DDT (estrogenic), 4% p,p'-DDD, and 3.5% p, p'-DDE (androgen ), stimulated reproduction in B6D2F1/J hybrid (progeny of C57Bl/6J X DBA/2J) mice at low dietary levels of 5, 10, or 20 ppm, as evidenced by number born and weaned, whereas at higher levels, 30 - 120 ppm, generally inhibited reproductive parameters (Ledoux et al, 1977; Kelce et al, 1995).

Pregnant CF-1 mice orally dosed over a 4-log range of 0.02 - 200 ug/kg body weight/day on gestational days 11-17 of pregnancy with either o, p'-DDT, methoxychlor, or DES in corn oil had their offspring examined at birth. The offspring which were exposed to their respective chemicals

28 during the fetal period of development exhibited significant alterations in the number of live pups born per litter, sex ratios of the litters, anogenital distances of male and female offspring at birth (a bioassay for fetal androgen action), and body weight of offspring at birth and during the first 5 days of postnatal life. In most cases the effects observed at the higher dose levels were opposite to the effects observed at the lower dose levels (Palanza et al, 2001).

In New Jersey, bald eagles, once numbering 22 pairs, declined with the introduction of the chlorinated pesticides such as DDT, to just one nesting pair in 1970. With the federal ban on the use of DDT in the United States in 1972 and the restoration efforts by biologists in the NJ Division of Fish and Wildlife's Endangered and Nongame Species Program the bald eagle population increased moderately to 14 nesting pairs in 1997 and to a record 27 active nests in 2001, with most located in southern New Jersey (Smith et al, 2001). The bald eagle is currently listed as a threatened species by the federal government (proposed for de-listing) and as an endangered species by the state of New Jersey. While the population is improving, some nesting pairs in the Delaware Bay still experience reproductive failure. Eggs from these non-producing pairs were examined and found to contain significant levels of DDT breakdown products, DDE, DDD, as well as PCBs, in the Racoon Creek and Stow Creek areas of Delaware Bay. The measured levels were similar to concentrations associated with reproductive impairment in eagle populations from other areas (Clark et al, 1998).

Several DDT-related chemicals, previously thought to be estrogenic (uterotrophic), were investigated for androgen receptor binding activity. In androgen receptor competitive binding assays, p, p'-DDE was shown to be the most potent competitive inhibitor of androgen binding to the specific androgen receptor (AR) binding site. Once bound, p, p'-DDE acts as an antiandrogen by inhibiting the transcription of androgen-dependent genes. P, p'-DDE binds the androgen receptor 200 times more effectively than to the estrogen receptor. Together with other information, these data suggest that the androgen receptor, rather than the estrogen receptor, is the site of hormone blockade by persistent pollutants such as p, p'-DDE. This illustrates that androgen mimics as well as estrogen mimics may affect endocrine process even in females. This complicates testing and assessment of potential endocrine disrupting environmental chemicals (Kelce et al, 1995 and Chapin et al, 1996).

Pyrethroid insecticides are the most used agents for indoor pest control and consequently pose potential for human and pet exposure. The estrogenic potential of several synthetic pyrethroid compounds (sumithrin, fenvalerate, d-trans allethrin, and permethrin) was investigated with an in vitro test that used the MCF-7 human breast carcinoma cell line and measured pS2 mRNA levels as an end point. Nanomolar (nM) concentrations of sumithrin or fenvalerate induced pS2 expression to levels comparable to 10 nM 17-B-estradiol. The estrogenic activity of sumithrin, but not that of fenvalerate, was abolished by co-treatment with an antiestrogen. Both sumithrin and fenvalerate induced cell proliferation of MCF-7 cells in a dose-response manner, indicating that they were capable of causing estrogen-dependent cell proliferation. These findings indicate pyrethroids should be considered to be capable of endocrine disruption and their potential to affect endocrine function in humans and wildlife should be investigated (Go et al, 1999).

An EPA Science Advisory Panel, in June 2000, reviewed an extensive data set for atrazine, the most intensively applied pesticide in the U.S. (70 million pounds active ingredient per year) and

29 concluded that the endocrine mode of action (MOA) is responsible for both cancer and non-cancer (endocrine disrupting) effects observed in test animals. The proposed cancer MOA is that atrazine alters the hypothalamic-pituitary-gonadal axis which effects the secretion of hormones. This results in the prolonged secretion of ovarian estrogen which leads to an increased formation of mammary tumors in a certain strain of rat. For the noncancer endpoint, they have concluded there is emerging evidence that the same MOA, perturbation of the hypothalamic-pituitary-gonadal axis, delays pubertal landmarks, increases abortions at doses that affect maternal health, causes enlarged prostates in offspring, and causes delay of onset of puberty in several strains of test animals (Risk Policy Report, 2000).

Recently, the possibility was raised that ED effects could occur with chemical mixtures, at concentrations low enough that individually the chemicals would not be anticipated to cause an adverse effect (i.e., synergistic interactions). The interactive effects of a mixture of aldicarb (0, 10 ppb), atrazine (0, 10 ppb), and nitrate (0, 28 ppm) on endocrine, immune, and nervous system function was investigated in two species of mouse, Peromyscus maniculatus and outbread Swiss Webster mice, ND4 strain (Porter et al, 1999). The concentrations of the chemicals used were of the same magnitude as current maximum contaminant levels (MCLs) allowed in drinking water and similar concentrations of these chemicals are found in some locations in the United States in groundwater. Thyroid hormone, immune, and behavior changes were evaluated. For the most part effects were not seen with exposure to individual chemicals. For mixtures, however, effects were noted for increased aggression with mixtures of atrazine/nitrate and aldicarb/atrazine/nitrate. Effects were also observed on body weight with mixtures of aldicarb/nitrate, atrazine/nitrate, and aldicarb/atrazine/nitrate, free thyroid index and on plaque forming (antibody production) with mixtures of aldicarb/nitrate, atrazine/nitrate, and aldicarb/atrazine/nitrate. Aldicarb/ nitrate also affected terminal spleen weight. It was not clear how or why nitrate in combination with pesticides may be more active and produce some endocrine disruptor related effects.

A child health assessment study was conducted in the Yaqui Valley of Sonora Mexico where agricultural pesticides have been used for approximately fifty years. Earlier investigations demonstrated neuromental and neuromuscular deficits in exposed children aged 4-5 years, along with suggestions that illness occurred in these children more frequently. The health status of children living in this area was compared to that health status of children in a control area. Parents were asked to recall the number of time illness occurred in their children during the past three months. Most of the lesser exposed children in the control area had either no or one episode of illness compared to the mode of six to eight episodes for exposed valley children. Some of the illnesses in valley exposed children were associated with direct pesticide exposure such as rashes and gastrointestinal problems. No cases of asthma were reported, while the high incidence of infectious disease among the exposed children usually involved the respiratory system (Guillette, 2000). In a similar study between pesticide exposed and non-exposed children, while height and weight were alike, differences existed in developmental skills, as measured through play behaviors. Neuro-muscular deficits, in terms of coordination and stamina were found in children from the agricultural communities. The heavier exposed children exhibited neuromental deficits, as measured through the use of drawing and memory problems (Guillette, 2000).

30 10. GROWTH PROMOTERS

Diethylstilbestrol (DES) bears marked similarities in structure to DDT and other DDT-related chlorinated pesticides. DES was widely used in steers and capons as a growth promoter which resulted in their gaining weight and muscle mass more quickly, thus providing those who used it an economic advantage by decreasing the time required to finish animals for market. The endocrine disrupting effects of DES have been discussed previously.

11. ORGANOTIN COMPOUNDS

Synthetic organotin compounds, mainly triphenyltin and tributyltin, are widely used in marine environments as biocides in anti-fouling paints on ships' hulls and other underwater structures and are also used in agricultural pesticides, , and plastic stabilizers. In the 1980s some restrictions were placed on these compounds after it was noted that they were capable of inducing an imposex condition, - formation of a penis and vas deferens in female gastropods at concentrations as low as a few ng/L. (Sekizawa, 1998 and Harino et al, 2000).

Organotin compounds are thought responsible for the 100% imposex observed in the rock shell mollusc at 22 sites in the Seto Inland Sea and seven sites of the Sanriku region of Japan. The Relative Penis Length Index, defined as the ratio of mean penis length in females to that in males, exceeded 50 at many sites. That is the females had penises substantially larger that the males. Sterile individuals whose oviducts were blocked by vas deferens formation, a male reproductive tissue, were also extensively observed (Horiguchi et al, 1998).

Imposex in the dioecious snail (Nassarius obsoletus Say) was studied by exchanging samples of snails between a yacht basin and a 'clean' locality where the natural population was normal. Imposex was induced in some normal snails kept in a marina and suppressed, but not lost in abnormal sails from a marina kept at a clean location. Similar positive results were obtained in laboratory studies (Smith, B. S., 1981). Imposex was rated in the dioecious snail Nassarius obsoletus Say as the fraction of the population affected and the intensity of expression in bearer snails along a transect ranging from Rhode Island to Georgia. Imposex scores were significantly higher in all marinas studied (Smith, B. S., 1981). In the laboratory imposex can be caused by exposure to parts per billion concentrations of tributyltin oxide, tributyltin resinate and tributyltin chloride in female mud snails Nassarius obsoletus Say. Occurrence of such abnormalities in nature can, therefore, suggest organotin pollution (Smith, B. S., 1981).

Concentrations of organotins were measured in the muscle of 11 species of fish in the Port of Osaka and Yodo River in Japan. Tributytin (TBT) and triphenyltin (TPT) were detected in the range of 0.011 - 0.182 mg/kg wet weight and < 0.001 - 0.130 mg/kg wet weight, respectively. Concentrations of TBT in muscle were higher than TPT. Concentrations of organotins in fish from sea areas were higher than those in rivers. Tributytin in the fish was found to bioaccumulate by a factor of 500-8000. Concentrations of butyltins and phenyltins in organs of three fish species were analyzed. TBT was higher in liver, brain, and muscle of white croaker and yellowtail. Sex differences were not noted and there was no correlation of TBT to lipid content. Although the

31 concentration of TBT in the brain was lower than in other organs its presence indicates that TBT can pass the blood-brain barrier (Harino, et al, 2000).

The International Maritime Organization is expected to agree soon to a treaty banning the use of TBT on ship hulls. The environmental organization Greenpeace last summer found elevated levels of TBT contamination in harbor sediments in Greece, Italy, France and Spain. They also determined from testing paint on hulls of sea vessels that in excess of 35 percent were already using alternatives to TBT paint (ENS, 2000).

12. PHARMACEUTICAL COMPOUNDS

The potential environmental impact of pharmaceutical agents as a group has received very little attention. The primary reason they should be looked for in the environment is that, as medical substances, they were developed with the specific intention of producing a biological effect. Very little is known regarding routes of environmental exposure to medical substances, however, excretion of metabolized or unmetabolized pharmaceuticals will likely to lead to their occurrence in wastewater and in water bodies receiving wastewater. The medical substance clofibrate, a cholesterol lowering agent, is increasingly being reported in various ground and tap water samples in Europe up to 165 ng/L (Halling-Sorensen et al 1998). Recently, clofibric acid, the pain reliever naproxen, and the hormone estrone, a metabolite of estrogen, were detected at three sites along the Mississippi River, at four sites around Lake Pontchartrain, and in Tulane University's tap water. Assays revealed a minimum of 10 part per trillion (ppt) of each drug at least once at every site. Estrone in tap water averaged 35 ppt, with a high of 80 ppt. Environment Canada has detected similar pollutants in its 1998 nationwide survey of sewage-treatment effluent. At some sites, estrone reached 400 ppt and ethinylestradiol hormone, a synthetic estrogenic steroid used in oral contraceptives, was 14 ppt. At exposures of 0.1 ppt or 10 ppt some Japanese Medaka male fish became intersex, exhibiting both male and female reproductive tissues. At 1,000 ppt exposures to either estrone or ethinylestradiol, males were transformed into females (Raloff, 2000). British biologists in the early 1990s, noted that most fish taken from the River Lee had testes laden with eggs, something definitely not normal for male fish. It was determined later that male fish were being feminized by estrogenic compounds in sewage effluent from birth control pills (Kaiser, 1996).

Antibiotics used as growth promoters and feed additives in feedlots and in fish farms are also likely to end up in the environment. Compounds and their metabolites used in birth control pills, treatment of osteoporosis, and postmenopausal patients are usually very potent hormonal agents that may survive sewage treatment and consequently find their way into the environment (Halling- Sorensen et al 1998). Their presence in the environment should be examined.

13. DIESEL GASES

Exposure to high concentrations of diesel exhaust fumes masculinizes rodent fetuses during pregnancy. Exposure of pregnant female rats exposed to high concentrations of diesel exhaust fumes, both with and without particles, resulted in delayed or abnormal development of testes,

32 ovaries, and thymuses. Groups of pregnant rats were exposed to atmospheres containing 5.6 mg/meter3 particulate matter or no particulate matter, 4.1 ppm nitrogen dioxide, and 8.1 ppm nitrogen oxide during days 7-20 of pregnancy. Additional groups of pregnant rats were treated with aromatase inhibitors or testosterone to help clarify the process whereby diesel exhaust exerts its toxicity. Anogenital distance was significantly greater in male and female fetuses in exhaust- exposed groups than in control groups. Maternal levels of testosterone and progesterone were significantly higher and lower, respectively, in pregnant rats exposed to total and filtered exhaust. Serum adrenocorticotropic hormone and urinary 17-hydroxycorticosteroids did not differ among pregnant groups of animals indicating that elevated testosterone did not result from elevated maternal adrenal function. The authors speculate that inhibition of aromatase activity and synthesis caused by exhaust inhalation may have played an essential role in the accumulation of testosterone. As the effects were seen from exposure to exhaust both with and without particulates, the gaseous phase must have included the relevant toxicants. The relevance of these findings to environmental exposure levels is speculative as workplace exposures are about 20 percent of the concentrations of diesel exhaust used in this study and residential exposure levels near freeways are about 0.2 percent (Watanabe and Masayuki, 2001). C57Bl-strain female mice, exposed to 0.3, 1.0, or 3.0 mg diesel exhaust particles (DEP)/M3 for 4 months (12 hr/day, 7 days/wk) including gestation exhibited abnormal delivery and their young exhibited abnormalities in growth and sexual maturation. Among the findings were estrous females who were exposed to 1.0 mg DEP/M3 had significantly lower uterine weights than control unexposed estrous females. Anogenital distance (AGD) was significantly less in 30 day-old males whose dams were exposed to 0.3 mg DEP/M3 (Tsukue, et al, 2002). Ultra structural changes were observed in Leydig cells of mice exposed to DEP at 0.3 mg /M3 for 12 hr/day, up to 6 months. Daily sperm production per gram of testis was dose- dependently decreased with DEP exposure of 0.3, 1.0, and 3.0 mg DEP/M3, respectively. The no- observed-adverse-effect level (NOAEL) was observes with approximately 30 micrograms DEP/M3, which is lower than the WHO-recommended exposure limit (Yoshida, et al, 1999).

E. IMPACTED ANIMALS

1. SNAILS

See discussion of ORGANOTIN compounds.

2. MOLLUSKS

See discussion of ORGANOTIN compounds.

3. FROGS

In the United States, public attention on endocrine disruption was aroused when school children on a field trip to Lake Suer, Minnesota in 1995, discovered malformed frogs. Endocrine disruption as apparently manifested through malformed frogs became a general interest story which aroused

33 widespread public interest. The frog deformities noted included missing feet, missing and/or extra legs, missing eyes, one eye smaller than the other, webbing between the hind legs, club feet, missing toes, and various combinations of the above. This led to an unprecedented explosion in frog-related websites and groups of people searching for deformed frogs across North America and around the world (http://mncs.k12.mn.us/html/projects/ frog/frog.html). Other URLs on this same subject are: http://www.mpm.edu/collect/vertzo/herp/atlas/welcome.html, (Wisconsin Herpetology Homepage); and http://www.npwrc.usgs.gov/narcam (North American Reporting Center for Amphibian Malformations). These URLs contain many additional links to other websites that contain additional information on various aspects of this and related endocrine disruptor issues. Deformed frogs were also reported in three Canadian provinces and 38 U.S. states (Conlon, 1997). Frog surveys have been set up in Minnesota, Wisconsin, Michigan, Iowa, Missouri, Illinois, Indiana, Ohio, and Canada. The popular explanation for occurrences of frog deformities was that some environmental contaminant in the water was perturbing development of these amphibians. Suspected causes were chemicals like PCBs, herbicides, and pesticides, and later parasites and mutations (MPCA, 1997).

While it is likely the defects observed are developmental abnormalities rather than inheritable deformities, their cause remains uncertain. However, frogs with the same or similar defects have been noted previously in the historical literature. This leads one to conclude that, to some degree at least, this is not an entirely recent phenomenon and therefore is not likely to be caused only by synthetic organic chemicals, since the dawn of organic chemistry occurred about 150 years ago and synthetic pesticides and herbicides are relatively recent discoveries (Bounias, 1998).

Evidence suggests that similar malformations can occur when parasites infect the developing tadpole causing a physical disruption to limb formation and other structural processes (Sessions and Ruth, 1990). Other research suggests that infections by fungal species, hitherto unobserved in amphibians, may be related to such deformities (Kaiser, 1998). Nonetheless, some studies suggest a link between contaminants in the water of the frogs' environment and the deformities. Similar deformities were observed in studies in which frogs were grown in the suspect water after the water had been filtered to remove potential biological sources of infection (Merrsman, 1998; Burkhart et al, 1998). The interpretation of these studies is, however, in question, as the species studied may be effected by naturally occurring and nutrients in the test water in a way that naturally occurring species are not (Souder, 1997). Overall, the extent and cause of these deformities is unclear.

The following is a brief summary of the incidence of frog malformation data as reported to NARCAM for New Jersey and bordering counties in New York, Pennsylvania, and Delaware as of February, 2003. In New Jersey, Camden County data from each of three different months at the same site contain reports of three malformed specimens of Fowler's Toad with one or more missing digits and two normal specimens. Cape May County reported 50 normal specimens, representing three species of frog, and no malformed specimens. Middlesex County reported 5 normal and one malformed specimen in one species of frog. Passaic County reported about 150 normal and 2 malformed specimens in one species of frog. Sussex County reported 126 normal and two malformed specimens representing four species of amphibians. Morris County reported 5 normal and two malformed specimens, one each from two species of frog. In summary, New Jersey data contained reports of 338 normal and only 10 malformed specimens of amphibians, which, due to

34 caveats described earlier, is probably biased toward the high side of the expected real value. In New York, Sullivan County, in one species with the number of normal frogs not reported there were two malformed specimens. In Pennsylvania, Bucks County, in one species with the number of normal frogs not reported there was one malformed specimen. In New Castle County, Delaware, there were no normal or malformation data reported.

The effects of dibutyl phthalate (DBP) on gonadal sex differentiation were examined by exposing genetically male tadpoles of Rana rugosa to dilute solutions of DBP at concentrations of 0.1, 1, or 10 micro molar during days 19-23 after fertilization, which is the critical period for differentiation in this species. Positive controls consisted of tadpoles exposed to dilute solutions of 17 beta- estradiol (E2) at concentrations of 0.01, 0.1, or 1 micro molar during the same period. The internal structure of the tadpole gonads was examined at necropsy on day 40. The gonads of the control tadpoles all had the typical structure of testes. In contrast, 0.01, 0.1 and 1 micro molar E2 treatments caused the undifferentiated gonads of 18, 63, and 100% of the tadpoles, respectively, to develop into gonads of complete or partial ovarian structure. After 0.1, 1, and 10 micro molar DBP treatments, 0, 7, and 17% of tadpoles, respectively, were similarly affected. While these findings suggest that DBP was about 1,000-fold less potent than E2, nevertheless DBP is a potentially environment hormone that has the potential to disrupt the pathways of testicular differentiation in genetically male animals (Ohtani, et al, 2000).

Xenopus laevis tadpoles exposed for 48 hours during sexual differentiation to atrazine at a concentration of 21 microgram/L resulted later in life in a 57% reduction in testicular volume. Additionally, primary spermatogonial cell nests, which represent germ cells for the life of the frog, were reduced by 70%. Nursing cells, which provide nutritive support for developing germ cells, declined by 74%. Testicular resorption was observed in 70% and aplasia or failure of full development of the testis occurred in 10% of the atrazine-exposed tadpoles. Atrazine-exposed ovaries had a significant increase of secondary oogonia. Atresia, or oogonial resorption of both primary and secondary oogonia, was also significantly increased. These results suggest that the primary germ cells, which constitute the total germ cells in the ovary for the reproductive life of the organism were reduced 18% more than the controls following a 48-hour exposure period. Together the effects reported suggest that a pulse exposure of 21 microgram/L of atrazine during sexual development could significantly reduce reproduction during the reproductive life of these frogs (Tavera-Mendoza, et al, 2002a; Tavera-Mendoza, et al, 2002b).

The effects of atrazine were examined on the sexual development of African clawed frogs (Xenopus laevis), a very well studied frog species. Larvae were exposed to 0.01-200 ppb atrazine by immersion throughout larval development and later examined for gonadal histology and laryngeal size at metamorphosis. Atrazine at 0.1 or greater ppb induced hermaphroditism and demasculinized the larynges of male frogs. When male X. laevis frogs were exposed to 25 ppb atrazine they exhibited a 10-fold decrease in testosterone levels. The authors hypothesized that atrazine induced aromatase which is involved in the conversion of testosterone to estrogen. This hypothesized disruption in steroidogensis could explain the demasculinization of the male larynx and the production of hermaphrodites. The effective atrazine levels used in these studies, comparable to realistic environmental exposures, suggest that other amphibian species exposed to atrazine in the wild could be at risk of impaired sexual development and consequently, either alone or together with other environmental endocrine disruptors, may be a factor in global amphibian

35 declines. It should be noted, that these studies were repeated four times and, in total, atrazine exposure at these levels was replicated 51 times in this laboratory with similar results. The recommended levels of application for atrazine ranges from 2,500,000 - 29,300,000 ppb, whereas the maximum allowable contaminant level for atrazine in drinking water is 3 ppb. Currently, short- term exposures of 200 ppb are not considered a health risk. Atrazine had been as high as 21 ppb in ground water, 42 ppb in surface water, 102 ppb in river basins in farm areas, up to 224 ppb in midwestern streams, and 2,300 ppb in tailwater pits in agricultural areas in the Midwest. Therefore, the likelihood that wild amphibians are exposed to levels determined to cause adverse effects in this study is extremely high (Hayes, et al, 2002).

Evidence exists that environmental contaminants can alter sex ratios and prevalence of intersex gonads (gonads containing both male and female sex cells) in cricket frogs (Acris crepitans). The prevalence of intersex gonads was assessed by examination of 341 cricket frogs collected during 1993-95 from over 20 sites throughout Illinois (these frogs are also found in New Jersey in or near permanent shallow water). The intersex rate in this species was determined to be 2.7% and was not associated with the presence of 37 herbicides or 44 insecticides and metals. However, it was noted that there is a weak association (p=0.07) between the detection of atrazine and the intersex individuals. A comparison of frogs from sites with point sources of (PCB) and polychlorinated dibenzofuran (PCDF) contamination to frogs from reference sites showed significant relationships between sex-ratio reversal and PCB and PCDF contamination. This reversal favored males over females, which is the opposite of the sex ratio in control ponds and highly statistically significant. This correlation between organochlorine contamination and sex ratio reversal suggests PCBs and PCDFs can influence cricket frog sexual differentiation (Reeder et al, 1998).

4. FISH

Early in the 1990s, British biologists noted that fish taken from the River Lea, near London, contaminated with sewage, had testes laden with eggs (Kaiser, 1996; Jobling et al, 1998, and Rodgers-Gray et al, 2001). Later, it was demonstrated that young male rainbow trout placed in a wire basket/cage near the outfall of a waste treatment plant produced high levels of , a protein involved in egg-laying that is normally only found in female fish. After a period of time it was noted that the male fish had taken on female physical characteristics. The assumption made and reported at that time was that some environmental chemical possessing estrogenic activity dumped into the waste stream was responsible. This study was widely reported as an indication of the dangers of hormone-disrupting chemicals. Subsequent researchers collected sewage effluent from three treatment plants and isolated compounds likely to act like estrogen in fish. They discovered that the estrogenic compounds isolated were not industrial pollutants, but rather three hormones found in females: 17-B-estradiol, estrone, and ethinyl estradiol. This last compound, while in very low concentrations, is a potent synthetic hormone used in birth control pills. Isolation of these compounds was unexpected because prior to excretion by the kidney a glucuronide or sulfate group is added by the body thereby inactivating the compound. It appears that during sewage treatment these conjugating groups were removed, thus restoring the biological activity to these estrogenic chemicals (Kaiser, 1996).

36 Complete, permanent, and functional male-to-female sex reversal in Japanese Medaka was accomplished after a onetime embryonic exposure to o, p'-DDT. O, p'-DDT was micro injected into the egg yolks of these fish at fertilization to mimic the maternal transfer of lipophilic contaminants to the embryo. An embryonic exposure of 227 ng/egg o, p'-DDT resulted in sex reversal of genetic males to a female phenotype in 86% of the cases. Histology of the gonads confirmed active ovaries in all of the feminized males (XY females). This demonstrated that a weakly estrogenic pesticide, o, p'-DDT when given during the critical period of gonadal development can profoundly alter sexual differentiation (Edmunds, et al, 2000).

Vitellogenin is a yolk precursor occurring in all egg-laying animals. It is produced naturally in the liver in females under the control of estrogens secreted by the ovary. Vitellogenin is also produced in male fish after exposure to exogenous estrogens (Clemens, 1978, as cited by Purdom et al, 1994). A nationwide survey was conducted in England involving exposure of fish to effluent from 15 sewage-treatment plants (Purdom et al, 1994). Induction of vitellogenin levels occurred in fish in all non-control groups.

It has been demonstrated that 4-nonylphenol or oil refinery treatment plant effluent induced vitellogenin synthesis and zona radiata proteins (contained in the vitelline envelope surrounding the fish embryo during development) in a dose-dependent manner in juvenile Atlantic salmon after 4- week exposures to 1, 10, and 50 percent mixtures of effluents (Arukwe et al, 1997).

Exposure of male rainbow trout (Oncorhynchus mykiss) to four alkylphenolic chemicals, octylphenol, nonylphenol, nonylphenoxy-carboxylic acid, and nonylphenoldiethoxylate in water, caused synthesis of vitellogenin, and a concomitant inhibition of testicular growth. The magnitude of the estrogenic effects was dependent on the estrogenic potency of the chemical, the stage of reproductive development of the fish, and the concentration of the chemical in the water (Jobling et al, 1996).

The U.S. Geological Survey performed a reconnaissance of sex steroid hormones in common carp to assess whether endocrine disruption may be occurring in fish in United States' streams. 17-B- estradiol (E2), 11-ketotestosterone (11-KT), vitellogenin, and gonadal histopathology were measured in adult carp in a wide range of environmental settings across the nation involving 23 streams and 2 impoundments. In addition, organochlorine pesticides and polychlorinated biphenyls in fish tissue were evaluated, as well as phthalates, , polycyclic aromatic hydrocarbons in bed sediment, and dissolved pesticides in water. While no significant regional differences in steroid hormones were detected in male carp, the female carp from the northern and southern mid- continent were significantly different from other regions in levels of E2 and/or 11-KT. Within all regions, there were significant differences between sites for one or both hormones for both sexes of fish. Contaminants with significant correlation with the biomarkers, 17-B-estradiol, 11- ketotestosterone, and vitellogenin were organochlorine pesticides, phenols, and dissolved pesticides. The strongest association common to both males and females was a negative correlation between the hormone ratio E2/11-KT and dissolved organochlorine pesticides. The site-to-site differences in biomarkers and the presence of significant correlations between biomarkers and contaminants are evidence that fish in some streams may be experiencing endocrine disruption. Better information is needed to determine whether endocrine disruption is actually occurring and if

37 there are developmental effects on individuals or populations of carp or other species (Goodbred et al, 1997).

5. ALLIGATORS

In 1980, a spill of mainly DDT and dicofol, organochlorine pesticides occurred in Florida's Lake Apopka. Coincident with this spill was a decline in the population of juvenile alligators. The rate of successful hatching of alligator eggs was markedly reduced, the reproductive tract morphology seriously altered, and the hormone levels of surviving juveniles seriously disrupted. Female alligators had plasma estradiol 17-B concentrations approximately twice that of normal control alligators from nearby Lake Woodruff, the control lake. The ovaries from Lake Apopka alligators contained abnormally high numbers of polyovular follicles and multinucleated oocytes. Male alligators showed poor organization of the testes and abnormally small phalli. In addition, juvenile male alligators had plasma testosterone concentrations three times lower than corresponding control males. These data suggested the gonads of juvenile alligators from Lake Apopka were permanently modified before hatching so that normal production of steroid hormones was not satisfactorily initiated, thus making normal sexual maturation unlikely (Guillette, Jr. et al, 1994). Prenatal exposure to environmental chemicals functioning as hormones was proposed as responsible for these effects. In competition binding assays with [3H] 17-B-estradiol, some DDT metabolites showed inhibition of [3H] 17-B-estradiol binding to the estrogen receptors. A combination of showed an additive decrease in [3H] 17-B-estradiol binding to estrogen receptors. Chemicals mixed in the same concentration as those found in the alligator eggs were tested for their ability to compete with estrogen at estrogen receptor sites. P, p'-DDD and trans- nonachlor, two chemicals found in Lake Apopka, interacted with the estrogen receptor but chlordane and toxaphene did not, thus suggesting that the competition was chemical-contaminant specific. Combinations of these chemicals decreased labeled binding activity of 17-B-estradiol in assays in a greater than additive manner, suggesting synergism (i.e., in a greater than additive fashion).

In a competitive binding assay using American alligator estrogen receptors and a synthetic progesterone, most chemicals did not reduce binding to progesterone receptors but endosulfan, alachlor, and kepone inhibited binding. These results provided evidence that environmental chemicals bind the estrogen and progesterone receptors from the American alligator supporting the hypothesis that the reproductive abnormalities may be related to the modulation of endocrine- related responses (Vonier et al, 1996).

6. TURTLES

Temperature can override the sex genotype in some species of lizards, and the presence of male or female genitalia in turtles can therefore be determined by the incubation temperature during embryonic development. Temperature determines sex by influencing steroid hormone metabolism and/or sensitivity to steroid hormones. Steroidogenic enzyme inhibitors or exogenous steroid sex hormones can override or enhance temperature effects on sex determination. Androgens and inhibitors of androgen metabolism induce testis differentiation at temperatures which normally

38 produce females. Nonaromatizable androgens and aromatase inhibitors induce testis differentiation at female-producing temperatures, whereas aromatizable androgens and estrogens induce ovarian differentiation at male-producing temperatures. Natural estrogens and temperature synergize to produce more females than expected if estrogens and temperature were purely additive on sex determination. Weak natural estrogens estrone and 17-B-estradiol synergize with a low dose of the more potent estriol to reverse gonadal sex during the critical period of sexual differentiation. The results suggest that weak environmental estrogens may also be synergistic with stronger natural estrogens (Bergeron et al, 1999). Aromatase inhibitors, which can stop the metabolism of testosterone to estrogen, are capable of converting females to males in turtles (and chickens) (Jeyasuria, P., and A. R. Place, 1998).

The sexual characteristics of gonads and genitalia is determined in the red-eared slider turtle by the incubation temperature with female development requiring endogenous estrogen produced by elevated egg incubation temperature. Exogenous estrogens can cause sex reversal in presumptive males. When exogenous estrogen was applied to newly laid eggs at doses as low as 400 pg/egg (40 ng/kg) and incubated at this temperature which normally produces predominately if not exclusively males, sex reversal occurred in 14.4% of the animals. These results provide a simple dose response model which suggests that chemicals which act mechanistically like estradiol may show no threshold dose. If so, even low doses of such chemicals may carry risk for sex reversal (Sheehan et al, 1999).

The induction of plasma vitellogenin in male adult turtles (Trachemys scripta) and frogs (Xenopus laevis) with injections of 17-B-estradiol, diethylstilbestrol, or o, p'-DDT demonstrated that vitellogenin may be a useful biomarker of xenobiotic estrogen activity, hence a measure of exposure, in wild populations of male reptiles and amphibians (Palmer, B. D., and S. K. Palmer, 1995).

7. HUMANS

a. THELARCHE

Premature breast development (thelarche) is the growth of mammary tissues in girls less than 8 years of age without other manifestations of puberty. Puerto Rico has the highest known incidence of thelarche in the world (e.g., 18.5 times higher in Puerto Rico than in the state of Minnesota). This elevated incidence of thelarche, which continues today, was first noted by pediatric endocrinologists in Puerto Rico in 1979. Since that time, many hypotheses have been proposed and investigated, however, the cause of this elevated rate of thelarche remains unknown, as do the long- term consequences of the aberrant premature sexual development in this population of young girls. Theories under consideration for this elevated rate of thelarche include the association with ovarian cysts, premature endogenous production of sexual hormones, and environmental contamination by pharmaceutical waste products (Colon, et al, 2000).

While the amounts of substances such as phthalate esters, alkyl phenols, and surfactants in commercial products used for packaging, storing, and preserving food used in Puerto Rico is

39 unknown, it can be assumed to be potentially significant because of the high level of consumption of dietary products in plastic containers imported to the island. The serums of Puerto Rican girls with thelarche were chemically analyzed for pollutants. Serum samples from 41 thelarche patients and 35 control subjects were analyzed. No pesticides or their metabolites were detected in the serum of the study or control subjects. Significantly elevated levels of dimethyl, diethyl, dibutyl, and di- (2-ethylhexyl) phthalate (DEHP) and its major metabolite mono- (2-ethylhexyl) phthalate were identified in 28 (68 %) samples from thelarche patients. Only one individual in the control sample showed significantly elevated levels of di-isooctyl phthalate. The phthalates identified have been considered to be potential endocrine disruptors in animal studies. While the findings of this study cannot be used to show causality for thelarche in Puerto Rican girls, they do suggest a possible association between plasticizers with known estrogenic and antiandrogenic activity and premature breast development in a human female population. While, this is the first analytical evidence of the presence of plastic additives with known estrogenic activity in girls with thelarche, the authors point out this does not prove causation (Colon, et al, 2000).

DEHP daily consumption from all sources is estimated at 5.8 mg in the United States and 2.1 mg in Japan. The principle sources of exposure for children are ingestion of contaminated formulas, food, and water from contact with plastic wrappings and containers and mouthing of plastic toys and pacifiers (Colon, et al., 2000).

During the last five decades of the 20th century there has been a noted worldwide increase of testicular cancer. In Northern Europe testicular cancer is high in Denmark and Norway, intermediate in Sweden, and low in Finland and the Baltic countries. A number of studies suggest a decline in sperm count during this same period. Sperm concentration of army conscripts from Norway and Sweden was compared to those from Lithuania and Estonia (average age 18.2 - 20.6 years). Testicular cancer incidence rates from 1996 were inversely related to median sperm concentration. In addition, in Norway, a positive correlation was found between low birth weight and reduced sperm count, as previously observed between low birth weight and testicular cancer. This suggests an important role of the intrauterine environment on later adult reproductive function (Haugen, et al, 2000).

b. SEX RATIO

Sex ratio is a measure of the number of males to females and in humans is defined as the number of boys born per 100 girls. Normally, it is expected to always exceed 100. Some 125 males are conceived for every 100 females, yet only 106 boys are born for every 100 girls (Alan et al, 1997). The male embryo/fetus can, therefore, be considered more fragile. Between 1930 and 1990 the Canadian sex ratio increased for the first two decades and this was attributed to improvements in health care. However, starting in 1970 the ratio dropped in every region, with the most significant decrease of 5.6, twice the average for the rest of Canada, occurring in the Atlantic Provinces. Subsequent examination of the sex ratio in the United States indicated that the ratio had also dropped by about one boy per 1000 births. Decreases such as these are also being noted in Sweden, Germany, Norway, and Finland. Several investigators are attributing these decreases to environmental factors. These decreases, when reported as changes in sex ratio, do not attract very much attention, however, due to the population sizes of the countries involved, they reflect

40 decreases of 8,600 and 38,000 births for Canada and the United States, respectively (Davis et al, 1998).

Recently, it was noted that while male births showed a pattern of decreasing there was an increasing rate of male reproductive defects (Davis et al, 1998). From 1970 to 1993, the rate of hypospadias (developmental anomaly in males in which the urethra opens on the underside of the penis or in the space between the anus and scrotum) has nearly doubled in all regions of the United States. During this same period testicular cancer rates have also increased (Parkin et al, 1997, as cited by Davis et al, 1998).

Dibromochloropropane, DBCP, a nematocide, was shown in early toxicology studies to have special impact on the testes. DBCP production workers in California and Israel were found to be sterile as a result of their occupational exposure evidenced by decreased sperm counts and increased gonadotrophin levels. Follow-up studies showed some recovery of testicular function, however, in their otherwise healthy offspring there was a predominance of females (Goldsmith, 1997).

Preliminary evidence suggests an alteration of the in offspring of parents most highly exposed to TCDD from the accidental release of 2,3,7,8 TCDD in Seveso, Italy in 1976. During the 9-month period following the accident, there were 26 male and 48 female children born for a resulting sex ratio of 54. This highly unusual event was statistically significant (p<0.001). From 1985 to 1994, 60 male and 64 female children were born for a sex ratio of 94, which is not a statistically significantly altered sex ratio. During the first 8 years after the accident nine couples who had more than 100 parts per trillion (ppt) of TCDD in their serum delivered 12 daughters and no sons (p<0.001). Four couples whose blood dioxin concentrations were below 100 ppt had children that included males. Serum samples collected between 1977 and 1996 showed a statistically significant increased probability of female births with increasing TCDD concentrations in the serum samples from the fathers. This effect started at concentrations less than 20 ng/kg body weight. Fathers exposed when younger than 19 years old sired significantly more girls than boys resulting in a sex ratio 0.38 (Mocarelli, et al, 2000). Currently, there is no proposed mechanism for the sex-ratio change associated with TCDD exposure.

c. MALE FERTILITY

A number of researchers have reported that the number of sperm in human male ejaculate is decreasing and that this decease is reflected by an increase in . The evidence for decreasing ejaculate sperm numbers is controversial, as regional differences have been detected within the same country (Heinze, 1999).

In 1992, it was reported that a significant global decline of about 1 % per year in sperm density occurred between 1938 and 1990 (Carlsen et al, 1992). Reanalysis of the studies examined by Carlsen controlling for abstinence time, age, percent of men with proven fertility, and method of specimen collection, found significant declines in sperm density in the United States and Europe/Australia. Results showed a decline in sperm density in the United States of about 1.5 %/year while Europe/Australia had approximately 3 %/year. No declines however were noted in

41 non-Western countries based on limited data (Swan, et al, 1997). More recently, the data set has been expanded to 101 studies from 1934 to 1996. Results from examination of this enhanced data set are consistent with those of Carlsen et al and the earlier work of Swan, suggesting that the trends reported are not dependent on the particular studies included by Carlsen and that the observed trends previously reported for 1938-1990 are also seen in data from 1934-1996. This work demonstrates that the studies initially used by Carlsen et al did not represent a biased selection of the English language literature as some had earlier suggested (Swan et al, 2000).

VII. ENVIRONMENTAL TRANSPORT OF ENDOCRINE DISRUPTIVE CHEMICALS

The widespread dispersal of endocrine disruptors (EDs) in the environment follows the same pathways as other chemicals in the environment. Dispersal occurs directly through incidental activities involved with the manufacture, transportation, use, and disposal of bulk commodity chemicals, consumer products, agricultural products and , and sewage (through metabolites in urine). Secondary dispersal is also important, and occurs through air transport. Extensive long- term use in the Great Lakes region for urban, industrial, and agricultural purposes has resulted in significant long range transport and regional inputs of land-applied chemicals, some with endocrine disrupting activities, and subsequent deposition in the waters of the Great Lakes and other regional water bodies. Airsheds can be contaminated by both long range transport and regional inputs. This, in turn, has led to contamination of the water, sediments, fish, and wildlife by a variety of persistent compounds (Simcik, et al, 1997; Pearson, et al, 1997a; Pearson, et al, 1997b; Fox, 1992; and NRC, 1999). This was recently demonstrated at Fumee Lake in the Upper Peninsula of Michigan. This lake has little direct impact from human activities, except for access by foot travel. Elevated concentrations of PCBs, DDTs, chlordane, and dieldrin in small mouth bass taken from this isolated lake are believed to be from atmospheric inputs and are similar to concentrations of these organochlorine contaminants found in finfish taken from Lake Superior. Concentrations of PCBs in Fumee Lake small mouth bass are sufficiently high to be of concern for the eagles and/or mink that consume them (Henry et al, 1998). Long-range transport of environmental contaminants has also been documented to occur from the Great Lakes to the northeast and from the mid- latitudes to Polar Regions.

IX. ALTERATIONS IN THE POPULATION DISTRIBUTIONS OF VARIOUS ENDOCRINE-RELATED PARAMETERS

One of the gravest concerns regarding exposure to endocrine-disrupting chemicals is how these chemical exposures may adversely affect the development and response of individuals in subtle ways not easily identifiable on the individual level but could be carried over to the population level by causing shifts in the population response curve to various endocrine-mediated functions as measured on continuous scales by a variety of parameters. Examples of parameters that may be adversely affected by endocrine-mimicking chemicals in animals and man are age of onset of menarche and menopause in humans (and corresponding reproductive periods in vertebrates), coordination of endocrine function, fertility, gender-related behavior, abnormal incidence of

42 disease, and response to diseases. As an example, potential adverse effects of decreasing the age at onset of puberty may lead to a shift toward earlier age at first pregnancy with resulting changes in social structure in the population, insufficient maternal physical development to carry a pregnancy to term successfully, or small-for-gestational-age infants with attendant increased neo-natal mortality. In addition, early onset of sexual maturation can have significant psychological impacts on the individual as well as psycho-social and societal impacts.

As was noted earlier, in the section on human impacts, Puerto Rico has the world's highest incidence of premature breast development and decreasing age of onset of puberty but not menarche in very young girls. While the etiology for this elevated incidence of thelarche is not known, some aspect of endocrine disruption is widely suspected. Among the environmentally associated causes investigated to date are contaminants in food such as phthalates; dietary factors, such as, consumption of soy-based diets; parental health factors, such as ovarian cysts; and estrogenic components in foods such as meat.

X. RELATIONSHIP OF ESTROGENIC SUBSTANCES TO CANCER

A. BREAST CANCER

In some cancers, estrogenically active compounds may accelerate or exacerbate the progression of cells that have been previously initiated (cancer-activated). Exposure to "environmental estrogens" in pesticides and other chemicals has been proposed as a cause of increasing breast cancer rates. Several studies, of varying quality, have reported higher blood levels of 1,1-dichloro-2, 2-bis (p- cholorphenyl) ethylene (DDE) (a metabolite of DDT), and polychlorinated biphenyls (PCBs) in patients with breast cancer than in controls (Hunter et al, 1997). A well designed study, however, using large numbers of patients, showed that median levels of DDE and PCBs were significantly lower among women diagnosed with breast cancer than controls. This study, has, however, proven controversial, and its conclusions have been questioned (vom Saal and Hansen, 1998; Talbott, et al, 1998; and Gammon, et al, 1998). A study by Gammon et al, 2002 reported few associations between the risk of breast cancer and serum levels of environmental toxins, such as DDT, DDE, PCBs, chlordane and dieldrin, measured in women living on Long Island, NY at the time of diagnosis. The study, however, had significant limitations, which weakened the ability to draw the policy-based conclusion regarding the potential role of endocrine disrupting contaminants. The conclusions are cautionary statements, such as, "These data do not rule out the possibility, however, that breast cancer risk is elevated by high organochlorine exposures several decades earlier that, through variation in individual metabolism, now measure as low body burden levels." The possibility that breast cancer risk may be elevated by exposure to organochlorine exposures several decades earlier, is at the heart of the endocrine disruptor hypothesis. Exposure during critical times in development may cause developmental errors, which in the present case may increase the likelihood of cancer later in life.

Work by Warner et al, 2002 demonstrated that significant links exist between dioxin exposure and breast cancer in Seveso, Italy. Exposure to TCDD occurred as the result of an industrial explosion

43 in Seveso that resulted in the highest known population exposure to TCDD. The women studied ranged in age from infants to 40 years old in 1976 and resided in the most contaminated areas resulting from the explosion. The Seveso Women's Health Study (SWHS) cohort is comprised of 981 women whose sera was collected and archived soon after the explosion. For each woman's serum, TCDD exposure was measured using high-resolution mass spectrometry. Cancer cases were identified during interview and confirmed by medical records. At time of interview, 15 women (1.5%) had been diagnosed with breast cancer and had serum TCDD levels ranging from 13 to 1960 parts per trillion. The median value was between 15 and 20 ppt for 11 pooled serum samples collected from women residing in an unexposed zone at the time of the explosion. Cox proportional hazards modeling showed the hazard ratio for breast cancer associated with a 10-fold increase serum TCDD levels was significantly increased to 2.1 (95% confidence interval, 1.0 - 4.6). It was concluded that individual serum TCDD is significantly related with breast cancer incidence among women from the SWHS cohort. Therefore, probability of breast cancer decades after accidental exposure was much more elevated in women that had been exposed to elevated TCDD levels resulting from the explosion, as evidenced by their increased serum TCDD levels measured in serum samples taken shortly after exposure.

B. PROSTATE CANCER

Benign prostatic hyperplasia, the abnormal multiplication or increase in the number of normal prostate cells and hence an increase in size of the prostate gland, is very common in men over 50. The incidence of prostate cancer in the U.S. population is increasing. African-American males have approximately a 60 % higher baseline prostatic cancer incidence rate in the U. S. than corresponding white males. During 1973 to 1988, the incidence rate for prostate cancer increased 2x and 1.5x, in white and African American males, respectively. From 1988 to 1992 the incidence rates further increased an additional 1.7x and 1.8x, for whites and blacks, respectively. Overall, these 1992-93 prostate cancer rates correspond to 3.3x and 2.4x the 1973 prostatic cancer rates for whites and blacks, respectively. From 1992-93 to 1995, the incidence rates for both whites and blacks decreased about 30 % and 20 %, respectively. The 1995 data, the last year available, offer limited evidence that the rate of decrease was diminishing for white and perhaps also for black males (Ries, et al, 1998).

Since the prostate gland encircles the male urethra, prostatic hyperplasia leads to variable degrees of bladder outlet obstruction. Incomplete bladder emptying causes stasis and predisposition to infection with secondary inflammatory changes in the bladder and the upper urinary tract. Prolonged obstruction can produce hydronephrosis (distention of the pelvis and calices of the kidney with urine, with accompanying atrophy of the parenchyma of the organ), compromise renal function, and lead to calculus formation (bladder stones), as well as progressive urinary frequency, and incontinency.

Dihydotestosterone (DHT) accumulation in the prostate gland serves as the endocrine mediator of this hyperplasia of the prostate. DHT uptake may be enhanced by an increased binding of androgen. However, the process is accelerated by estrogens, which enhances the level of androgen receptors in the gland (Hadley, 1996).

44 The American Cancer Society projects 220,900 new prostate cancer cases and 28,900 deaths from prostate cancer in the United States during 2003. One in six men will be diagnosed with prostate cancer during his lifetime and 1 in 32 will die from the disease. African-American men are more likely to have prostate cancer and die from it than white or Asian men. Eighty percent of these prostatic cancers are androgen-dependent. That is, androgens, such as testosterone, allow the cancer to proliferate (Hadley, 1996). Castration and pharmacological doses of estrogen are treatments that have been used to lower androgen levels. Castration is not acceptable to many men and cardiovascular disease may ensue from the high doses of estrogens. Hence, exposure to environmental estrogens and/or androgens by these susceptible populations of men may have profound adverse consequences (Earlier, data were presented demonstrating that exposure to low doses of estradiol or DES during fetal development in mice resulted in increases in prostate weight, whereas, high doses resulted in decreased prostate weights). It has been hypothesized that the increased frequency of prostatic cancer is related to such exposures although other explanations are possible, such as better detection and diagnosis.

Fertility of 354 patients with prostatic cancer and 597 controls was examined as part of a population-based case-control study in Alberta, Canada. The mean fertility of patients was not significantly different from the mean for the control group. The sex ratios were significantly different, however. Fifty-six percent of the patients fathered sons compared with 50.1% for the control group. This difference in sex ratio remained after adjusting for ethnic group, level of education, and age at first marriage, which were found to be risk factors for prostatic cancer (Hill et al, 1985). In conjunction with studies discussed previously, this suggests that disruption in hormone balances (in this case, due to disease) can effect sex ratio among newborns.

XI. OTHER MALE EFFECTS

The incidence of testicular cancer, cryptorchidism (a developmental defect characterized by failure of the testes to descend into the scrotum), and hypospadias (developmental defect in the male in which the urethra opens on the underside of the penis or on the perineum) has been shown to be increasing in young males during the 1960s, 1970s, and 1980s (Coleman et al, 1993, Forman and Moller, 1994, Bergstron et al, 1994, as cited by Weidner et al, 1998). More recent data from countries participating in the International Clearinghouse for Birth Defects Monitoring Systems indicate hypospadias increases were most marked in two American systems and in Scandinavia and Japan. Increases leveled off in many systems after 1985 and increases were not seen in less affluent nations. Clear increases in cryptorchidism were seen in two U. S. systems and in the South American system, but not elsewhere (Paulozzi, 1999). Exposure to estrogen during pregnancy resulted in increased incidence of cryptorchidism and hypospadias in male offspring of laboratory animals (Grocock et al, 1988 and Vorherr et al, 1979, as cited by Weidner et al, 1998). Parental occupation in the farming and gardening industry was investigated in a Danish case-control study where 6,177 cases of cryptorchidism, 1,345 cases of hypospadias, and 23,273 controls, born from 1983 to 1992 were investigated. Risk of cryptorchidism (but not hypospadias) was significantly increased in sons of women working in gardening (adjusted odds ratio = 1.67; 95% confidence interval, 1.14-2.47). The risks were not increased in sons of men working in farming or gardening. The authors suggest the increased risk of cryptorchidism among sons of female gardeners could

45 suggest an association with maternal exposure to occupationally related chemicals (Weidner et al, 1998).

The contribution of maternal diet to the incidence of hypospadias was examined in 7,928 boys born to mothers taking part in the Avon Longitudinal Study of Pregnancy and Childhood. There were 51 hypospadias cases. There were no significant differences in the proportion of hypospadias cases among mothers who smoked, consumed , or in any aspect of their previous reproductive history (such as previous pregnancies, number of miscarriages, use of contraceptive pill, time to conception, and age at menarche). Hypospadias incidence was determined to be statistically significant in mothers who practiced vegetarianism during pregnancy versus controls who did not practice vegetarianism or take iron supplements (adjusted odds ratio of 4.99 (CI, 2.10 - 11.88) and with omnivores who supplemented their diet with iron (adjusted odds ratio 2.07 CI, 1.00 - 4.32 (North and Golding, 2000). These results raise the possibility that plant-based endocrine disruptors in the diet can increase the risk of hypospadias.

XII. ROLE OF GOVERNMENT - FEDERAL, STATE, AND INTERNATIONAL ORGANIZATIONS

A. FEDERAL GOVERNMENT

Since 1996, appropriate agencies within the federal government have made a concerted effort in developing and implementing a comprehensive action plan that identified testing programs designed to provide credible answers for the many questions involved with the endocrine disruptor issue. The Food Quality Protection Act and the Safe Drinking Water Act amendments of 1996 required the EPA to develop screening and testing strategies to determine whether certain substances may have hormonal effects (i.e., potential endocrine disruptors) in humans by August, 1998, implement screening and testing by August, 1999, and report progress to Congress by August, 2000.

The EPA's Office of Prevention, Pesticides and Toxic Substances (OPPTS) established an Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) in 1996 (U.S. EPA, OPPTS, 1999b). Membership in EDSTAC is composed of approximately 40 representatives from the following stakeholder groups: the U.S. Environmental Protection Agency; other Federal Agencies; State/Public Health Organizations; Chemical, Pesticides, and Consumer products industries; other small companies, worker protection/labor groups; National Environmental/Environmental Justice/Public Health Groups; and research scientists. EPA's Office of Pollution Prevention and Toxic Substances (OPPTS) took the lead for the EPA on the endocrine disruptor issue. The Office of Water is involved in endocrine disruption issues as part of its responsibilities for implementing the Safe Drinking Water Act and the Office of Research and Development (ORD) is taking a leading role in determining the research needs related to endocrine disruption for all federal agencies. Other federal agencies involved are the Department of the Interior; Fish and Wildlife Service; the National Biological Service, Department of Health and

46 Human Services; Food and Drug Administration; National Toxicology Program; Center for Disease Control; National Center for Environmental Health; National Institute for Environmental Health Sciences; National Institute for Occupation Safety and Health; Agency for Toxic Substances and Disease Registry; and Department of Agriculture (Keystone, 1997).

The EDSTAC proposed a scheme for screening almost 87,000 chemicals and mixtures for potential effects on the endocrine system in early February, 1998. The panel's draft recommendation proposed the testing of most synthetic chemicals in certain classes, except for drugs, for interference with thyroid, estrogen, and androgen hormones in both humans and wildlife. In August 1998, EDSTAC's finalized recommendations for a screening program were issued before it disbanded. EPA then used these recommendations to design the Endocrine Disruptor Screening Program (U.S. EPA, 2001b). The scheme proposes three parts that would rely on automated and relatively inexpensive screening tests to test the ability of chemicals to bind to estrogen, androgen, or thyroid receptors. These chemicals would also be tested in short-term assays on live animals, such as fish, frogs, and rats. Any chemical with equivocal or positive results in short-term tests would be tested in more detailed tests, including a two-year, two-generation study in rodents. If one or more short-term assays and a two-year study demonstrated adverse effects the chemical would be considered an endocrine disruptor and potentially taken off the market. How these tests would be paid for is not clear, however, public funds will undoubtedly be needed to carry out this ambitious program (Hileman, 1998).

To handle the vast quantities of data generated by the Endocrine Disruptor Screening Program an Endocrine Disruptor Priority-Setting Database was developed and refined (US EPA, 2001c).

The Endocrine Disruptor Methods Validation Subcommittee (EDMVS) was formed to ensure the scientific basis of the screening tests and test validation process. This group oversees the evaluation of the screen/test's ability to achieve its stated purpose and ensuring that it can be performed reliably and consistently in different laboratories. Validation is a time and resource intensive activity. At present, EDMVS has meetings planned through March 2003 (U.S. EPA, 2002).

It is not known how data generated in screening and testing procedures will be interpreted and used in a regulatory context. Testing the huge number of chemicals being considered may generate equivocal evidence in some cases. How this evidence is evaluated and acted upon may generate controversy. As presently envisioned, the Tier One Screening Battery does not assess disruption from early developmental exposure. Consequently, EDSTAC is recommending that EPA take affirmative steps in collaboration with stakeholders to develop a protocol for a full life cycle developmental exposure screening assay that assesses embryonic exposure and evaluation of adult offspring.

As part of the screening process, the EPA conducted a feasibility study on the high throughput pre- screening process to evaluate the method's ability to indicate whether a chemical has the potential to interact with the endocrine system. The results of this study indicates that the process requires further development before implementation. Consequently, the EPA is considering other methods to serve a screening function, including a Quantitative Structure-Activity Relationship computer

47 simulation model to screen chemicals for their ability to interact with the endocrine system based on the molecular structure of the chemical (U.S. EPA, 2000).

The number of mixtures possible with a large universe of chemicals is daunting and could lead to "paralysis by analysis." To avoid this, EDSTAC recommended a priority to the kind of mixtures to be considered. They are: contaminants in human breast milk; phytoestrogens in soy-based infant formula; mixtures most commonly found at hazardous waste sites; pesticide/fertilizer mixtures; disinfection by-products; and gasoline, a complex mixture of volatile organic chemicals (EDSTAC Final Report, 1998). Some possible criteria for selection for testing, while not yet agreed to, could be Integrated Exposure/Effects, Exposure Only, Effects Only, and Specially Targeted Categories. Examples of Integrated Exposure/Effects Compartment priorities are: chemicals found in human biological samples; chemicals found in wildlife samples; highest volume chemical releases from industrial sites; commonly occurring chemicals found at hazardous waste sites; cosmetics, food additives, and related substances with FDA jurisdiction; chemicals that have a significant occupational exposure; and chemicals to which there is widespread environmental exposure (EDSTAC Final Report, 1998).

Current estimated costs for testing a chemical in the Tier 1 screening process range from $221,800 to $373,600. Tier 2 testing costs are estimated to be $1,249,500 per chemical (EDSTAC Final Report, 1998). From these figures of projected costs per chemical, it is evident that the entire endocrine disruptor testing program will be very expensive in time and money required to complete as presently envisioned in the EDSTAC report recommendations.

How will the gathered data be used? The screening data will be used to identify and characterize hazard (potential to cause harm). Integration and interpretation of the hazard data and information will be used in performing a hazard assessment. A determination of how much wildlife or humans are likely to be exposed to the chemical will be used in forming an exposure assessment. The final step in the process is the risk assessment where all the previous parts are integrated about how potentially harmful a chemical is with the likelihood that someone or something will be exposed to it. Based on the risk assessment, risk managers can then determine how best to regulate the chemical (U.S. EPA, 2001d).

EDSTAC calls for a communication and outreach strategy and recommends that the EPA be ready to address the following questions. What should be communicated? To whom should information be communicated? How should information be communicated, and when should information be communicated? In addition, consideration should be given to how this information, once obtained, could potentially impact our society, business, and industrial life. It is of the utmost importance that information generated by EDSTAC and the testing process is not misused to label chemicals as "endocrine disruptors" prior to the development of the needed evidence to support such a claim. Misuse of such information could be very damaging to economic vitality of an industry and deflect society's attention to potentially wrong chemicals of concern. A highly condensed summary of the main points of the Executive Summary of the EDSTAC Final Report may be found in the APPENDIX.

Agencies of the federal government have the relevant resources, information databases, and technical expertise available, together with input from the states, industry, and academia, to

48 determine what tests constitute a proper endocrine disruptor/mimic test battery, and to conduct the basic research and screening analyses to define and focus the recognition of specific endocrine disruption substances. However, the funding allocated for this effort is clearly inadequate for the task at hand. The overall coordination of the screening and testing effort should be guided by the agencies involved. These agencies should serve as a central gathering or repository point for the many kinds of data collected from the screening and testing activities and serve as a distribution point for the data summaries and progress reports from the many chemicals involved. The federal agencies should monitor the assessment work to ensure that the content of the reports is factual, of uniform quality, and unbiased.

Currently it is difficult to obtain information on the quantities of relevant chemicals produced and used in the United States. The manufacturers claim that much of the information is proprietary. However, the last routine comprehensive U.S. government report on the quantities of chemicals produced was in 1994. The unavailability of production should be remedied.

B. STATE GOVERNMENT

The state governments are in the best position to know those conditions unique to their state, peoples, and businesses. That is, states should be in the best position to predict likely areas and populations at-risk from endocrine disruption. They should also be able to identify endocrine disruptor substances produced, used and discharged in their jurisdictions that may have a significant local impact which may not necessarily be identified on the national level. States are in the best position to know what may exacerbate or ameliorate conditions impacting endocrine disruption within their borders. Active programs state-wide should be designed, implemented, and maintained that are capable of detecting potential problems which already exist, and recognizing the potential for problems prior to their developing.

If a state determines that it is being impacted by an adverse effect from endocrine disruption activity, it should have the first opportunity to determine what remediation efforts/options should be employed to ameliorate the situation. The federal government, however, should be available as a resource center to the states by offering expertise in a variety of basic research activities and remediation strategies and methodologies. The choice of preferred control and/or remedial methodologies and degree of effectiveness would be expected to differ from situation to situation or state to state due to differences in a variety of site specific factors, such as environmental conditions, life styles differences, economic issues, and number of people and ecological communities impacted. Due to these factors, it is quite unlikely that an appropriate strategy will be a one-size-fits-all proposition.

At present, New Jersey, DEP, DSRT is involved in a number of research projects to assess the status of endocrine disruption in New Jersey. Briefly, their titles are: a New York/New Jersey Harbor Estuary Project as part of the federal Toxic Trackdown project for Newark Bay; Development of a Method to Assess In Utero Exposure of Infants to Environmental Contaminants in conjunction with Dr. Mark Robson, Environmental Occupational Health Sciences Institute (EOHSI)/UMDNJ-RWJ Medical School/Rutgers University; and Investigation of Endocrine Disruption Effects of Atrazine on New Jersey Frogs, with Dr. Mark Robson EOHSI/UMDNJ-RWJ

49 Medical School/Rutgers University. Others academics actively engaged in endocrine disruptor research include Dr. Keith Cooper and Dr. George Lambert, EOHSI/UMDNJ-RWJ Medical School/Rutgers University. Later, more will be said concerning specific projects.

C. INTERNATIONAL

Many nation states have instituted programs to study and assess endocrine disrupters. The size of these individual programs is usually mirrored by the size and affluence of the country involved. The main international organizations involved are the World Health Organization and the Organization for Economic Co-operation and Development (OECD). OECD is an international organizational of 29 industrialized countries, predominantly from Europe. The group has been very active in the harmonization of toxicology preclinical testing guidelines and is taking a leadership role in the testing of chemicals for endocrine disruption. OECD involvement in ED started in 1996 and has increased since then. Today OECD is one of the major organizations involved in ED testing, worldwide. In February 1998, they established a Work Group on Endocrine Disrupter Testing and Assessment (EDTA) that defined an endocrine disrupter as an exogenous substance that causes adverse health effects in an intact organism, or in its progeny, consequent to changes in endocrine functions. The OECD testing scheme is tiered similar to that of the U.S. and includes initial screening endpoints such as estrogen/androgen (ant) agonism, male/female sexual development in pubertal animals to cover thyroid activity, steroid synthesis and estrogen androgen (ant)agonism. Higher tiered tests are much more involved and time consuming (OECD, 2000).

The need to structure international cooperation and frameworks for collaborative efforts to advance ED science on both sides of the Atlantic without duplication of effort was the subject of the European Union/United States Transatlantic Co-operation in Human and Environmental Health Experts Panel Meeting on Opportunities for Collaborative EU/US Research Programs conference in Ispra, Italy in 1999 (EU/US EDC Report, 1999). Some of the more achievable and critical research needs outlined included: 1) expansion of international efforts to standardize and validate screening and testing methods for EDs, 2) determine the range of normal values for critical endocrine parameters in wildlife, 3) international assessment of the status of marine mammals, 4) determination of the extent of the ED problem - what species are affected, 5) how are EDs affecting humans and wildlife, 6) assessment of what compounds are affecting organisms through endocrine mechanisms of action, 7) determination of whether current methods of studying the effects of chemicals are adequate, 8) assessment of whether the assessment of chemicals that act through an endocrine disrupting mechanism of action require different risk assessment protocols, 9)determination whether there is a causal relationship between exposure to EDs and specific health and ecological endpoints, 10) assessment of the effects of complex mixtures on the endocrine system, 11) and determination of the most sensitive responses to exposure to EDs. In addition, there was general agreement that establishment of common facilities and research support functions was necessary in order to minimize the degree of duplication, and to facilitate coordination of effects.

50 XIII. ENDOCRINE DISRUPTOR POSITIONS TAKEN BY NATIONAL SOCIETIES

A. NEHA POSITION ON ENDOCRINE DISRUPTERS (1997)

The National Environmental Health Association (NEHA) adopted a position on endocrine disruptors on July 2, 1997 (Gist, 1998). The group outlined examples of known and potential endocrine disruptors together with a list of species thought to have been affected by endocrine disruptors. Humans are included on this species list. Agents identified as potentially affecting humans in their report include polychlorinated biphenyls and associated contaminants, halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxins, diethyl-, and sex hormones.

The NEHA recommends additional research in the field of developmental biology of hormonally responsive organs, encourages inclusion of screening assays for hormonal activity in the testing of products for regulatory purposes, making reproductive effects and teratogenicity prime factors in evaluating health risks, and increasing public awareness and that of the scientific and medical communities of the presence of endocrine disruptors (Gist, 1998).

B. TERATOLOGY SOCIETY POSITION PAPER: THE DEVELOPMENTAL TOXICITY OF ENDOCRINE DISRUPTORS TO HUMANS (1999)

The Summary and Recommendations state:

"It is the position of the Teratology Society that the data presently available do not support a consensus view that chemicals present in the environment are contributing to observed increases in human developmental disorders which are potentially endocrine related. This conclusion is based on the facts that background historical data are lacking on many of these endpoints, that knowledge of geographical and time trends is very limited, and that exposure assessments have been virtually absent from the published literature. Nevertheless, there is considerable evidence that such effects have been found in some wildlife populations and in numerous, well-designed laboratory toxicology studies. Clearly chemicals with endocrine-disrupting actions have profound and predictable effects on the development of animals exposed during the perinatal period. Some of these chemicals are persistent in the environment and are present in the human body and conceptus. In particular, low level exposures to PCBs have been linked to delayed neurological development in several populations around the world (although the mode of action remains to be established). Additionally, in a number of countries, phytoestrogens are a major source of exposure to non- pharmaceutical, non-environmental based chemicals that can interact with the endocrine system. While a number of beneficial attributes to the health of adults are associated with intake of phytoestrogens, virtually no studies of potential developmental effects in fetuses and children are available. On the assumption that responses of experimental animals to chemicals predict hazard to humans, extrapolation of the experimental findings raises serious and controversial questions and indicates that concern for similar effects in humans is warranted. Therefore, the Teratology Society is also of the view that endocrine disruption remains a viable hypothesis for some of the effects

51 observed in humans and that additional research is needed to address the issue with greater certainty. Among these needs are:

Case-control studies of congenital malformations of the reproductive tract, with emphasis on potential exposures to endocrine-disrupting chemicals.

Determination of the nature and magnitude of observed geographical variations in reproductive tract malformations, and testicular cancer, and their relationship, if any, to exposure to endocrine-disrupting chemicals.

Measurement of internal dose in populations likely to be exposed to endocrine-disrupting chemicals.

Mechanistic studies to characterize the determinants of the shape of the dose-response from levels found in environmental to maximally-tolerated dosages.

Comparison of risks from man-made versus naturally-occurring endocrine-active chemicals.

Evaluation of potential interactive effects among endocrine-disrupting chemicals with dissimilar modes of action" (Barlow, et al, 1999).

C. NATIONAL ACADEMY OF SCIENCES REPORT: HORMONALLY ACTIVE AGENTS IN THE ENVIRONMENT (1999)

The National Academy of Sciences (NAS) released a report in 1999 entitled Hormonally Active Agents in the Environment. This report was produced in response to a request by the United States Environmental Protection Agency, the Department of the Interior, the Centers for Disease Control and Prevention, and the United States Congress to independently evaluate the scientific evidence regarding potential adverse human health effects of various endocrine disruptors, referred to in this report as hormonally active agents (HAAs). The work of the committee was first and foremost, a critical review of the literature on the subject and the work was divided up among subcommittees. Consensus within these groups, where achieved, was reached only by going through many iterations. As might be imagined, in some areas, notably the area of reproduction and development, including the issue of declining sperm production in human populations it was extraordinarily difficult to achieve consensus. The executive summary and other information on Hormonally Active Agents in the Environment are available on the internet at http://books.nap.edu/books/0309064198/ html/1.html#1.

Among the HAAs Committee's findings and recommendations are:

Mechanism of Action: They found that the specific mechanism of action for most associations reported for HAAs and various biologic outcomes is not well understood. The committee in general does not believe that HAAs can function only through receptor binding and recommends

52 research be conducted to distinguish between direct and indirect effects and between primary and secondary effects of HAAs.

Developmental Effects and Reproduction: Adverse reproductive and developmental effects have been observed in humans, wildlife, and laboratory animals as a consequence of HAA exposures. 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. At this time, increased incidence of human hypospadias, cryptorchidism, and testicular cancer cannot be linked to exposures to environmental HAAs. Retrospective trend analyses of human sperm concentration over the past 50 years remain controversial. How data from past studies are pooled and analyzed leads to different conclusions and there is not consensus on doing this.

Laboratory studies in rats, mice, guinea pigs, and rhesus monkeys have demonstrated that exposure during development to a variety of concentrations of HAAs (DDT, methoxychlor, PCBs, dioxin, bisphenol A, octylphenol, butyl benzyl phthalate, dibutyl phthalate, , and vinclozolin) can produce structural and functional abnormalities of the reproductive tract. Reproductive and developmental abnormalities have been observed in populations of fish exposed to effluents from sewage treatment plants and paper mills and polluted waters of the Great Lakes. Abnormalities observed include intersexes in trout, increased egg and fry mortality in trout and salmon, thyroid enlargement in salmon, and changes in plasma sex-steroid concentrations, decreased egg and gonad size, and delayed sexual maturity in white suckers. Laboratory experiments with specific HAAs found in those effluents and polluted waters have produced effects consistent with those wildlife observations. Agents producing wildlife-type effects in the laboratory include: sewage-treatment- plant effluents, ethinylestradiol, alkylphenol ethoxylates, dioxin and structurally related compounds, PCB-contaminated food, and B-sitosterol. Laboratory studies are also consistent with some reproductive and developmental abnormalities (skewed sex ratios, behavioral modifications, and morphologic abnormalities of the gonads) observed among North American gull populations. Similarly defects in alligators from Lake Apopka are consistent with structural and functional reproductive abnormalities that occur following prenatal exposure of laboratory rodents to estrogenic and antiandrogenic chemicals.

The committee recommends wildlife and human populations should continue to be monitored for abnormal development and reproduction. Wildlife populations that decline should be investigated with regard to chemical contamination. Human populations suspected of being affected by HAAs should be studied in prospective and cross-sectional studies using cohorts tracked from conception through adulthood.

Neurologic Effects: In humans cognitive and neurobehavioral studies of mother-infant cohorts exposed accidentally to high concentrations of PCBs and PCDFs and/or eating contaminated fish and other foods containing mixtures of PCBs, dioxin, and pesticides (e.g., DDE, dieldrin, and lindane) provide evidence that prenatal exposure to these HAAs can affect the developing nervous system. Monkeys similarly exposed to PCBs in utero and during lactation have cognitive function deficits, and rats and mice exposed prenatally to PCBs suffer impaired locomotor ability and learning.

53 The Committee recommends that human populations suspected of being affected by HAAs have longitudinal tests conducted on developmental milestones from conception through adulthood and that a standardized set of criteria be established to study neurobiologic and social development.

Immunologic Effects: Several HAAs affect diverse elements of the immune system in experimental animals. Evidence exists of suppression in the immune system due to 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 feeding on PCB contaminated fish in the Baltic Sea. This is believed to be the reason for increased incidences of bacterial and viral infections in seals. In humans data on immunologic effects of HAAs are inadequate to support any definite conclusions. The Committee recommendation is that 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. The immunologic activity of HAAs currently in use (e.g., endosulfan and lindane) should be investigated in laboratory studies.

Ecologic Effects: Environmental HAAs probably have contributed to declines in some wildlife populations, such as fish and birds of the Great Lakes, alligators of Lake Apopka, and possibly diseased and deformed mink in the United States, river otters in Europe, and marine mammals in European waters. Contaminants and inbreeding may have contributed to poor reproductive success of the endangered Florida panther and in the increased embryonic mortality of the snapping turtle in the Great Lakes. Biologic communities of the Great Lakes, encompassing all trophic levels from plankton to top predators, have undergone vast changes in the last century. These changes 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. The degree to which HAAs contributed to population size changes of individual species and to changes in the community structure is difficult to account for quantitatively. Epidemiologic and experimental evidence exists that persistent, bioaccumulative HAAs, (also referred to as persistent organic pollutants - POPs) have produced adverse effects on wildlife, but whether the primary effect is mediated by hormonal activity remains to be determined.

The Committee recommends that long-term studies of populations exposed to HAAs be undertaken 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 are needed to understand how chemical exposures affect long-term ecologic attributes in natural systems.

Exposure, Dosimetry, and Screening and Monitoring: Determination of the risk posed by environmental HAAs to humans and wildlife is difficult as exposure to these agents has not been routinely monitored, and effects that may be attributed to background concentrations could be complicated by endogenous hormones, pharmacologic estrogens (e.g., contraceptives containing hormones or hormone analogs), and naturally occurring HAAs (e.g., phytoestrogens) that are ubiquitous in the environment. Synthetic HAAs have been detected in all environmental media. HAAs can persist in the media for years and can spread to contaminate other areas far from the original site of contamination. Certain human populations have been found to contain relatively high exposures to HAAs, from diets that include frequent consumption of contaminated foods, especially fish, or foods containing phytoestrogens.

54

Understanding the relationships between exposure, adsorption, disposition, metabolism, excretion, and response is important for predicting whether exposure to an agent will be adverse. Once inside an organism, the transport of an HAA to potential target organs is influenced by its binding affinity to plasma proteins. Lipophilic HAAs will cross the and can have effects on the developing organism. HAAs can accumulate in adipose tissues and serve as reservoirs or depots. Differences among species and between adult and developing organisms can have important implications in assessing toxicity studies and/or in extrapolating from one species to another. Additionally, biologic responses to some HAAs may be greater at lower doses than at higher doses. There are no generally accepted, adequately validated methods for routine identification or monitoring exposures to HAAs and most of the in vitro and in vivo screening assays do not address the full range of putative actions of HAAs.

The Committee recommended better monitoring of contaminated media to determine environmental concentrations of HAAs and to assess persistence and recycling HAAs in and between various environmental media. Background concentrations of HAAs in human adipose tissue and blood, and other biota need to be established. 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 further investigation, especially at environmental concentrations. Dose-response relationships of recognized HAAs need to be investigated at those concentrations in 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 which are covered elsewhere in this paper.

XV. DISCUSSION

There are many data gaps in our knowledge of mechanism of action, low dose toxicology, environmental fate and transport, identification of Environmental Disruptor substances, and their impact on health and the environment. In a very general way, the environmental community can be divided into two camps. One camp says that policy and regulatory action (some combination of restricting the use or banning of particular chemicals for certain and/or all uses) should not be initiated until the significant data gaps have been bridged. The other camp says that society would be irresponsible to delay prudent action until all significant data gaps have been resolved. In resolving this difference of approach, it may be beneficial to reflect on how society acted with respect to cancer. Society acted slowly at first in addressing cancer as a health issue, but in the end has spent huge amounts of time, effort and resources to cure this disease. Much remains to be learned about cancer, its causes, control and cure. However, most would agree that steps taken to control the causes of cancer, including environmental cancers, have been prudent and worthwhile. This approach would also probably be justified concerning Eds as a potential environmental health problem. We should move forward in measured steps, based on what we know now. The

55 European Union/US Expert Panel noted areas for improvement in the knowledge base; these are cited earlier in this report. These recommendations provide a good framework for achieving progress in this area.

XV. NEW JERSEY-SPECIFIC RECOMMENDATIONS Based on evidence presented here, and elsewhere, it seems clear that at some level detrimental alterations have occurred and continue to occur in sectors of our environment from chemical substances that act via an endocrine or endocrine disruptor pathway. While the evidence for such effects is most pronounced and best documented in wildlife species, it is seems reasonable to conclude that analogous effects may be occurring in the human population at this time. Based on this premise, the following New Jersey-specific recommendations are presented.

A. GENERAL

It is recommended that the NJDEP lead a broad-based effort to adopt a policy for addressing endocrine disruption. This policy should be flexible in recognizing the dynamic nature of our understanding of endocrine disruption as it unfolds with continuing research. At the same time it must address specific activities within the knowledge base as it exists today and develops in the future. The policy should involve components that support continuing ED-related projects, surveillance where indicated, keeping current with results of endocrine disruptor screening research programs, and additional research initiatives where appropriate. One way this could be facilitated is establishing an Endocrine Working Group within New Jersey to set up a proposed course of action ; membership could include faculty from colleges and universities, experts as appropriate from NJ’s chemical and pharmaceutical companies, and environmental groups, and representatives from NJDEP and the Department of Health and Senior Services..

B. INDICATOR DEVELOPMENT/IMPLEMENTATION

At present there is no human breast-milk monitoring program in the United States. New Jersey could lead the way in establishing such a program that would establish a means of estimating body burdens of persistent organic pollutants (POPs) in mothers and breast-fed infants or children. Data derived from this program would provide baseline information on body burdens for women during the sensitive infant developmental period, and with the use of breast milk consumption data, provide noninvasive estimates of POPs levels in infants and children. Further, a breast-milk monitoring program would help identify hot spots of POP contamination and congener patterns to help determine the sources of the contaminants. Additionally, it would help to identify at-risk populations of mothers, infants, and children for follow-up interventions and health outcome studies. Time-trend data can serve as a useful and effective indicator of potential environmental health effects, can identify new POPs of emerging concern, can help assess the effectiveness of regulatory strategies to limit exposures to POPs, and would complement other air, soil, and water monitoring activities.

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C. ENVIRONMENTAL SURVEILLANCE

Establishment of an environmental surveillance project is recommended for locating ED chemicals in ground and surface waters in the state; chemicals would be prioritized/ranked by concentration and potency, as identified by the EDSTAC screening process or peer-reviewed scientific literature. Such chemicals may have originated from natural sources, or from industrial, agricultural, or residential activities, including discharges from waste treatment plants.

Such surveillance activities should first occur in areas where chemical concentrations in the environment are most likely to be elevated either from chemical manufacturing activities or chemical usage. Identification of the potential high ED chemical usage areas may be aided with the use of the Toxic Release Inventory information. Some areas may be waterways that are impacted by either current or historical high industrial activity or high population density, e.g., the Newark Bay complex; areas where water reuse is high which may tend to concentrate chemicals in the water; and/or where sewage treatment plant discharges and drinking water treatment plant intakes are in close proximity to each other, e.g., Passaic River basin, and/or areas where runoff may impact surface or ground water (golf courses, agricultural fields). While the discharges from point sources are more obvious, discharges from non-point sources may be at least as or even more important.

Examples of potential candidate chemicals for such surveillance might include pesticides (e.g., atrazine), components of personal care and cleaning agents (e.g., nonylphenol polyethoxylates), and over the counter (OTC) drugs (e.g., nonsteroidal anti-inflammatorys - aspirin, ibuprofen, etc.) or excreted pharmaceuticals or their metabolites (e.g., cholesterol lowering compounds or hormone preparations), and/or components from consumer goods (e.g., flame retardants - ethers).

The NJDEP Division of Science, Research, and Technology (DSRT) has examined the presence of some non-regulated chemicals in New Jersey waters. This effort was not specifically targeted toward ED substances and consequently cannot provide a complete picture of the presence of ED chemicals in drinking and source waters statewide (Rosen et al, 1995; Marhaba and Lippincott, 2000; Marhaba, 2000; Marhaba et al, 2000a; Marhaba et al, 2000b). This work should be expanded to include additional water resources of the state. More recently, the U.S. Geological Survey reported on a pilot sampling of pharmaceuticals, hormones and other organic wastewater contaminants in 139 sites in U. S. waters. These results included four sites in New Jersey (Kolpin et al, 2002). These efforts were preliminary and as such were not designed to detect all groups of potential ED chemicals.

Anomalies in biota noted in the wild, like those reported in a statewide monitoring program by the Bureau of Fresh Water and Biological Monitoring, should be investigated as to their cause. In this program, malformed antennae and mandibles (mouthparts) were noted as being significantly elevated in benthic invertebrates known as chironomids at certain sampling locations (NJDEP, 2002). These small macroscopic animals inhabit sediment layers in rivers and streams. These abnormalities have been noted in particular locations at repeated five-year intervals. The question

57 that should be investigated is whether such malformations are caused by a toxic (potentially endocrine disrupting) pollutant or by some genetic abnormality.

ENDEX, the Environmental Data Exchange, is a NJDEP Internet-based clearinghouse initiative, fashioned to enhance and support environmental management decision-making. This will create a forum for information sharing that will combine the advantages of the Internet with the NJDEP Geographic Information System. Once complete, ENDEX will centralize integrated data collection, analysis, and data sharing among New Jersey's environmental community. This statewide data reporting and analysis database will allow funneling of pertinent information concerning potential shifts in wildlife populations, incidence information on malformed or deformed species, and diseased aquatic and terrestrial species so the appropriate technical people and policy makers in government are kept informed of trends and developing problems (NJDEP, 2000). ENDEX should, therefore, be actively used to identify likely areas of significant ED chemical occurrence in NJ, and likely areas of ecological and human population impact.

D. RESEARCH

1. A research effort should be supported for the improved detection of endocrine disruptor chemicals in complex environmental matrices like soil, sediment, biota, and water. In these environmental matrices there are often large numbers of substances that can act as interferences with the identification of the target chemicals. This interference can be due to many different factors, such as the presence of other chemicals that act to mask or co-elute with target chemicals, and/or physical interference due to physical adsorption on sediments. Efforts in improving ED detection should be encouraged and supported at NJ research institutions so as to ensure that the analytical capabilities necessary to carry out New Jersey-specific assessments are available.

2. The NJDEP Division of Fish & Wildlife has conducted studies on the effects of organochlorine contamination on reproduction in eagles and ospreys in the Delaware Bay. The purpose of these studies is to determine the effect of contaminants on wildlife species; they should be continued and possibly expanded to other species.

3. Increased emphasis needs to be placed on the ultimate environmental fate of ED chemical discharges from all sources (industrial and non-industrial) entering our waterways and environment. This will require additional information on the environmental chemistry of a pollutant not only as initially discharged into the environment, but also how it may degrade and interact with other chemicals in the environment including during its passage through public and industrial wastewater treatment plants. A heightened awareness needs to be placed on determining the ecological ED properties of so-called inert ingredients, such as dispersants and emulsifiers, in formulations of pesticides and industrial and consumer products as they may have more profound ecological effects than the so-called active ingredients in certain products.

4. The effect of the concentrations of potential ED contaminants on sensitive sentinel species in NJ such as fish and amphibians needs to be investigated and evaluated. Some surface waters in the state are very heavily utilized. During low flow conditions in the summer and under drought conditions, the majority of the Passaic River flow is probably derived from sewage treatment plant

58 discharges. What is the cumulative effect on contaminant loading of endocrine disruptor chemicals as water moves downstream one community after another, or one industry after another discharging additional contaminants into the same water? Monitoring of endogenous sentinel species throughout the state should be inaugurated. If not monitored, the changes or shifts in populations that may have already occurred and the gender makeup within these populations cannot be known.

5. While a great deal of information is available on high dose effects of toxic compounds, basic research is needed on the biologic mechanisms involved at low concentrations of synthetic chemicals found in the environment for long durations of time that are typical of the environmental exposures that aquatic and wildlife species experience. Educational institutions in the state should be encouraged to increase investigation efforts in this area in direct collaboration with State government.

D. OTHER ACTIVITIES

1. It would be prudent to establish an endocrine disruptor resource directory. This directory should contain contacts of government, industry, and academia people with expertise on endocrine disruptor chemical issues. The directory would be provided to appropriate program staff and managers.

2. Monitor the federal activities concerning the endocrine disruption issue (EDSTAC) and adjust State action plans accordingly.

3. EPA is required by law to maintain a list of contaminants that may in the future be considerated for regulation. This list is known as Contaminant Monitoring List for drinking water that currently contains 35 chemicals that must be monitored by public water systems. They are considered "unregulated contaminants" since EPA has not established drinking water standards for them. Monitoring for another list of 12 chemicals established by EPA is voluntary at the discretion of the State. It is recommended that New Jersey add the other 11 of these 12 chemicals to its existing list (one of the 12, methyl tertiary butyl ether or MTBE, a gasoline additive is already regulated by NJ) of required testing as well and other such chemicals that are also detected by the same required analytical methods but not tracked electronically and/or not interpreted. Of the other 11 chemicals on this list, three are known to have endocrine-disrupting effects. They are acetochlor and perchlorate, which have thyroid effects and 4,4'-DDE, which has antiandrogenic effects.

XVI. EXAMPLES OF RECENT AND ONGOING ENDOCRINE DISRUPTOR RELATED NEW JERSEY RESEARCH PROJECTS

A. NJDEP, Division of Science, Research & Technology

59 1. New York/New Jersey Harbor Estuary Project

As part of the federal Toxic Trackdown project for Newark Bay, several tributaries that feed into the bay were monitored with PISCES units. PISCES units are passive water monitoring units consisting of a brass enclosure with a semipermeable membrane on one side. The units contain hexane and are deployed at geolocated reference points of interest for two weeks. Organic constituents in the water where these passive monitoring units are placed can pass through the semipermeable membrane and become dissolved in the hexane. When recovered, the hexane is extracted and analyzed by state-of-the-art analytical methodology for chemical classes of PCBs, pesticides, PAHs, and dioxin congers. Upon agreement between Dr. George Lambert of Rutgers/ EOHSI, and Dr. R. Lee Lippincott of NJDEP/DSRT, two PISCES extracts from two locations on the Raritan River were evaluated for endocrine disruption potential using Dr. Lambert's Yeast Estrogen Screening (YES) bioassay. Initial results for these samples show they contain no alpha estrogen receptor activity but significant beta estrogen receptor activity using the YES screening bioassay (Lippincott, 2001). The exact relevance of various alpha and beta estrogenic receptors is too new to be completely understood. Further, there is a lack of understanding of which chemicals will interact with which particular estrogenic receptor or why/how this will occur. Undoubtedly in the future much more remains to be learned about the mechanism and energetic of these chemical/receptor interactions.

2. Development of a Method to Access In Utero Exposure of Infants to Environmental Contaminants

A pilot range-finding study is currently underway in conjunction with EOHSI (Dr. Mark Robson), and St. Peter's Hospital in New Brunswick to provide data on levels of several potential endocrine disrupting chemicals in various tissue/media collected at birth that are indicative of gestational exposure. Such data have not previously been reported. This study has the potential for the development of reference (i.e., normal) levels of fetal exposure, and indicators of elevated exposure among specific populations.

3. Investigation of Endocrine Disruption Effects of Atrazine on New Jersey Frogs

In response to recent laboratory-based reports that extremely low level exposure to atrazine can result in feminization of male frogs, a field study is currently underway in NJ to determine whether frogs in ponds receiving atrazine from high-use agricultural applications have abnormalities predicted from the laboratory studies. This study is being conducted collaboratively between DSRT and EOHSI (Dr. Mark Robson).

B. Rutgers University, Environmental and Occupational Health Sciences Institute (EOHSI)

Dr. Keith Cooper is evaluating the capacity of environmental agents to disrupt developmental processes in Fundulus and the Japanese Medaka (Oryzias latipes).

60

Dr. George Lambert and his laboratory staff are developing non-invasive biomarkers for humans that are a measure of exposure to environmental agents, including endocrine disruptors.

In utero exposure to endocrine disruptors, and atrazine effects on frogs in high-use areas (Dr. Mark Robson). See NJDEP/DSRT research section.

C. Princeton University

A Princeton University senior thesis project, modeling mass balance of chemicals and domestic pharmaceutical use in New Jersey waters, indicates a potential problem with current assumptions (personal communication with Roselle Safran, 1999). At the present time, links to potentially applicable research, and channels for collaborative research in this area with Princeton University could likely be expanded to the mutual benefit of the State and the University.

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89 XVIII. APPENDIX I - GLOSSARY

Ah receptor, a gene regulatory protein, involved in cell signaling pathway, that xenobiotics like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds PCBs etc. (that are potent immunogenic, reproductive, and developmental toxicants) bind to, to inappropriately modulate gene expression. Through this binding ability these compounds alter gene expression and affect cell proliferation and differentiation. It is not known which tissues are most sensitive to these compounds and how this sensitivity varies during critical developmental periods (Gasiewicz, 2002).

An agonist is a chemical that has an affinity for and stimulates physiologic activity at a cell receptor, which is normally stimulated by another naturally-occurring substance (Dorland's, 1988).

Androgens are substances, such as the testicular hormone testosterone, that are conducive to masculinization. In males, it is responsible for male secondary sex characteristics, such as a masculinized body build, minimal distribution of fat over muscles, minimal indenting of waist, increased growth of body hair on the groin, arms, chest, back, and face, and lowering of the voice (Dorland's, 1988).

An antagonist is a substance that inhibits the action of another substance. Competitive antagonists are substances which compete with another substance for binding to a cell receptor or to a specific site on an enzyme and in so doing inhibit the action or metabolism of that substance. Competitive antagonists are often, but not always, a physical analogue of the substance with which they compete.

Bioaccessibility, in animals, refers to the solubility of a chemical in the liquid phase of the .

Bioavailability is a measure of the potential for a chemical to enter into either ecological or human receptors. For consideration of EDs, it is specific to the receptor, the route of entry, time of exposure, and the matrix containing the contaminant. Entry into receptors, ecological or human, normally requires satisfying three conditions: Opportunity, exposure of the receptor to the matrix in which the contaminant resides; Potential Availability, the fraction of the contaminant that is not irreversibly sequestered or bound to the matrix in which it resides; and Assimilative Capacity, the ability of an organism to assimilate the potentially available fraction of the contaminant. No bioavailability exists, and therefore no contaminant exposure and risk occur without satisfying all three of these conditions (Reible, 2002).

Endocrine means secreting internally; applied to organs and structures whose function is to secrete into the blood or lymph a substance (hormone) that has a specific effect on another organ or part (Dorland's, 1988).

The endocrine system is the system of ductless glands that secretes into the circulatory system biochemical substances called hormones which coordinate and control many basic developmental, reproductive, and other body processes. Organs having endocrine function include the

90 , pituitary, thyroid, parathyroid, adrenal glands, the gonads, the pancreas, the paraganglia, and perhaps the pineal body. The gut and the lung also have endocrine functions (Dorland's, 1988).

Estrogen is a generic term for estrus-producing steroid compounds, usually referred to as female sex hormones. However, their activity is not confined to females. In humans, estrogen is formed in the ovary, adrenal cortex, testis, and fetoplacental unit. Estrogen has various functions in both sexes. In females, it is responsible for the development of female secondary sex characteristics, such as breast development and indenting of the waist at puberty. During the menstrual cycle it acts on the female reproductive tract to produce an environment suitable for the fertilization, implantation, and nutrition of the early embryo (Dorland's, 1988).

Ex vivo means outside the living body, refers to removal of an organ for reparative surgery or other testing procedures, after which it is returned to the original site (Dorland's, 1988).

Homeostasis is a compensatory tendency towards stability in the normal body states of an organism. Homeostasis is achieved by a system of sophisticated integrated control mechanisms activated by positive or negative feedback, e.g., a high level of carbon dioxide in extracellular fluid triggers increased pulmonary ventilation, which in turn causes a compensatory (homeostatic) decrease in carbon dioxide concentration (Dorland's, 1988).

Hormone feedback is a regulatory control mechanism that promotes homeostasis in the organism. Secretion of many hormones is regulated at physiologic levels through negative feedback, wherein elevated hormone concentrations inhibit further hormone secretion.

Hormones are biochemical messengers secreted by one cell type into the blood stream where they travel some distance to another cell type where they bind to specific specialized sites on those cells called receptors. Once bound to receptor sites, hormones trigger various activities in these cells. Hormones may be delivered by endocrine, neuroendocrine, neurocrine, paracrine, or pheromonal routes. General classes of hormones include peptide, thyroid, steroid, neurotransmitter, neuropeptide, chalone, peptide growth-stimulating factors, and pheromones. All hormones are not found in common among all species of animals; however, many are, and cross-species hormone activity is not uncommon. The hormonal activity that is shared between species may be of equivalent activity or may be different. There are about 82 known vertebrate hormones, of which at least 75 are found in man. (Hadley, 1996).

The immune system is a complex system of cellular and molecular components with the primary function of distinguishing self from not self and defense against foreign organisms or substances in the body (Dorland's, 1988).

In situ, in the natural or normal place; confined to the site of origin without invasion of neighboring tissues.

In vitro tests are conducted outside an organism in an artificial laboratory environment but may employ cells, biological fluids, and other components of organisms.

91 In vivo tests are conducted in whole, intact, living organisms. In vivo tests at some level are usually preferred in safety assessment testing when the results need to be extrapolated to intact animals, such as man.

MCF-7 human breast carcinoma cell line, stands for Michigan Cancer Foundation, seventh trial , for establishing a self-perpetuating cell line from a woman with breast cancer. MCF-7 cells are used extensively in in vitro tests to determine which test chemicals either accelerate or retard growth of these cancerous cells and aid in the understanding of the basic mechanisms involved in cancer.

The nervous system is an extensive organ system which, along with the endocrine system, coordinates the adjustments and reactions of an organism to internal and environmental conditions (Dorland's, 1988).

Neurocrine denotes an endocrine influence on or by the nerves, pertaining to a neurosecretion (Dorland's, 1988).

Neuroendocrine refers in particular to the interaction between the nervous and endocrine systems and to hormones synthesized in the nervous system and ultimately secreted by an endocrine gland, such as vasopressin (Dorland's, 1988).

Odds ratio of a disease is the ratio of the odds that the disease will develop in an exposed person to the odds that it will develop in a nonexposed person (also called the cross products ratio) (Tulloch, 2001).

Paracrine denotes a type of hormone function in which a hormone synthesized in and released from endocrine cells binds to its receptor in nearby cells and affects their function. Paracrine also denotes the secretion of a hormone by an organ other than an endocrine gland (Dorland's, 1988).

Pheromones are substances secreted outside the body by an individual and perceived by smell or olfaction by a second individual of the same species causing a specific behavioral reaction in the percipient (Dorland's, 1988).

The sometimes referred to as the master gland, is about the size of a pea in humans, located at the base of the brain. In man, it consists of two lobes, the anterior lobe and the posterior lobe. The anterior pituitary lobe secretes Thyroid Stimulating Hormone (TSH), two - Follicle Stimulating Hormone (FSH), and Luteinizing Hormone (LH), Prolactin (PRL), Growth Hormone (GH), Adrenocorticothrophic Hormone (ACTH), and Alpha Melanocyte Stimulating Hormone. The posterior pituitary lobe secretes Antidiuretic Hormone (ADH) and Oxytocin.

Trophic hormones stimulate release of other hormones, e.g., TSH, LH, and FSH.

The uterotrophic assay is traditionally used to test for the estrogenicity of a substance. The uterus contains a high concentration of estrogen receptors making it one of the most sensitive tissues to estrogen. When exposed to estrogen, the uterus demonstrates two basic responses: it swells by influx of water into the lumen of the uterus, and it increases in weight by growth of the endometrial

92 layer. In this assay, ovariectomized mice (mice with the ovaries removed) are dosed with a test compound. Several days later the uteri are removed and weighed. Changes in the uterus to body weight ratios, compared to control animals, are taken as an indication of estrogenic or anti- estrogenic effects.

93 XVIII. APPENDIX II - SUMMARY OF THE ENDOCRINE DISRUPTOR SCREENING AND TESTING ADVISORY COMMITTEE FINAL REPORT AND RECOMMENDATIONS

This is a condensed summary of the main points of the Executive Summary of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) two volume Final Report, released August 1998.

I. Introduction

The EDSTAC was formed due to concerns of the presence of endocrine disruptors in food, water, or other environmental media, and the potential risk they pose to humans and wildlife. In 1996, the Food Quality Protection Act (FQPA) and Amendments to the Safe Drinking Water Act (SDWA) required the U.S. EPA to develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator may designate.

The term endocrine disruptor the EDSTAC report describes, rather than defines, as an exogenous chemical substance or mixture that alters the structure or function(s) of the endocrine system and causes adverse effects at the level of the organism, its progeny, populations or subpopulations of organisms, based on scientific principles, data, weight-of-evidence, and the precautionary principle.

II. Endocrine Disruptor Screening and Testing Advisory Committee

A. EDSTAC formation and Structure

The EPA EDSTAC committee was composed of individuals representing stakeholder groups and scientific expertise. Members consisted of scientists and other representatives from the EPA, other federal and state agencies, various sectors of industry, water providers, worker protection organizations, national environmental groups, environmental justice groups, public health groups, and research scientists.

Scope

The primary scope of the Committee was to develop recommendations for a screening and testing program for endocrine disrupting chemicals. The EDSTAC interpreted this to include not only the FQPA and SDWA provisions, but also those of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), The Toxic Substances Control Act (TSCA), and the Federal Food, Drug, and Cosmetic Act (FFDCA). As a result the EDSTAC recommended the scope of work should: Address both human and ecological effects: It should include screening for adverse effects to wildlife, as well as humans, as the overall well-being of wildlife can be an indication of the overall health of the environment in which humans live.

94 Examine effects to estrogen, androgen, and thyroid hormone-related processes. These three primary hormone systems were recommended because they are important hormones in both humans and wildlife with a large body of relevant data, screening assays, and tests from which to select.

Evaluate endocrine-disrupting properties of both chemical substances and common mixtures. The universe of chemicals to prioritize for endocrine disruptor screening numbers more than 87,000. This list includes chemicals listed in the TSCA Inventory, active pesticide ingredients, commodity chemicals, naturally occurring non-steroidal estrogens, food additives, cosmetics, and nutritional supplements. In addition, EDSTAC recommends testing six distinct types of mixtures to determine whether they have endocrine effects, different from those of the individual component chemical, which can only be detected when tested as a mixture.

III Chapter Three - Conceptual Framework and Principles

A. Conceptual Framework Overview

The EDSTAC recommended a tiered approach to be most effective in utilizing available resources to detect endocrine-disrupting chemicals and quantifying their effects. The key elements of the tiered approach include: initial sorting, priority setting, Tier 1 Screening (T1S), and Tier 2 Testing (T2T).

Initial Sorting. Using available data, chemicals would be sorted into one of four categories. The first would lead to a "hold box" for those chemicals not likely to interact with the estrogen, androgen, and thyroid hormone systems and no further action would be taken on this group. The second category is chemicals without sufficient data to make a determination to proceed to T2T or hazard assessment. These chemicals enter the priority setting and the T1S portions of the Endocrine Disruptor Screening and Testing Program. Most chemical substances and mixtures entering the program are anticipated to enter this category. The third category contains chemicals with sufficient existing data to move directly to T2T. The fourth category is chemicals with sufficient existing data to move directly to hazard assessment. The program also has a voluntary bypass scenario whereby the owner of a chemical could voluntarily go to T2T without completing the full T1S battery of tests.

Priority Setting. This refers to the need to set priorities for the chemicals that move into the second category, T1S. Prioritization is to be based on exposure-related information, effects-related information, and statutory criteria, and then phased into the program so as not to choke the available limited laboratory testing and screening resources.

Tier 1 Screening. T1S is designed to detect chemicals and mixtures capable of interacting with the estrogen, androgen, and thyroid hormonal systems (EAT). Completion of this stage will result in a decision to move the chemical into T2T or an indication that no further testing is necessary. In the latter case, the chemical would proceed to the "hold box."

Tier 2 Testing. T2T will determine whether a chemical or mixture exhibits endocrine-mediated adverse effects and to identify, characterize, and quantify those effects for EAT hormones. If no endocrine-mediated adverse effects are observed, the chemical would move to the "hold box." If

95 effects are observed, the information collected during testing would be used in the Hazard Assessment process.

"Hold Box. This term is used in the EDSTAC report to mean that either no or no further screening and testing are necessary. As the testing process continues EDSTAC has set forth criteria for periodic evaluations of chemicals and mixtures in the "hold box" to determine whether new or additional screening and testing may be necessary.

B. Recommended Principles and Guidance

EDSTAC provided several sets of principles to guide the development of the screening and testing program including:

Provisions to Bypass Tiers. When information warrants such a move, a chemical substance or mixture could bypass one or more testing tiers.

Proactive Effort to Generate Adequate Information. When existing information is inadequate to determine whether a chemical or mixture should proceed to the next tier, there should be an active process for generating the needed information.

Moving Through the Program. Criteria and assumptions for deciding when a chemical should move from one tier to the next should be in place prior to initiation of screening and testing.

EDSTAC also provided principles to -guide overall development of screening and testing; -guide decisions for selection of screens and tests; and, -guide the design and use of the T1S and T2T battery.

IV. Chapter Four - Priority Setting

As an aside: The universe of chemicals that require testing exceeds 87,000 and EDSTAC acknowledges the testing program will be heavily driven and constrained by the EPA's statutory authority. (This is not reflected in the Executive Summary.)

A. Initial Sorting and Phased Approach to Screening and Testing the Universe of Chemicals

Due to the large number of chemicals involved and for testing considerations the EDSTAC recommends a phased approach. This approach identifies high priority chemicals and permits them to proceed through the program first, followed by medium priority chemicals, and then low priority chemicals. EDSTAC recommends the chemicals initially be sorted into the following four categories

1. Chemicals (primarily polymers with a molecular weight greater than 1,000 Daltons, which are unlikely to cross biological membranes and hence unlikely to be available to cause endocrine- mediated effects - estimated to be 25,000 polymers) that are unlikely to have endocrine disrupting effects that enter the "hold box.

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2. Chemicals with insufficient data that will undergo High Throughput Pre-Screening and T1S and possibly T2T.

3. Chemicals with sufficient data to bypass T1S and go directly to T2T.

4. Chemicals with sufficient data that will to go directly to hazard assessment.

EDSTAC estimates that 25,000 polymers greater than 1,000 Daltons will meet Category 1 criteria. Additionally, EDSTAC estimates the number of chemicals that fall into Categories 3 and 4 will not exceed 1,000. The remaining 62,000 chemicals will need to be considered for screening and testing, if necessary.

B. Information Useful for Prioritization

Exposure- and effects-related information will be used to prioritize chemicals for T1S. Exposure- related information includes: biological sampling data for humans and other biota; environmental, occupational, consumer product, and food-related data; data on environmental releases; production volume; and fate and transport data and models.

Sources of data on effects-related information are: toxicological laboratory studies and databases; epidemiological and field studies and databases; predictive biological activity or effects models; and results of high throughput pre-screening.

C. High Throughput Pre-Screening

EDSTAC recognizes that biological effects data are lacking or incomplete for most chemicals under consideration. Therefore it recommends that the T1S be conducted using an automated high- speed method. This technique is called "high throughput pre-screening" (HTPS) and is recommended for (1) all chemicals with current production volumes greater than 10,000 pounds per year (about 15,000 chemicals); (2) all pesticide active ingredients and formulation inerts; and (3) all chemicals that are proposed to bypass either T1S or both T1S and T2T for any reason.

EDSTAC recommends the T1S in vitro transcriptional activation assays be modified and validated for use in the high throughput mode. The HTPS assays should provide information on the ability of test chemicals to bind to estrogen, androgen and/or thyroid hormone receptors; be used with exposure- and effects-related data to prioritize chemicals for T1S; improve QSAR models; and provide information for the design of tests in T2T for chemicals bypassing T1S.

EPA has initiated a feasibility demonstration pilot program to assess the feasibility of HTPS.

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D. Endocrine Disruptor Priority Setting Database

EDSTAC recommends the EPA to complete and maintain a relational Endocrine Disruptor Priority Setting Database (EDPSD). The EDPSD would extract existing data from other sources on fate, transport, and toxicity; data not readily available, such as, from the National Health and Nutritional Examination Survey and the Hazardous Substances Emergency Events Surveillance; and incorporate HTPS data and improved QSAR models.

E. Recommended Approach to Priority Setting

1. Compartment-Based Approach to Priority Setting for T1S

EDSTAC recommends an open and simple priority setting approach by the EPA that allows for chemicals of concern with less data to be included in the higher priority rankings.

Four broad categories are recommended to be developed that (1) would be based on integration of exposure, effects information, and criteria; (2) rely only on exposure-related information and criteria; (3) rely only on effects-related information and criteria; and (4) focus on special compartments of chemicals.

2. Special Compartments of Chemicals

EDSTAC recommended a number of specially targeted compartments for purposes of priority setting which would include:

Nominations

EDSTAC will give high priority to chemicals with widespread exposure at the national level. Also, it sets as a goal that chemicals with disproportionately high exposure to identifiable groups, communities, or ecosystems could have a parallel but separate priority setting process whereby chemicals with regional or local exposure could be nominated to receive a priority for T1S.

Mixtures

EDSTAC felt that mixtures needed special attention during the sorting and prioritization stages. Where possible, the EPA should determine the feasibility to screen representative samples of mixtures including: contaminants in human breast milk; phytoestrogens in soy-based infant formula; mixtures of chemicals commonly found at hazardous waste sites; pesticide/fertilizer mixtures; disinfection byproducts; and gasoline. EDSTAC acknowledges the technical feasibility of screening and testing mixtures is by no means certain.

Naturally Occurring Non-Steroidal Estrogens (NONEs)

These are natural products from plants (phytoestrogens) and fungi (). These occur widely in foods and have the potential to act in additive, synergistic, or antagonist manner with

98 other hormonally active chemicals. EDSTAC recommends representatives from the seven chemical classes on NONEs be tested in the EDSTP.

3. Recommended Approach to Priority Setting for T2T

EDSTAC recommended that priority setting for T2T for chemicals that bypass T1S is done as follows: Food-use pesticides could use the EPA schedule established for tolerance reassessments and pesticide re-registration under FQPA. For all other chemicals voluntarily bypassing T1S testing should be done in a manner that expedites, but does not delay, testing.

EDSTAC did not make explicit recommendations for setting priorities for chemicals that produce positive results in T1S and must move forward into T2T.

V. Chapter Five - Screening and Testing

A. Tier 1 Screening

1. Recommended Screening Assays

The screening tier of tests is designed to detect whether a chemical or mixture may interact with the endocrine system for estrogen, androgen, and thyroid hormones. The T1S assays should

-maximize sensitivity in order to minimize false negatives; -include animals with differences in metabolism; -detect all known modes of actions for these endocrine hormones; -include a range of taxonomic groups among the test organisms; and -include sufficient diversity in endpoints, to permit weight-of-evidence conclusions.

Recommended assays include:

In Vitro Assays -Estrogen Receptor Binding/Reporter Gene Assay -Androgen Receptor Binding/Reporter Gene Assay -Steroidogenesis Assay with minced testis

In Vivo Assays -Rodent 3-day Uterotrophic Assay -Rodent 20-day Pubertal Female with thyroid -Rodent 5-7-day Hershberger Assay -Frog Metamorphosis Assay -Fish Gonadal Recrudescence Assay

These tests are to function as a whole, but must first be validated prior to inclusion in the T1S battery. To address concerns about effects from prenatal/pre-hatch exposure that might not be detected from pubertal or adult exposures EDSTAC recommends development of a protocol for a

99 full life cycle developmental exposure screening assay that can be subjected to validation and standardization.

EDSTAC also suggest examining possible alternatives to the above group of tests. Alternative tests recommended as possible alternatives are: In Vitro Assay -Placental Aromatase Assay

In Vivo Assays

-Modified Rodent 3-day Uterotrophic Assay (intraperitoneal dosing) -Rodent 14-day Intact Adult Male Assay with thyroid -Rodent 20-day Thyroid/Pubertal Male Assay.

If these tests are as sensitive as the assays proposed for the T1S battery, they might replace some of the recommended assays for costs, time, and complexity considerations.

2. Criteria for Evaluating Tier 1 Results

EDSTAC recommends a weight-of-evidence approach be used to evaluate T1S results to make decisions concerning what chemicals would go on to T2T which would include: (1) the balance of positive and negative responses observed in in vitro and in vivo assays; (2) the nature and range of the biological effects observed; (3) shape of the dose-response curves; (4) severity and magnitude of the effects induced; and (5) presence or absence of response in multiple taxa.

Evaluation of these data would result in a decision either that the chemical needs no further STET and can move to the a HOLD BOX or that the chemical needs to be tested in Tier 2.

B. Tier 2 Testing

1. Test Selection

The purpose of T2T tests is to determine whether a chemical exhibits endocrine-mediated adverse effects and to identify, characterize, and quantify those effects for EAT hormones. These tests must include the most sensitive developmental life stage, identify the specific hazard caused by the chemical and establish a dose-response relationship, and include a range of taxa.

The following tests are recommended to be included: Two-Generation Mammalian Reproductive Toxicity Study; Avian Reproduction Test; Fish Life Cycle Test; Mysid Life Cycle Test; and an Amphibian Development and Reproduction Test. The outcome of the T2T if negative will supersede a positive outcome in T1S. These tests remain to be validated and standardized before incorporation into the screening and testing program.

100 2. Low Dose Issues in T2T

EDSTAC recommends a project be performed to resolve the underlying uncertainties and controversy concerning the adequacy of conventional toxicology study designs for assessment of endocrine active substances, with regard to low dose selection and the identification of no- observed-adverse-levels (NOAEL).

C. Validation, Standardization, Methods Development, and Research

EDSTAC believes that validation and standardization of the recommended screens and tests are essential for implementation of the EDSTP. It further believes that the process is of such importance that it be placed on an accelerated schedule.

VI. Chapter Six - Communications and Outreach

A. Principles to Guide a Communications and Outreach Strategy

Good communication is essential for the success of the screening and testing program. Particular care needs to be exercised to prevent the potential misuse of information generated by the EDSTAP. The EPA should develop a strategy for clear and accurate communication to all stakeholders during the program concerning the limitations that must be placed on the results as well as the meaning and implications of the decisions made by the Agency based on these results.

B. Basic Features of a Communications and Outreach Strategy

The EPA strategy should address: (1) what should be communicated; (2) to whom it should be communicated; (3) how it should be communicated; and (4) when it should be communicated. Further, this process should be open to all interested parties and groups. The information may be specifically tailored to special target audiences, such as farm workers. A variety of ways should be used to communicate to differing audiences. In addition, a tracking system should be available to enable interested parties to easily determine the status of any chemical.

VI. Chapter Seven - Compilation of EDSTAC Recommendations

This chapter contains all the Committee's recommendations made in Chapters Three, Four, Five and Six.

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