United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/9-90/053
Dec. 1990
£EPA
Strategy for
Environmental Health
Research at EPA
EPA
HEALTH
RESEARCH
APPLIED
RESEARCH
BASIC
RESEARCH
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EPA/600/9-90/053
December 1990
Strategy for Environmental Health
Research at EPA
Lawrence W. Reiter Ken Sexton
Health Effects Research Laboratory Office of Health Research
Research Triangle Park, NC 27711 Washington, DC 20460
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC 20460
Printed on Recycled Paper
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FOREWORD
The U.S. Environmental Protection Agency (EPA) is increasingly recognized as both a scientific and a regula-
tory agency. Yet the role of scientific research in developing effective regulatory policy and its contributions to in-
formed decision-making are often obscured by the more easily understood social, economic, and political issues
that tend to dominate the public debate. The fact remains, however, that it is the documented or suspected interre-
lationships among pollution sources, environmental concentrations, human exposures, and associated health effects
that form the justification for policy decisions about safeguarding public health.
Research sponsored by EPA is frequently discussed and dissected along two sometimes conflicting dimen-
sionsprogrammatic and scientific. Programmatic refers to the various regulatory programs that tend to view re-
search through the prism of their statutory mandates and deadlines. The regulators primarily look to research to
provide specific data that will be immediately relevant in meeting statutory deadlines. The scientific perspective,
on the other hand, is more holistic and longer term. The scientists tend to view research as a way to elucidate un-
derlying chemical, biological, or physical mechanisms that will eventually lead to a better understanding of key re-
lationshipsfor example, the relationships between exposure and dose or between dose and effect. Scientists see
their work as a series of incremental steps that will ultimately improve our ability to estimate true risk, while regu-
lators view research as a tool to produce precise and timely answers to specific regulatory questions. Scientists di-
vide their work along disciplinary (e.g., toxicology, epidemiology, clinical) or health outcome (e.g., genetic,
reproductive, pulmonary, neurologic) lines, while the regulators see things along media (e.g., air, water, soil, food)
or legislative (e.g., Clean Air Act, Toxic Substances Control Act) lines.
It is our intent in this document to lay out a strategic health research plan that is responsive to EPA's regula-
tory needs, but also consistent with the underlying scientific issues. A major goal is to show clearly that there is a
common set of health research issues that cuts across media and regulatory programs. And furthermore, to make it
clear that all the regulatory programs will benefit from a comprehensive and integrated health research program
that addresses the major uncertainties in health risk assessment.
Ken Sexton, Sc.D.
Director
Office of Health Research
Lawrence W. Reiter, Ph.D.
Director
Health Effects Research Laboratory
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Table of Contents
EXECUTIVE SUMMARY vii
SECTION ONE: INTRODUCTION
1.1 OVERVIEW 1-1
1.2 LEGISLATIVE MANDATE 1-1
1.3 RESEARCH TOPICS 1-3
1.4 ORGANIZATION OF HERL 1-7
1.4.1 Office of the Director 1-7
1.4.2 Neurotoxicology Division 1-7
1.4.3 Genetic Toxicology Division 1-7
1.4.4 Environmental Toxicology Division 1-8
1.4.5 Developmental Toxicology Division 1-8
1.4.6 Human Studies Division 1-8
1.4.7 Research and Regulatory Support Division 1-9
1.5 STRUCTURE OF THIS REPORT 1-9
SECTION TWO: CURRENT AND FUTURE RESEARCH DIRECTIONS BY DIVISION AT
THE HEALTH EFFECTS RESEARCH LABORATORY
2.1 OVERVIEW: HEALTH EFFECTS RESEARCH LABORATORY 2-1
2.1.1 HERL Research Program 2-1
2.1.2 Laboratory-Specific Research Needs 2-2
2.1.2.1 Hazard Identification 2-3
2.1.2.2 Dose-Response Assessment 2-4
2.1.3 Research Plan 2-5
2.1.3.1 Neurotoxicology 2-5
2.1.3.2 Genetic Toxicology 2-6
2.1.3.3 Pulmonary Toxicology 2-7
2.1.3.4 Immunotoxicology 2-8
2.1.3.5 Pharmacokinetics 2-9
2.1.3.6 Developmental and Reproductive Toxicology 2-10
2.1.3.7 Epidemiology (Human Studies) 2-10
2.2 NEUROTOXICOLOGY DIVISION 2-11
2.2.1 Divisional Program 2-13
2.2.2 Division-Specific Research Needs 2-13
2.2.2.1 Hazard Identification 2-13
2.2.2.2 Dose-Response Assessment 2-13
2.2.2.3 Chemical-Specific Data 2-13
2.2.2.4 Biological Markers 2-13
2.2.2.5 Pollutant Mixture '.'.'.'.'.'.'.'. 2-14
2.2.3 Research Plan 2-14
2.2.3.1 Hazard Identification 2-14
2.2.3.2 Dose-Response Assessment 2-15
2.2.3.3 Chemical-Specific Data '.'.'.'.'.'.'.'. 2-17
2.2.3.4 Biological Markers 2-17
2.2.3.5 Pollutant Mixtures 2-17
III
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2.2.4 Emerging Issues 2-18
2.2.4.1 Microorganisms 2-18
2.2.4.2 Environmental Agents and Neurodegenerative Disease 2-18
2.2.5 Summary 2-18
2.2.5.1 Hazard Identification 2-18
2.2.5.2 Dose-Response Assessment 2-18
2.3 GENETIC TOXICOLOGY DIVISION 2-19
2.3.1 Divisional Program 2-19
2.3.2 Division-Specific Research Needs 2-20
2.3.2.1 Hazard Identification 2-20
2.3.2.2 Dose-Response Assessment 2-21
2.3.2.3 Chemical-Specific Data 2-21
2.3.2.4 Biological Markers 2-22
2.3.2.5 Pollutant Mixtures 2-22
2.3.3 Research Plan 2-22
2.3.3.1 Hazard Identification 2-22
2.3.3.2 Dose-Response Assessment 2-23
2.3.3.3 Chemical-Specific Data 2-25
2.3.3.4 Biological Markers 2-25
2.3.3.5 Pollutant Mixtures 2-26
2.3.4 Emerging Issues 2-26
2.3.4.1 Nongenotoxic Cancer 2-26
2.3.4.2 Susceptible Populations 2-27
2.3.4.3 Microorganisms 2-27
2.3.5 Summary 2-27
2.3.5.1 Hazard Identification 2-27
2.3.5.2 Dose-Response Assessment 2-27
2.4 ENVIRONMENTAL TOXICOLOGY DIVISION 2-28
2.4.1 Divisional Program 2-29
2.4.2 Division-Specific Research Needs 2-29
2.4.2.1 Hazard Identification 2-29
2.4.2.2 Dose-Response Assessment 2-29
2.4.2.3 Chemical-Specific Data 2-31
2.4.2.4 Pollutant Mixtures 2-31
2.4.3 Research Plan 2-31
2.4.3.1 Hazard Identification 2-31
2.4.3.2 Dose-Response Assessment 2-31
2.4.3.3 Chemical-Specific Data 2-35
2.4.3.4 Biological Markers 2-36
2.4.3.5 Pollutant Mixtures 2-36
2.4.4 Emerging Issues 2-36
2.4.4.1 Biologicals/Microorganisms 2-36
2.4.4.2 Natural Versus Synthetic Fibers 2-36
2.4.4.3 Ultraviolet Radiation 2-36
2.4.4.4 Bioavailability of Chemicals from Soils or Other Matrices 2-36
2.4.5 Summary 2-36
2.4.5.1 Hazard Identification 2-36
2.4.5.2 Dose-Response Assessment 2-37
2.4.5.3 Chemical-Specific Data 2-37
2.5 DEVELOPMENTAL TOXICOLOGY DIVISION 2-37
2.5.1 Divisional Program 2-38
2.5.2 Division-Specific Research Needs 2-39
IV
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2.5.2.1 Hazard Identification 2-40
2.5.2.2 Dose-Response Assessment 2-41
2.5.2.3 Chemical- or Agent-Specific Data 2-42
2.5.3 Research Plan 2-42
2.5.3.1 Hazard Identification 2-42
2.5.3.2 Dose-Response Assessment 2-44
2.5.3.3 Chemical- or Agent-Specific Data 2-44
2.5.4 Emerging Issues 2-45
2.5.4.1 Improvements in Risk Assessment 2-45
2.5.4.2 The Female as a Susceptible Population 2-45
2.5.4.3 Nontraditional Exposure Regimens 2-46
2.5.4.4 Age and Toxicity 2-46
2.5.4.5 Biotechnology 2-46
2.5.5 Summary 2-46
2.5.5.1 Hazard Identification 2-46
2.5.5.2 Dose-Response Assessment 2-46
2.6 HUMAN STUDIES DIVISION 2-47
2.6.1 Divisional Program 2-47
2.6.2 Division-Specific Research Needs 2-48
2.6.2.1 Hazard Identification 2-48
2.6.2.2 Dose-Response Assessment 2-48
2.6.2.3 Chemical-Specific Data 2-49
2.6.2.4 Biological Markers 2-50
2.6.2.5 Pollutant Mixtures 2-50
2.6.3 Research Plan 2-50
2.6.3.1- Hazard Identification 2-51
2.6.3.2 Dose-Response Assessment 2-52
2.6.3.3 Chemical-Specific Data 2-53
2.6.3.4 Biological Markers 2-54
2.6.3.5 Pollutant Mixtures 2-55
2.6.4 Emerging Issues 2-56
2.6.4.1 Indoor Air 2-56
2.6.4.2 Immunotoxicology 2-56
2.6.5 Summary 2-56
2.6.5.1 Hazard Identification 2-56
2.6.5.2 Dose-Response Assessment 2-57
SECTION THREE: CURRENT AND FUTURE DIRECTIONS IN HEALTH RESEARCH BY
REGULATORY PROGRAM
3.1 OFFICE OF AIR AND RADIATION 3-1
3.1.1 Office Programs 3_2
3.1.1.1 Ambient Air Quality Program 3_2
3.1.1.2 Air Toxics Program 3_2
3.1.1.3 Mobile Sources Program 3.3
3.1.1.4 Indoor Air Program 3.3
3.1.1.5 Global Atmospheric Program 3.3
3.1.1.6 Radiation Program 3.4
3.1.2 Office-Specific Research Needs 3.4
3.1.2.1 Ambient Air Quality Program 3.4
3.1.2.2 Air Toxics Program 3.5
3.1.2.3 Mobile Sources Program 3.5
3.1.2.4 Indoor Air Program 3.5
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3.1.2.5 Global Atmospheric Program 3-6
3.1.2.6 Radiation Program 3-6
3.1.3 Research Plan 3-6
3.1.3.1 Ambient Air Quality Program 3-6
3.1.3.2 Air Toxics Program 3-7
3.1.3.3 Mobile Sources Program 3-8
3.1.3.4 Indoor Air Program 3-9
3.1.3.5 Global Atmospheric Program 3-9
3.1.3.6 Radiation Program 3-9
3.2 OFFICE OF WATER (DRINKING WATER) 3-9
3.2.1 Office-Specific Research Needs 3-11
3.2.1.1 Source Water 3-11
3.2.1.2 Water Disinfection and Disinfection By-Products 3-11
3.2.1.3 Nondisinfectant Additives and Distribution System Contaminants 3-12
3.2.2 Research Plan 3-13
3.2.2.1 Source Water 3-13
3.2.2.2 Water Disinfection and Disinfection By-Products 3-14
3.2.2.3 Nondisinfectant Additives and Distribution System Contaminants 3-15
3.3 OFFICE OF WATER (WATER QUALITY) 3-15
3.3.1 Office-Specific Research Needs 3-16
3.3.2 Research Plan 3-16
3.4 OFFICE OF PESTICIDES AND TOXIC SUBSTANCES 3-16
3.4.1 Office-Specific Research Needs 3-18
3.4.1.1 Federal Insecticide, Fungicide, and Rodenticide Act 3-18
3.4.1.2 The Toxic Substances Control Act 3-20
3.4.2 Research Plan 3-21
3.4.2.1 Pesticides 3-21
3.4.2.2 Toxic Substances 3-24
3.5 OFFICE OF EMERGENCY AND REMEDIAL RESPONSE 3-25
3.5.1 Office-Specific Research Needs 3-27
3.5.1.1 Site Evaluation Program 3-27
3.5.1.2 Removal Program 3-27
3.5.1.3 Remediation Program 3-28
3.5.1.4 Post-Closure Program 3-28
3.5.2 Research Plan 3-28
3.5.2.1 Site Evaluation Program 3-28
3.5.2.2 Removal Program 3-28
3.5.2.3 Remediation Program 3-29
3.5.2.4 Post-Closure Program 3-30
3.6 OFFICE OF SOLID WASTE 3-30
3.6.1 Office-Specific Research Needs 3-31
3.6.1.1 Waste Characterization 3-31
3.6.1.2 Hazardous Waste 3-31
3.6.1.3 Nonhazardous Waste 3-31
3.6.2 Research Plan 3'31
3.6.2.1 Waste Characterization 3-31
3.6.2.2 Hazardous Waste 3-32
3.6.2.3 Nonhazardous Waste 3-32
VI
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EXECUTIVE SUMMARY
In the past 20 years, major environmental legislation has given the U.S. Environmental Protection Agency
(EPA) the regulatory tools it needs to protect our environment and public health. Environmental protection, how-
ever, requires more than legislative vehicles; appropriate regulatory decisions based on those laws must be founded
on scientific data concerning the scope and magnitude of health risks associated with the environmental hazards to
which the public is exposed. For most pollutants, that link between environmental exposures and adverse health
effects is not yet clear. Moreover, the difficulty facing decision makers in weighing the known costs of pollution
reduction or prevention against the ambiguous public and health benefit is becoming more acute in the face of se-
verely constrained public resources.
EPA's health research program is well situated to address this informational gap through an integrated research
strategy that assimilates and builds on related work in other federal agencies, as well as in the scientific community
at large. Only at EPA is there a direct interface between the researchers, risk assessors, and risk managers for forg-
ing the most scientifically sound regulatory decisions. To function in this environment, EPA health scientists must
be cognizant of important breakthroughs in the basic biologic sciences (e.g., genetics, molecular biology) and capa-
ble of applying these scientific advances to problems facing the Agency. Conversely, they must be knowledgeable
about regulatory activities and able to recognize and conceptualize the basic research questions raised by contem-
porary environmental issues. In short, they function at the interface between basic and applied research. The Of-
fice of Health Research, and more specifically, the Health Effects Research Laboratory (HERL) is the focal point
for these research efforts.
To address the broad range of environmental contaminants covered under various legislative statutes, HERL
research must assist EPA in evaluating the health risks for diverse environmental agents. While the chemical and
physical composition of these pollutants differs significantly, the evaluation of their health effects must address a
common set of issues: exposure, or the extent to which humans are exposed to pollutants in the environment; dose,
or the relationship between the exposure and the dose of the pollutant received at the site(s) of toxic action within
the body; and effect, or the health impact from the pollutant dose. These fundamental issues form the risk assess-
ment paradigm that underlies the research needs of all EPA regulatory program areas, and therefore they are central
to the entire HERL program. These issues can be further subdivided into seven research topics that are addressed
in the HERL program: exposure, dose-response, hazard identification, chemical-specific data, pollutant mixtures,
and biological markers research as well as human data development.
HERL is functionally organized along the lines of the key scientific specialties in environmental health re-
search. This document presents a strategic research plan for HERL that merges three key components: regulatory
program concerns, key research topics, and HERL scientific divisions. The remainder of this report explains how
these three components are synthesized in the HERL research strategy. In sum, the document explains the direc-
tion of EPA's health research program over the next five years, and the reasons for the prioritizations made by each
division.
vii
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SECTION ONE
INTRODUCTION
1.1 OVERVIEW
The Health Effects Research Laboratory (HERL)
within the Office of Health Research (OHR) is the focal
point of the U.S. Environmental Protection Agency's
(EPA's) effort to understand the human health impacts of
environmental pollutants. The regulatory program offices
within EPA use HERL's research resultseither directly
or after they have been incorporated by the Office of
Health and Environmental Assessment (OHEA) into a risk
assessmentto develop regulations that effectively and
efficiently safeguard public health. HERL develops its re-
search strategy and plans based on the needs of the regula-
tory programs and on the scientific uncertainties that must
be resolved to fulfill those needs. HERL's scientific re-
search is used, therefore, to protect public health from the
effects of pollutants.
1.2 LEGISLATIVE MANDATE
EPA's and HERL's authority to conduct environ-
mental health research is derived initially from the major
federal laws mandating broad programs to protect public
health and the environment. Each of these laws, including
the Clean Air Act, the Safe Drinking Water Act, the Clean
Water Act, the Toxic Substances Control Act (TSCA) the
Federal Insecticide, Fungicide, and Rodenticide Act (FI-
FRA), the Resource Conservation and Recovery Act
(RCRA), and the Comprehensive Environmental Re-
sponse, Compensation and Liability Act (CERCLA), re-
quires that EPA develop regulatory programs to protect
public health. Several regulatory programs established
under each statute require specific consideration of health
effects:
Air Quality - National Ambient Air Quality Stand-
ards (NAAQS) are based on human health endpoints.
State programs and federal emissions limits are de-
signed to achieve those standards. Special emissions
limits are established for pollutants that are hazardous
to human health (i.e., toxic air pollutants). Radiation
standards and radon guidance are also designed to
protect human health. The health effects of pollutants
generated by the combustion of alternative fuels and
indoor air pollutants are emerging concerns.
Drinking Water - National drinking water standards
(Maximum Contaminant Levels, or MCLs) are based
directly on human health endpoints. Health Adviso-
ries provide human health recommendations for some
pollutants not covered under MCLs.
Water Quality - EPA develops ambient pollutant
limits (criteria) for surface waters based in part on hu-
man health endpoints. For permitting decisions, the
states or EPA may require testing of effluents for tox-
icity. Regulations for sludge disposal are based on
health risks.
Toxic Substances - Health data collected on new and
existing chemicals are used to determine whether to
implement restrictions on the manufacture, use,
and/or disposal of toxic chemicals.
Pesticides - The pesticide registration, reregistration,
and special review programs involve the evaluation of
toxicity and other health-related information to assess
the effects of pesticide products.
Hazardous and Nonhazardous Wastes - Toxicity
data are used to help determine which wastes are
regulated as hazardous. Regulations for facilities that
accept waste and restrictions on land disposal of
waste are designed to protect the health of popula-
tions near disposal sites.
Superfund Waste Sites - Emergency response and
cleanup actions are designed to protect the health of
human populations near waste sites.
Because each major environmental statute requires
the development of health-protective programs, the stat-
utes include specific legislative authority for EPA to con-
duct health effects research. The sections of each statute
authorizing health research, and examples of environ-
mental exposures covered under this research, are pre-
sented in Table 1-1. Based on these legislative authorities,
HERL formulates its research strategy to provide scientific
results that facilitate informed decision-making and rule-
setting under each regulatory program.
1-1
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Table 1-1:
MAJOR ENVIRONMENTAL LEGISLATION ADMINISTERED BY THE U.S. ENVIRONMENTAL PROTECTION
AGENCY, SPECIFIC AUTHORIZATION FOR THE EPA TO CONDUCT HEALTH RESEARCH, AND EXAMPLES OF
RELEVANT ENVIRONMENTAL EXPOSURES
Enabling Authorizing Language Authorizing EPA
Legislation Section to Conduct Health Research
Examples of Relevant
Environmental Exposures
Clean Ak Act Title I. The Administrator is given broad authority to conduct re-
(CAA) Part A., search relating to the "causes, effects, extent, prevention,
Sec. 103 and control of air pollution." Special emphasis should be
given to research on the short- and long-term effects of
air pollutants on public health and research to "improve
our knowledge of the contribution of air pollutants to the
occurrence of adverse effects on health, including, but
not limited to, behavioral, physiological, lexicological,
and biochemical effects."
Part B, The Administrator is given authority to conduct studies
Sec. 153 on "biomedical, or other research and monitoring...to as-
certain any direct or indirect effects upon the public
health and welfare of changes in the stratosphere, espe-
cially ozone."
National Ambient Ak
Quality Standard Pollut-
ants (e.g., ozone, nitro-
gen, sulfur dioxide,
carbon monoxide, inhal-
able particles, lead)
Hazardous Air Pollutants
(e.g., benzene, formalde-
hyde, styrene)
Safe Drinking
Water Act
(SDWA)
Resource Conser-
vation and Re-
covery Act
(RCRA)
Part E, "The Administrator may conduct research...of physical
Sec. 1442 and mental diseases and other impairments of man result-
ing directly or indirectly from contaminants in water, or
to...improve methods to identify and measure the health
effects of contaminants in drinking water."
Subtitle The Administrator shall conduct-research, investiga-
H, Sec. dons, experiments...and studies relating to any adverse
8001 (A) health and welfare effects of the release into the environ-
ment of material present in solid waste.
Contaminated Drinking
Water (e.g., lead, chlorin-
ated solvents, trihalo-
methanes, pathogenic
bacteria and viruses, natu-
ral organics)
Hazardous and Munici-
pal Wastes (e.g., contami-
nated soil, water, and air
from hazardous, munici-
pal, or medical wastes)
Solid Wastes (e.g., sew-
age sludge, incinerator
ash)
Superfund Chemicals
(e.g., contaminated soil,
water, and air from Su-
perfund sites, with em-
phasis on high priority
chemicals listed in Sec-
tion 313 of CERCL A)
1-2
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Table 1-1: Continued
Enabling Authorizing Language Authorizing EPA
Legislation Section to Conduct Health Research
Examples of Relevant
Environmental Exposures
Superfund
Amendments
Reauthorization
Act
(SARA)
Sec. 209
Sec. 403
Clean Water Act
(CWA)
Part
1254,
Sec. 104
Toxic Sub-
stances Control
Act (TSCA)
Sec. 10
The purposes of this section are to establish a comprehen-
sive and coordinated Federal (SARA) program of re-
search..^ improve the scientific capability to assess,
detect and evaluate the effects on and risks to human
health from hazardous substances."
"...The Administrator of the EPA shall establish a re-
search program with respect to radon gas and indoor air
quality... The research program required under this sec-
tion shall include...research related to the effects of in-
door air pollution and radon on human health..."
"...the Administrator shall conduct research on the harm-
ful effects on the health and welfare of persons caused
by pollutants in water..."
"...research undertaken by the Administrator and directed
toward the development of rapid, reliable, and economi-
cal screening techniques for carcinogenic, mutagenic,
teratogenic, and ecological effects of chemical sub-
stances and mixtures."
Indoor Air Pollutants
(e.g., environmental to-
bacco smoke, emissions
from building materials
and unvented combus-
tion appliances)
Contaminated Surface
and Ground Water (e.g.,
industrial effluents, ur-
ban/rural runoff, munici-
pal and hazardous wastes)
Contaminated Wetlands,
Near Coastal Regions,
and Oceans (e.g., sewage
outfalls, industrial efflu-
ents, dumping of wastes,
spills)
Toxic Substances (e.g.,
manufactured/ processed
substances such as PCBs,
asbestos, solvents, etc.,
excluding pesticides)
Federal Insecti-
cide, Fungicide,
and Rodenticide
Act (FIFRA)
Sec. 20 "The Administrator shall undertake research...to carry
out the purposes of this Act..."
Pesticides (e.g., dinoseb,
nitrofen, alar, captan, car-
baryl)
1.3 RESEARCH TOPICS
To address the broad range of environmental contami-
nants covered under the statutes, HERL research must as-
sist EPA in evaluating the health risks for diverse
environmental agents including automotive and diesel ex-
haust, power plant emissions, pesticides and other toxic
chemicals, hazardous waste, municipal solid waste, natu-
rally occurring and genetically engineered microorgan-
isms, drinking water disinfectants and associated
by-products, and ionizing and nonionizing radiation.
While the chemical and physical composition of these pol-
lutants differs significantly, the evaluation of their health
effects must address a common set of questions:
1-3
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Exposure - How and to what extent are humans ex-
posed to the pollutant in the environment (i.e., route,
magnitude, frequency, duration)?
Dose - What is the relationship between this expo-
sure and the dose of the pollutant received at the
site(s) of toxic action within the body?
Effect - What is the health impact of the pollutant
dose?
These fundamental research questions underlie the re-
search needs of all EPA regulatory program areas, and
therefore are central to the entire HERL program. The
questions can be further subdivided into seven research
topics (four principal topics and three cross-cutting topics)
that are addressed in the HERL program:
1. Exposure Research defines the route, magnitude,
frequency, and duration of exposure of humans to en-
vironmental pollutants. Most exposure research ef-
forts are carried out by another office within the
Office of Research and Development (ORD), the Of-
fice of Modeling, Monitoring Systems, and Quality
Assurance (OMMSQA), which is currently focusing
its efforts on investigating the exposures experienced
by individuals and populations during normal daily
activities, with emphasis on identifying high-exposure
groups. HERL efforts in this area focus on the devel-
opment and validation of biological markers for expo-
sure, effects, and susceptibility in human populations;
these efforts involve substantial interaction with ORD
monitoring laboratories.
2. Hazard Identification Research develops, refines,
and validates approaches and methods for identifying
potential human health hazards. HERL is developing
techniques to determine causal relationships between
environmental pollutants and adverse health outcomes
that are faster, more accurate, less expensive, and
more reliable than current options. This research in-
cludes the development of test methods for screening
and characterizing new and existing chemicals and
procedures to evaluate qualitative and quantitative re-
lationships between the chemical structure of pollut-
ants and their related biological effects
(structure-activity relationships [SARs]).
3. Dose-Response Research elucidates a) the relation-
ship between exposure (i.e., applied dose), dose at the
site of toxic action (i.e., target dose), and biological
effects; and b) the basic biological mechanisms re-
sponsible for the observed effects. HERL's research
in this area includes developing better methods to re-
late exposure to dose (i.e., physiologically based
pharmacokinetic models) and exploring the physio-
logical and biological mechanisms of toxicity, includ-
ing compensatory processes, that are crucial to
accurate extrapolation of research results (e.g., ex-
trapolating results from animals to humans, from high
to low dose, and from acute to chronic effects).
4. Chemical-Specific Research develops scientific data
on individual pollutants at the request of the regula-
tory program offices. HERL's efforts in this area in-
clude the evaluation of methods for screening and
characterizing agents for which critical data gaps have
been identified, generation of chemical-specific dose-
response data using laboratory animals, and human
clinical studies to investigate the acute effects of spe-
cific air pollutants. This research is generally short-
term (1-2 years) and aimed at filling data gaps
concerning specific chemicals or chemical mixtures
that are of immediate importance to a regulatory pro-
gram.
In addition to these four principal research topics,
three other research themes that cut across the four main
areas are of sufficient importance to be considered major
research topics within the HERL program.
5. Pollutant Mixtures Research clarifies the extent to
which synergistic, antagonistic, or additive interac-
tions cause the effects of exposure to a mixture of pol-
lutants to differ from the effect that would be
predicted based on the characteristics of the individ-
ual pollutant components. HERL efforts will focus
increasingly in this area because most common hu-
man exposures to environmental contaminants (e.g.,
urban air pollution, drinking water, incinerator emis-
sions) involve pollutant mixtures.
6. Biological Marker Research develops and validates
biological measurements that can be used to calculate
dose at the site of toxic action and to detect effects at
cellular and molecular levels. HERL will focus on
the development of biological markers in humans for
exposure, effects, and susceptibility. The use of these
techniques in epidemiologic investigations will facili-
tate more direct assessment of exposure and effects,
and thereby reduce related uncertainties.
7. Human Data Development consists of the collection
of information on exposure, dose, and effects in hu-
1 -4
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man populations. Scientists at HERL and elsewhere
in EPA develop and use human data to assess the
status of public health, to identify potential environ-
mental health problems, and to evaluate the efficacy
of risk reduction measures. Human data are used also
to identify and evaluate subgroups that are at higher
risk because of increased susceptibility or elevated
exposures, and to ascertain the degree to which ef-
fects observed in animals are analogous (or homolo-
gous) to those observed in humans. Research
activities in this area include epidemiological and
clinical studies and the establishment and mainte-
nance of exposure and disease registries.
The health effects concerns of all EPA's regulatory
program offices fall under these seven research topics.
The Office of Health Research recently undertook a sur-
vey of the regulatory program offices to determine the pri-
orities they assign to these topics. The results are pre-
sented in Table 1-2. The rankings by a given regulatory
program office represent the relative, not absolute priori-
ties of that office. Therefore, the assignment of a rela-
tively low priority for a given topic does not necessarily
mean that the topic is not important to the program office,
but only that the topic has a lower priority than the other
research areas.
The data presented in Table 1-2 reinforce the funda-
mental and essential nature of the seven research topics
across diverse regulatory programs. All seven regulatory
program offices rated dose-response assessment as a high
priority, while five rated the collection of human data a
high priority. Chemical-specific information and pollutant
mixtures were a high priority for four programs, hazard
identification and exposure assessment for three, and bio-
logical markers for two.
Table 1-2:
SUMMARY OF RESEARCH PRIORITIES BY REGULATORY PROGRAM
Regulatory Programs
Topics
Drinking Water Hazardous
Air Water Quality Toxics Pesticides Waste Superfund
Hazard xx
Identification
Dose-Response xxx
Assessment
Exposure xxx
Assessment
Chemical-Specific xxx
Information
xxx
xxx
xxx
xx
xxx
xxx
xx
xxx
xxx
XX
xxx
XX
xxx
xxx
xxx
xxx
xxx
xxx
Pollutant
Mixtures
Biological
Markers
Human Data
xx
xxx
xxx
xx
xx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
x = low priority
xx = medium priority
xxx = high priority
1-5
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OFFICE
of the
DIRECTOR
PY: 32
S/E: $2.2 m
R/D: $1.6m
PROGRAM MANAGERS
Associate
Director
for
Air and
Radiation
Associate
Director
tor
Hazardous
Waste and
Superfund
Associate
Director
lor
Toxics and
Pesticides
Associate
Director
tor
Water
DEVELOP-
MENTAL
TOXICOLOGY
DIVISION
ENVIRON-
MENTAL
TOXICOLOGY
DIVISION
RESEARCH &
REGULATORY
SUPPORT
DIVISION
HUMAN
STUDIES
DIVISION
NEURO-
TOXICOLOGY
DIVISION
GENETIC
OXICOLOGY
DIVISION
PY: 39
S/E: $2.5 m
R/D: $8.6 m
PY: 37
S/E: $1 9 m
R/D: $2.7 m
PY: 58
S/E: $2.9 m
R/D: $6.7 m
PY: 50
S/E: $2.4 m
R/D: $2.5 m
PY: 55
S/E: $2 7 m
R/D- $5.5 m
PY: 18
S/E: $1 1 m
R/D: $1.6m
Neuro physiological
PY Person Years
S/E Salaries and Expenses Budget in Milions (FYS8)
R/D Research and Development Budget n Milions (FYB9)
Fig. 1 -1: Health Effects Research Laboratory
1-6
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1.4 ORGANIZATION OF HERL
1.43 Genetic Toxicology Division
To effectively carry out its mission to provide health
research to support EPA's regulatory programs, HERL is
organized in accordance with the key scientific specialties
in the environmental health field (Figure 1-1). The func-
tional role of each of the divisions is summarized as fol-
lows:
1.4.1 Office of the Director
The Office of the HERL Director includes the Direc-
tor and Deputy Director, as well as four Associate Labora-
tory Directors (Program Managers), who ensure that the
scientific program is responsive to Agency needs; and the
Program Support Staff, who are responsible for budgetary
and administrative oversight. In general, this office over-
sees and coordinates the activities of the laboratory, in-
cluding interactions with other parts of EPA and outside
organizations.
1.4.2 Neurotoxicology Division
The Neurotoxicology Division plans, conducts, sup-
ports, and evaluates research to examine the effects of
physical and chemical agents on nervous system function.
In the course of examining toxicant-induced changes in
the developing and adult nervous systems, the program
touches on all levels of neural organization, including
functional and structural. Behavioral evaluations include
measurements of sensory, motor, and cognitive integrity.
Extensive efforts are underway to develop methods to
screen chemicals for neurotoxic potential and to develop
detailed information regarding the specificity of effects of
toxic chemicals.
Neurochemical research encompasses measurements
of exogenous chemicals in biological tissues and studies
of the influence of neurotoxicant exposure on endogenous
chemicals (e.g., neurotransmitters and their associated
synthetic and degradative enzymes, neurotoxic esterase,
nervous system specific proteins) and chemical processes
(e.g., energy metabolism, axonal transport). The neuro-
physiology program focuses on direct measurements of
nervous system activity, including evoked potentials,
EEGs, and seizure susceptibility. Measurements are gen-
erally made in vivo and are correlated with behavioral,
neuropathological, and biochemical indicators of neuro-
toxicity.
The research program of the Genetic Toxicology Di-
vision improves EPA's understanding of environmentally
induced mutagenesis and carcinogenesis, with special em-
phasis on pollutant mixtures and biological markers of ex-
posure and effects (e.g., biochemical, molecular, and
cellular indicators). The program encompasses research
on the metabolic activation and detoxification of environ-
mental pollutants that act through genetic mechanisms, as
well as studies on alternative mechanisms for mutagenesis
and carcinogenesis. The division has the capability to
evaluate the mutagenic and oncogenic potential of envi-
ronmental agents, both singly and in combination, through
stepwise application of structure-activity analysis, short-
term screening tests, and confirmatory bioassays for mut-
agenesis and carcinogenesis using laboratory animals.
The objective of mutagenesis research is to under-
stand the chemical induction of somatic and germ cell mu-
tations as a basis for improving risk assessments for
cancer, reproductive failure, and heritable mutations. The
carcinogenesis component of the research program is
aimed at achieving a better understanding of chemical car-
cinogenesis in order to improve cancer risk assessments.
Laboratory research is undertaken to examine the ability
of chemicals to influence the induction, promotion, and
progression of cancer; while theoretical studies are carried
out to relate the molecular structure of chemicals to poten-
tial biologic effects. Research on pollutant mixtures is de-
signed to develop, validate, and refine bioanalytical
methods to qualify and quantify human exposure, molecu-
lar dose, and associated genotoxic effects. The division
has the capacity to apply genetic bioassays in the field to
detect, identify, and compare potential human health haz-
ards from exposures to mixtures of pollutants.
Substantial efforts are also aimed at identifying and
validating biological markers for genotoxicants that can
provide quantitative data on exposure and dose, and that
can produce meaningful information about the relation-
ships among early biochemical changes, preneoplastic le-
sions, and cancerous lesions. This work will further the
field of molecular epidemiology and will aid in the devel-
opment of biologically based dose-response models for
mutagenicity and carcinogenicity.
A relatively new area is research concerning the envi-
ronmental release of naturally occurring and genetically
engineered products from the biotechnology industry.
Currently, there is a small program to identify the poten-
tial human health hazards that might arise from this tech-
1-7
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nology, with initial emphasis on the development of suit-
able screening tests.
1.4.4 Environmental Toxicology Division
The Environmental Toxicology Division conducts
both in vivo and in vitro animal research to determine the
potential for human health risks from a broad spectrum of
environmental pollutants involving different routes of ex-
posure. This research is designed to determine cause and
effect relationships at pollutant concentrations and expo-
sure patterns that mimic those occurring in the environ-
ment. Studies focus on the pulmonary and cardiovascular
systems, the immune system, the skin, and the liver. Al-
though much of the research involves inhalation toxicol-
ogy, exposure of animals to toxic substances by other
routes and the development of route-to-route extrapolation
methods are also important components of the research ef-
fort. These efforts reflect the division's commitment to
develop physiological models for describing the pharma-
cokinetic behavior of environmental chemicals. Experi-
mental studies to develop the database to predict
high-to-low dose, acute-to-chronic, and interspecies ex-
trapolations are being conducted that form the basis for
dosimetry models. In addition, the division emphasizes
studies to determine absorption, distribution, metabolism,
and elimination of toxicants.
1.4.5 Developmental Toxicology Division
The Developmental Toxicology Division conducts
biological research to detect, interpret, and extrapolate the
effects of environmental pollutants, singly or in combina-
tion, on reproduction and development. Major emphasis
is on the development of new and improved methodolo-
gies for the assessment of embryo and fetal toxicity, post-
natal functional deficits, and male and female reproductive
toxicity. Studies focus on the morphological, biochemi-
cal, and physiological assessment of germ cell function,
gonadal function, and embryonic development in both the
normal and abnormal situation. The environmental agents
currently under investigation include toxic substances,
chemical and microbial pesticides, air pollutants, drinking
water contaminants, and hazardous wastes.
The perinatal toxicology research conducted by a
branch of this division examines the potential develop-
mental toxicity when environmental exposure occurs
between fertilization and sexual maturation. The reproduc-
tive toxicology research conducted by the division is
aimed at defining the effects of environmental agents on
the functional integrity of the reproductive system, and de-
termining the significance of data generated in animal
studies to the human situation.
The reproductive and developmental biochemistry re-
search focuses particular attention on additives and con-
taminants found in drinking water. Efforts are directed
toward understanding the relationship between maternal
and developmental toxicity by varying exposure scenarios,
examining the consequences of changes in maternal ho-
meostasis and body burden during different developmental
stages, developing and validating novel techniques for
evaluating male reproductive health, and comparing the
results of in vivo and in vitro bioassays for developmental
effects.
1.4.6 Human Studies Division
The Human Studies Division conducts both clinical
and epidemiological investigations to improve the under-
standing of human health risks associated with environ-
mental pollution. Clinical studies are conducted on
research questions that are best approached under highly
controlled laboratory conditions, whereas epidemiologic
investigations rely on field studies in more natural set-
tings. The goal of many research projects is to better char-
acterize the similarities and differences between effects
observed in humans and those in animals, or between re-
sults of in vivo and in vitro tests.
The Clinical Research Branch emphasizes controlled
laboratory experiments to study primarily the health ef-
fects of inhaled air pollutants in humans. The research
emphasizes several different, but complementary, meas-
urements: the deposition, fate, and biologic effects of in-
haled gases and particles; pulmonary and cardiovascular
function; neurobehavioral function; pulmonary and sys-
temic immunity and host defenses. Study populations are
volunteers and include healthy adults, the elderly, chil-
dren, and patients (e.g., those with respiratory infections,
asthma, chronic obstructive lung disease, or ischemic heart
disease).
The Epidemiology Branch conducts research that in-
volves the collection of human data through field studies
or the analysis of existing databases. The research is fo-
cused on three major areas: 1) airborne pollutant expo-
sures (e.g., photochemical oxidants, hazardous air
pollutants) and their effects on human health; 2) the hu-
man health effects of exposures to pollutants in drinking
water, wastewater, and sewage; and 3) the human health
effects of exposures to hazardous substances (e.g., pesti-
cides, toxic substances, and hazardous waste materials).
1-8
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1.4.7 Research and Regulatory Support Division
This division has the following responsibilities: coor-
dinating multimedia, multidivisional projects; examining
current and future trends that are likely to affect the direc-
tion of environmental health research; synthesizing, sum-
marizing, and reviewing health effects information,
especially as it relates to the Agency's regulatory respon-
sibilities; and acting as liaison to the regional offices, state
and local agencies, and the public. The division also pro-
vides statistical and mathematical support to all compo-
nents of the laboratory.
1.5 STRUCTURE OF THIS REPORT
The previous discussion described the broad health
effects concerns of EPA's regulatory programs, identified
seven basic health research topics that encompass all of
these concerns, and described the functional organization
of HERL along the lines of key scientific specialties in en-
vironmental health research. HERL's research strategy
merges these three components: regulatory program con-
cerns, key research topics, and HERL scientific disci-
plines. The remainder of this report explains how these
three components are synthesized in the HERL research
strategy.
Figure 1-2 depicts the three components of the HERL
strategy as a three-dimensional matrix, with the research
topics serving as a link between the regulatory program
offices and the HERL research disciplines. This linkage
can be explained as follows: the environmental health con-
cerns of the regulatory programs all fall within the seven
research topics; the research questions of the scientific dis-
ciplines of HERL also fall within the seven research top-
ics. Thus, any cell in the matrix represents a research topic
that is both a concern of one of the EPA regulatory pro-
grams and addressed by HERL.
The remainder of this document explains this linkage
in more detail. Section Two explains how each research
division of HERL establishes its priorities and selects re-
search projects within the seven research topics. Section
Three explains how the concerns of EPA's regulatory pro-
grams under each research topic are addressed by the
HERL program.
Regulatory Issues
Fig. 1-2: Dimensions of OHR Research Program
1-9
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SECTION TWO
CURRENT AND FUTURE RESEARCH
DIRECTIONS BY DIVISION AT THE HEALTH
EFFECTS RESEARCH LABORATORY
* In this section, current and future research efforts
under the EPA health program are separated first by scien-
tific specialty and then by research topic (the z and y axes
in Figure 1-2). Section 2.1 provides an overview of the
HERL research program, outlines specific research needs
of program offices that must be met by the research pro-
gram, and introduces HERL efforts in the scientific dis-
ciplines relevant to these needs. Sections 2.2-2.6 detail
the work performed by the different HERL divisions
(neurotoxicology, genetic toxicology, environmental
toxicology, developmental toxicology, and human
studies). This discussion presents the scientific point of
view on the Agency's current and future health research
needs.
2.1 OVERVIEW: HEALTH EFFECTS
RESEARCH LABORATORY
2.1.1 HERL Research Program
OHR/HERL is responsible for supporting the
Agency's regulatory programs through a strong research
Fig. 2-1: The Interface Role of EPA Health Scientists
program in environmental health. The regulatory
programs require research to improve the accuracy of en-
vironmental health risk assessment. Addressing the needs
of various program offices involves projects that span the
gamut from short-term, applied research to more long-
range, basic research.
To run such a health program, EPA scientists must
have expertise in conducting research at all levels of func-
tion (i.e., molecular, intracellular, cellular, tissue, organ,
whole organism). They must be aware of important
breakthroughs in the basic biological sciences (e.g.,
genetics, molecular biology) and at the same time be
capable of applying these scientific advances to problems
facing the Agency. Conversely, they must be knowledge-
able about regulatory activities and able to recognize and
conceptualize the basic research questions raised by con-
temporary environmental issues. In short, they function
primarily at the interface between basic and applied re-
search, ensuring rapid and productive communication be-
tween these two ends of the research spectrum (see Figure
2-1).
HERL uses the risk as-
sessment paradigm outlined in
Section 1 to structure a health
research program that addres-
ses the regulatory needs of the
program offices. Assessing
the risk associated with ex-
posure to a particular pollutant
requires defining and charac-
terizing the factors that in-
fluence both the movement of
the pollutant from source to
dose levels in target organ(s),
and the response of those
organ(s) to the pollutant dose.
(Important examples of these
factors are listed in Figure 2-
2.) HERL's analysis of these
factors will enhance con-
2-1
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Route
Magnitude
Duration
Intensity
Absorption
Distribution
Metabolism
Elimination
Disease Mechanisms
Compensatory Mechanisms
Susceptibility
The products of the research pro-
gram include:
State-of-the-science methods for
use by the Agency in hazard
identification and the biologic
(dose) aspects of exposure as-
sessment
Models for extrapolation of dose
and effect needed for do^e-
response assessment
Application of methods and
models to collect scientific data
(measurements) on specific pol-
lutants requested by the regu-
latory programs
Fig. 2-2:
Factors Influencing Pollutant Movement and Dose-Response
fidence in its interpretation of data obtained directly from
human populations and will also lessen the uncertainty of
extrapolating risk to human populations from data ob-
tained from laboratory animals. The manner in which re-
search to understand the unknown events between
exposure and health effects supports the different com-
ponents of the risk assessment process is illustrated in Fig-
ure 2-3.
Within the risk assessment paradigm, the health re-
search program focuses on hazard identification and dose-
response assessment, which are the steps directly
concerned with the health consequences of human ex-
posure to pollutants. Research to improve hazard iden-
tification primarily involves efforts to develop
methods for screening (detection) and characterization
of health hazards (e.g., fate of agent within the body;
identification of putative targets) and provides
presumptive evidence of causality between exposure
and effects. Research to improve dose-response as-
sessment primarily involves efforts to develop predic-
tive models of dose (i.e., physiologically based
pharmacokinetic models) and effects (i.e., biologically
based dose-response models) that allow quantitative
integration and extrapolation of dose and effect. Sup-
port is also provided for the facets of exposure assess-
ment that rely on measures of applied and delivered
dose (e.g., body burden, biomarkers) in constructing
more precise models of actual human exposure.
2.1.2 Laboratory-Specific Research Needs
The various program offices in EPA give different
priority ratings to all the research topics outlined in Sec-
tion One except for dose-response assessment (see Table
1-2); the high-priority rating given this topic by all the of-
fices reflects a universal need for predictive models of
dose and effect. Within a particular discipline, the overall
balance of the research program at any point in time is
determined both by the program office needs and by the
state-of-the-science in that discipline. Often, fundamental
research is required to provide the framework by which an
issue of concern can be directly addressed.
Risk
Characterization
Exposure I Hazard Identification
Assessment
Dose-Response
Assessment
Exposure
Models
Fig. 2-3: HERL Support of Risk Assessment Needs
2-2
-------
For example, sensitive and reliable measures to char-
acterize health effects (i.e, hazard identification) are a pre-
requisite for developing predictive models. These
methods have been developed for some target systems and
processes (e.g., genotoxicity), but not for others (e.g., im-
munotoxicity). Thus, in some areas of health research
(e.g., developmental neurotoxicology), research efforts in
hazard identification must precede work on predictive
models"even though the development of such models is
the predominant goal of health research.
Following is a brief discussion of research needs
under the two topics of hazard identification and dose-
response assessment. These needs are addressed by all
divisions of HERL.
2.1.2.1 Hazard Identification
If no toxicologic data are available for a chemical, in-
vestigators must first focus their efforts on answering this
question: What health effects are produced by exposure to
the chemical? Answering the question involves develop-
ing and/or running procedures that screen for possible ef-
fects. Once researchers suspect a chemical produces a
particular type of health effect, efforts can turn to charac-
terizing the nature and defining the mechanism for these
effects. This research may pinpoint the target organ(s)
responsible for the effect(s), improve the sensitivity of the
quantitative dose-effect estimations, and assess the
similarity in expected response between the laboratory
species and humans (if the data were not obtained from
humans).
Hazard identification research has two primary goals:
1) to develop, refine, and validate methods for use in
screening and characterizing potential health hazard; and
2) to apply these methods to the characterization of
specific chemicals of interest to the regulatory programs.
Screening. HERL needs methods to evaluate the
potential for new and existing chemicals to cause potential
health hazards. These methods should be rapid, simple,
and economical to administer, while providing reliable
qualitative, presumptive evidence for a particular type of
health effect (e.g., genetic damage). Screening methods
have two general applications: 1) to determine if exposure
to a specific chemical can produce a particular health ef-
fect and whether further testing is required, or 2) to estab-
lish which of the large number of chemicals now released
into the environment should be tested first.
The development of screening methods to prioritize
chemicals for further testing is a prime example of a risk
assessment-related activity that benefits the program of-
fices but has limited applicability to setting regulatory
levels. Research in this area, therefore, is not a major
component of the health research program. Given the in-
creased Agency concern for noncancer health effects,
however, the development of in vivo methods that focus
on some of the newer areas of health concern (e.g., im-
munotoxicology) will be supported. Similarly, the
development of in vitro approaches to screening, especial-
ly in areas in which short-term, cost-effective tests have
not been available (e.g., neurotoxicology), will receive in-
creased attention. These in vitro models can also be used
by researchers examining cross-species mechanisms and
pharmacokinetics"both important components in devel-
oping predictive models of dose and effect.
HERL also needs to concentrate on developing
qualitative and quantitative relationships between chemi-
cal structure and related biological activity (i.e., structure-
activity relationships [SARs]). A better understanding of
SARs will aid in identifying/predicting health effects as-
sociated with new chemicals and setting priorities for
toxicity testing. Before SAR research can be more fully
developed, however, HERL needs better methods for iden-
tifying effects and better approaches for evaluating chemi-
cal structure.
Characterization. Developing methods to charac-
terize hazard is a much more demanding task than
developing screening methods because the former are used
to precisely define and verify relationships between ex-
posure and dose and between exposure and various health
effects. HERL needs approaches for specifying the tar-
get^) of toxicity, the progression of toxicity with dose and
time, the severity of given effects, and the interaction of
concurrent or sequential toxicities. Furthermore, for a
given target organ, researchers may need a number of
methods to characterize the variety of possible health out-
comes and their inter-relationships. These methods must
be sufficiently rigor-ous to associate single or multiple
mechanisms/targets with the expression of single or multi-
ple health effects. For example, as a result of either single
or multiple mech-anisms, a neurotoxicant may produce a
variety of outcomes (e.g., learning disabilities, visual im-
pairment, motor dysfunction).
The use of sensitive test methods to characterize
health outcomes can also serve as a major source of input
data for predictive models. Thus, because of the impor-
tance placed on these models, research related to hazard
2-3
-------
characterization (as opposed to screening) is a major com-
ponent of the health research program.
HERL also needs methods to characterize the dose
of a chemical to a target organ. The uptake, distribution,
and disposition of a chemical within the body is in-
fluenced by the concentration (dose) to which a person is
exposed, the duration of that exposure, and the portal of
entry for the pollutant. Analyzing these influences allows
better estimation of health effects in the various body sys-
tems. For example, the pulmonary function may be im-
paired by lower levels of an inhaled pollutant than the
reproductive function"not because the lungs are more sen-
sitive to the chemical than the reproductive tract, but be-
cause more of the chemical contacts the lungs (the portal
of entry). The growth of a pharmacokinetic program in
HERL will foster research efforts to develop these
methods for characterizing target dose.
2.1.22 Dose-Response Assessment
A dose-response assessment estimates the relation-
ship between the magnitude of the exposure to a pollutant
and the probability that the health effect in question will
occur. Often the data needed to make this assessment are
not available, and risk assessors must instead estimate the
relationship by extrapolating from data gathered under dif-
ferent conditions. Most exposures to humans involve low
doses and chronic exposures, while most available data on
various chemicals have been gathered on laboratory
animals, at high doses and with acute exposures. Alterna-
tively, either animal or human data may be available for a
given chemical, but the information was gathered in rela-
tion to a different route of exposure than that under current
examination.
Because of the paucity of appropriate available data,
and because of a lack of understanding of the underlying
biological mechanisms responsible for health effects, large
uncertainties hamper the risk assessment process. HERL
needs to develop, refine, and validate models which
predict the dose of a chemical that reaches the human tar-
get tissue as well as the health effects that will result. To
be accurate, these models must take the underlying
biological processes that are affected by chemical ex-
posure into account. Two types of models are needed:
Predictive Models of Dose. Over the last few years,
a goal of quantitative risk assessment has been to use
"delivered dose""the dose of proximate toxicants,
whether parent compound or metabolite, at the tissue
site of toxic action"rather than applied dose or am-
bient concentration. Determining this delivered dose
is an extension of exposure assessment, in that the
direct exposure of the target tissue is examined free of
the various physiological fate and transport processes
by which the body filters, attenuates, degrades, and
modifies compounds absorbed from its environment.
To reduce uncertainty in the risk assessment process,
however, scientists must analyze the effects of dif-
ferent conditions of exposure on the amount and pat-
tern of delivered dose. For example, being able to
define the delivered dose of a pollutant would be very
useful in comparing the toxic effects of exposures to
that pollutant by different routes of administration. In
this way, extraneous factors such as different degrees
or rates of absorption can be accounted for, resulting
in more meaningful comparisons. Research to deter-
mine ways to estimate tissue-level doses is also
necessary for progress in mechanistic biological
modeling of toxicity, which requires extensions of ex-
posure assessment to the internal sites where these
mechanisms occur.
These data are critical to the ultimate focus of this ef-
fort: the development of theoretical and computation-
al models to predict dose levels, which can then be
compared across species and across exposure
scenarios. In the past, researchers used compartmen-
tal models that reflected primarily mathematical con-
structs. The proposed research in this area will
generate data for pharmacokinetic models that use as
input physiological parameters of the system being
modeled as well as the molecular structures and reac-
tivities of the pollutants under consideration. Valida-
tion of these models will allow researchers to scale
exposure, dose, and effects data observed in one situa-
tion to different exposure conditions and/or popula-
tions.
Predictive Models of Effects. The ultimate goal of
dose-response assessment is to estimate the incidence
of a particular health effect at human exposure levels.
Extension of the dose-effect curve downward from
the levels at which laboratory animals are exposed to
the levels at which humans are usually exposed re-
quires an understanding of the various rates of attack,
repair, and propagation of damage across species.
HERL, therefore, needs to develop biologically
plausible dose-response models that consider the
potential for different biological mechanisms to elicit,
initiate, or contribute to the health effect of concern.
The primary focus of this research is to better under-
stand the role of various biological processes on
chemically induced injury and to produce models
2-4
-------
flexible enough to incorporate new information as it
is obtained.
Specific research areas include 1) intra- and inter-
species extrapolation (e.g., mechanisms of action,
species and subpopulation sensitivity); 2) extrapola-
tion of health effects across different exposure
scenarios; and 3) incorporation of recent mechanistic
concepts into predictive models.
2.1.3 Research Plan
The health research program is organized to address
the major categories of health effects that result from ex-
posure to environmental chemicals, including genetic,
developmental/reproductive, neurobiologic, pulmonary,
and immune effects. Limited programs also exist to
evaluate cardiovascular and hepatic effects. Recently,
HERL has also developed a formal program to evaluate
pharmacokinetics. In each scientific discipline, HERL
prioritizes research by integrating Agency needs (mission)
with scientific priorities dictated by the state-of-the-
science in that area. The following discussion introduces
the laboratory priorities and directives for each of these
disciplinary areas in relation to the two most important re-
search topics for determining health effects"hazard iden-
tification and dose-response assessment. The divisions
apply these methods and models to chemicals of
regulatory interest; information gathered in these efforts
falls under the research topic chemical-specific data.
Finally, the divisions examine some research topics that
cut across hazard identification and dose-response assess-
ment, such as biomarkers. Note that in the more detailed
discussions of the divisions' activities in the remainder of
the section, cross-cutting research efforts are outlined
under separate sections; however, in the summaries of
each division's activities, the cross-cutting efforts are in-
cluded under hazard identification and dose-response (as
appropriate).
2.1.3.1 Neurotoxicology
The neurotoxicology program is designed to identify,
quantify, and characterize the effects of environmental
pollutants on the adult and developing nervous system. In
order to evaluate these effects thoroughly, research efforts
encompass all levels of neural organization, including
whole animal, cellular, subcellular, and molecular.
Current interest in the use of noncancer endpoints for
risk assessment has focused considerable attention on
neurotoxicology. Various regulatory programs in EPA are
planning to require recently developed in vivo screening
tests to be used in routine testing. There is a clear need,
however, for in vitro tests to complement or replace the
more costly in vivo tests. The Agency is committed to
support research using in vitro technology and thus will
place an increased emphasis on these needs in
neurotoxicology.
A distinctive characteristic of the nervous system is
the complexity of the processes it mediates and controls.
Perhaps unlike any other area of health research,
neurotoxicology requires the development of a variety of
validated test methods that can be used to identify, quan-
tify, and characterize neurotoxic outcome following
toxicant exposure. Researchers will continue to develop
and validate test methods, especially those for evaluating
sensory, motor, and cognitive dysfunction produced by
environmental chemicals. Research in this program will
determine the extent to which current assessment proce-
dures can predict the appropriate neurotoxic outcomes in
humans. Studies on interactive effects of various classes
of neurotoxicants will also be conducted.
Neurotoxicity can be produced by a variety of
mechanisms that can be studied at several levels of
neuronal organization. Research, therefore, will focus on
understanding mechanisms of neurotoxicity and factors
that affect neurotoxic outcome (e.g., compensatory
mechanisms, exposure conditions). A related issue is that
neurotoxic outcome is dependent on the extent and site of
insult. Research will focus on understanding the relation-
ships between neurotoxicity measured at the various levels
of neuronal organization.
A first step in the development of predictive models
for neurotoxicity is a better understanding of extrapolation
issues related to age, species, route, and exposure
scenario. Research efforts will thus address the ability of
different test methods to predict effects across species and
as a function of various dosing scenarios and routes of ad-
ministration. Included in the area of species extrapolation
is the development of methods that provide a link between
what can be measured in humans and homologous animal
models. HERL also needs data concerning adaptation,
recovery of function, and developmental and delayed
neurotoxicity.
This research represents both new areas of emphasis
and growth areas for the existing program. Efforts to
validate methods for characterizing neurotoxic outcome
and for extrapolating these results to humans reflect a
general evolution in the state of the science. A major new
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area of research for the neurotoxicology program will be
in vitro test methods. Future research directions in
neurotoxicology can be highlighted as follows:
Hazard Identification
n Complete validation of primary in vivo screen for
routine neurotoxicological assessments
D Develop and validate in vitro tests to be used in
routine neurotoxicological assessments
n Validate methods to characterize neurotoxic out-
come, linking effects observed at the screening
level to effects quantified at the secondary levels
n Develop and validate methods and approaches to
identify and characterize developmental neurotox-
icity
a Develop and validate cellular and molecular
predictors for neurotoxicity
n Evaluate structure-activity relationships of neuro-
toxic agents using in vivo or in vitro procedures
Dose-Response Assessment
n Evaluate cellular and molecular mechanisms of
neurotoxicity
n Emphasize studies concerning extrapolation issues,
such as route-to-route, dosing scenario, species,
age, stress, and high-to-low-dose
n Understand mechanisms of compensation, recov-
ery of function, and delayed and/or long-term dys-
function produced by acute or intermittent
exposures
n Address the additivity hypothesis for combinations
of neurotoxicants on high-to-low-dose extrapola-
tion
2.1.32 Genetic Toxicology
The genetic toxicology program is designed to ex-
plore the influences of environmental pollutants on genetic
changes in somatic and germinal tissues (mutagenesis)
and the conversion of normal cells to neoplastic cells (car-
cinogenesis). Researchers analyze both direct and indirect
interactions with genetic material for environmental
chemicals, complex mixtures, and genetically engineered
microorganisms.
Historically, hazard identifications and risk assess-
ments in the Agency have emphasized cancer as a health
endpoint Considerable progress has been made in
developing both in vitro and in vivo methods for detecting
genotoxic chemicals and in understanding the relation-
ships between the genotoxicity and the carcinogenicity of
environmental chemicals. Within the framework of the
risk assessment paradigm, research emphasis is now shift-
ing toward the development of data that will allow the ex-
trapolation of results between in vitro and in vivo systems
and between rodent and human systems. In doing so, re-
searchers will also shift emphasis to in vitro and in vivo
human systems.
The induction of cancer involves many potential
mechanisms (e.g., gene or chromosomal mutation,
heritable changes in DNA transcription reflected in altered
gene expression) and multiple stages in tumorigenesis.
Researchers will therefore focus on understanding
mechanisms of carcinogenesis and the factors that modu-
late neoplastic changes. Because certain substances seem
to induce tumors without appearing to induce genotoxic
effects, HERL must also develop methods and models for
nongenotoxic carcinogens.
The development of predictive models will be
facilitated by the development of biomarkers for exposure
and effects. The division will continue to emphasize the
application of DNA adduct dosimetry in exposure assess-
ment for genotoxic chemicals. Efforts will also be
directed to understanding the relationship between
molecular markers (e.g., oncogenes and tumor suppressor
genes) and mechanisms of cancer development.
These research areas reflect a shift in emphasis from
the current genetic toxicology program. Less emphasis
will be placed on the application of short-term bioassays
and greater emphasis on understanding the issues related
to extrapolation that are crucial to dose-response modeling
and risk assessment. Future research directions in genetic
toxicology can be highlighted as follows:
Hazard Identification
a Develop computational tools to predict the
lexicological activity of chemicals based on
molecular structure and existing data
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n Develop in situ genetic tests (plants and animals)
for environmental monitoring and personal
monitoring methods to quantify human exposure to
genotoxic agents
n Improve gene cell assays for heritable genetic
damage
D Improve methods to detect and characterize the ef-
fects of nongenotoxic carcinogens
n Improve approaches to identifying genotoxic com-
ponents in complex mixtures, including interpreta-
tion of data, but decrease emphasis on actual
application (i.e., sample collection and chemical
characterization)
I Dose-Response Assessment
n Examine molecular mechanisms of cancer (both
for genotoxic and nongenotoxic chemicals)
n Explore the molecular basis of somatic and gene
cell mutation
n Develop models to extrapolate between in vitro
and in vivo systems and between rodents and
human systems
Cross-Cutting Issues
n Develop biomarkers of exposure and effect with
emphasis on molecular dosimetry and molecular
alterations
2.1.3.3 Pulmonary Toxicology
The pulmonary toxicology program, which is jointly
directed by the Environmental Toxicology Division and
the Human Studies Division, is designed to explore the ef-
fects of environmental pollutants on pulmonary function
in both laboratory animals and humans. The program's
major research components in both laboratory work with
animals and clinical and epidemiologic studies with
humans make it unique in HERL. The program em-
phasizes examination of the effects of inhaled pollutants,
including both gases and particles.
Historically, pulmonary effects"most notably, of
criteria pollutants"have played a prominent role in the
Agency's regulatory program in air. A variety of pul-
monary function tests are available for hazard identifica-
tion in both laboratory animals and humans. Human
clinical studies, however, are limited to examining acute
exposures at or near ambient concentrations. Consequent-
ly, major uncertainties still exist about the development of
chronic lung disease following long-term exposure to air
pollutants and the extent to which animal models of lung
disease predict the human response. A continuing major
focus of the pulmonary research program is the extrapola-
tion issues related to mechanisms of lung injury across
species and across exposure conditions.
The development of predictive animal models will be
facilitated by parallel studies in rodents, nonhuman
primates, and humans focusing on similar endpoints with
relevance to mechanisms of lung injury and host defenses.
These studies will be extended to address the relationship
between lung dosimetry and pulmonary effects.
Mechanistic studies in animals will also continue to focus
on the pathogenesis of toxicant-induced lung diseases.
The development of animal models for pulmonary disease
(e.g., emphysema, asthma, obstructive and restrictive lung
disease) will permit parallel studies with human subjects
to address the issue of susceptible populations. Research
to define biochemical markers that reflect early changes in
lung function (e.g., biomarkers of pulmonary immune
function and fibrotic changes in lung structure) will also
facilitate cross-species extrapolation.
HERL researchers in pulmonary toxicology will con-
tinue to emphasize the development of chemical-specific
information in support of the national ambient air stand-
ards (e.g., ozone, NO2, and acid aerosols) as well as
regulations for hazardous air pollutants (e.g., methanol,
volatile organics). These research areas represent the
progression of the program toward the development of
predictive models for dose-response assessment. For
laboratory animal studies, greater emphasis will be placed
on relating tissue dose to early indicators of chronic lung
disease. The human clinical studies program will ex-
perience some growth in the areas of lung biology and
dosimetry. Future research directions in pulmonary
toxicology can be highlighted as follows:
Hazard Identification
a Develop in vitro methods for evaluating pulmonary
toxicity using human cells
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a Develop and refine methods to characterize lung
dosimetry in humans including, deposition, absorp-
tion, metabolism, and elimination
Dose-Response Assessment
D Develop animal models for human lung disease
(e.g., emphysema, aging, asthma)
a Evaluate mechanisms of lung injury related to ex-
posure parameters (intensity and duration), chronic
lung disease, and role of inflammatory processes in
both laboratory animals and humans
D Evaluate concentration/time interactions
Chemical-Specific Data
D National Ambient Air Quality Standard pollutants
(e.g., ozone, NO2, acid aerosols)
n High-production-volume chemicals (e.g., metha-
nol, VOCs)
Cross-Cutting Issues
a Gather data on complex mixtures (e.g., alternative
fuels, indoor air)
n Develop biomarkers of lung injury
2.1.3.4 Immunotoxicology
Immunotoxicology is an emerging program in HERL
developed under the Environmental Toxicology Division
and the Human Studies Division, and it is designed to in-
vestigate the effects of environmental chemicals on im-
mune function and to relate these effects to increased risk
of infectious, neoplastic, allergic, and autoimmune dis-
ease. In its formative stages, this program focuses on
developing and validating both in vivo and in vitro
methods for hazard identification. This research will iden-
tify the most sensitive and predictive methods for screen-
ing and characterizing immunotoxicants and facilitate the
development of Agency test guidelines. Especially impor-
tant will be an understanding of the relationship of these
test methods to actual compromised immune function (i.e.,
increased susceptibility to disease) and the significance to
risk assessment.
This program will address the use of immunotoxicity
test results in risk assessment. A few tests will directly
measure (in vivo) host resistance, hypersensitivity, or
autoimmunity in animal models. The issues involved in
extrapolating the results to humans are the same as for any
other type of toxicity data (e.g., extrapolation from animal
to human or from high to low dose). For certain environ-
mental toxicants, analysis of the relevance of many rodent
exposure studies to human health effects awaits further in-
vestigation. HERL's ability to conduct immunotoxicologi-
cal testing in both human (in vivo and in vitro) and animal
models is unique. Well-designed studies conducted in
parallel in humans and animals are being developed and
applied. Epidemiological studies and research designed to
assess chemical immunomodulation in man are necessary
to confirm the animal data. This combined approach will
result in a better understanding of immunotoxic effects of
pollutants, and facilitate the risk assessment process based
on immunotoxicological data.
Research to validate test methods will include studies
to understand mechanisms of action for immunotoxicants,
local versus systemic effects as they relate to route of
toxicant exposure, species dependence, and genetic in-
fluence on susceptibility. Biomarkers will also be
developed that are predictive of enhanced susceptibility,
severity, and/or recovery from disease. The development
of host resistance models will lead to a better under-
standing of how exposure to toxic chemicals affects sus-
ceptibility to disease as a result of suppression or
unwanted stimulation of the immune system.
Future research directions in immunotoxicology can
be highlighted as follows:
Hazard Identification
D Develop models of infectious, neoplastic, and aller-
gic disease
D Develop and validate methods for screening and
characterizing immunotoxicants both in vivo and in
vitro
Dose-Response Assessment
n Examine mechanisms of immunotoxicity
D Develop models for evaluation of sensitive popula-
tions
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Cross-Cutting Issues
n Develop biomarkers that reflect immunocompe-
tency
2.1.3.5 Pharmacokinetics
The pharmacokinetics program within the Environ-
mental Toxicology Division is designed to explore the
quantitative relationships between exposure and integrated
dose to the target tissue. Research efforts include the
quantitative study of absorption, distribution, metabolism,
and elimination of pollutants and the use of mathematical
models to describe these processes. This program area,
which is new for HERL, will experience significant
growth in the coming years. The two major areas of re-
search will be characterizing dose to the target site for
various environmental chemicals (hazard identification)
and developing predictive models of dose (physiologically
based pharmacokinetic models) for dose-response assess-
ment
Researchers will focus on developing methods and
collecting data to characterize dose to the target site for a
variety of toxicant classes of special interest. These data
are needed to develop predictive dose models and to im-
prove the accuracy of risk assessment. Research efforts
will also develop and improve biomarkers for use in char-
acterizing dose to the target tissue. Efforts to collect more
experimental and physiological data relative to effective
dose will be coupled with studies on toxic mechanisms of
action to reduce uncertainties inherent in the use of exter-
nal exposure information for risk assessments.
Experimental dosimetry studies will also begin to ex-
amine the influence of route, duration, chemical matrix,
and rate of exposure on delivered dose to better under-
stand the uncertainties inherent in extrapolating data from
one exposure scenario and species to another. These data
will be used to evaluate theoretical dosimetry models to
predict target dose of inhaled gases and ingested or ab-
sorbed chemicals in humans and laboratory animals. Con-
sideration will be given to defining factors (both
physiological, physicochemical, metabolic, and anatomi-
cal) that influence pharmacokinetic parameters including
structure-activity relationships.
Mathematical formulations for the deposition of com-
pounds following oral, dermal, or inhalation exposure will
be evaluated using data collected from laboratory animals
and humans on absorption, metabolism, distribution, and
elimination of parent compounds and their metabolites.
These models will provide for better predictions of dose-
equivalency across species and across exposure condi-
tions.
Until the recent reorganization of HERL, small pock-
ets of pharmacokinetic research were ongoing but were
not well coordinated. The reorganization has resulted in
an integrated program in pharmacokinetics that will ex-
perience major growth in the coming years. Expansion of
the program will focus on developing the scientific exper-
tise to measure toxicants in biological tissue, evaluating
pharmacokinetic properties including their relationship to
health outcomes, and developing the predictive models
needed for dose-response assessment
Future research directions in pharmacokinetics can be
highlighted as follows:
Hazard Identification
n Develop data on pulmonary absorption in the lung,
including improved morphometric data
n Develop and validate in vivo and in vitro methods
for studying dermal and oral absorption
n Develop and validate in vitro and in vivo methods
for studying elimination (metabolism and excre-
tion) of toxicants
D Expand efforts to characterize dose to target tissue
Dose-Response Assessment
n Place increased emphasis on developing extrapola-
tion models for dose, particularly cross-species,
route-to-route, and high-to-low-dose and acute-to-
chronic-exposure
n Evaluate factors (e.g., age, sex, disease status) that
influence target dose to determine the appropriate-
ness of physiological, anatomical, and physico-
chemical parameters used in predictive models
a Evaluate influence of elimination processes, in-
cluding metabolism and excretion, on target dose
Cross-Cutting Issues
a Develop biomarkers of dose
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2.1.3.6 Developmental and Reproductive Toxicology
The developmental and reproductive toxicology pro-
gram is designed to explore the effects of environmental
pollutants on both development (i.e., interference with
developmental processes) and reproduction (i.e., the func-
tional integrity of both the male and female reproductive
systems).
In general, in vivo methods are available for detecting
developmental toxicity, but little is known regarding the
mechanism of this type of toxicity. Researchers will con-
tinue to develop and validate embryo organ cultures as in
vitro tools for examining biochemical and molecular
events associated with abnormal development. Research
in the developmental area will become increasingly
focused on interpretation and extrapolation of methods to
characterize developmental effects. Reproductive studies,
on the other hand, will focus more on methods validation.
An improved ability to interpret and extrapolate both
developmental and reproductive effects necessarily relies
on a better understanding of mechanisms. Efforts will be
directed toward understanding alterations in endocrine
control of reproductive function, reproductive dysfunction
(including early pregnancy loss), and mechanisms of
teratogenesis as well as toward examining the relevance of
maternal toxicity on fetal well-being. Predictive models
will also be improved by studies that link dose to the fetus
to developmental outcome. Also, research to understand
the pathological events in homologous developmental
models will improve interspecies extrapolation of
teratogenic effects.
These research areas reflect, to a large extent, the
natural progression of research in developmental and
reproductive toxicology from hazard identification to
dose-response assessment. Growth in this program will
occur primarily in the areas of mechanisms of
teratogenesis (structure/function), ovarian physiology, and
mathematical modeling of dose-response data. Less em-
phasis will be placed on the development of in vivo bioas-
says. Future directions in the program can be highlighted
as follows:
Hazard Identification
D Develop in vitro models for evaluating
mechanisms of developmental toxicants
n Systemically evaluate structure-activity relation-
ships for developmental toxicants
n Evaluate the relationships between gamete produc-
tion/function and pregnancy outcome
n Complete development of alternative reproductive
test
a Develop cell cultures to better identify direct-act-
ing reproductive toxicants
Dose-Response Assessment
a Evaluate physiological, cellular, and molecular
mechanisms of developmental and reproductive
dysfunction, in vitro and in vivo
D Evaluate quantitative dose models for developmen-
tal and reproductive toxicity
D Link administered dose to delivered dose to out-
come for both developmental and reproductive
endpoints
n Evaluate the influence of critical periods and
maternal toxicity on developmental outcome
n Identify and evaluate the models for susceptible
populations (including the aged)
D Elucidate the role of endocrine factors in reproduc-
tive toxicity
2.1.3.7 Epidemiology (Human Studies)
The epidemiology program is designed to explore the
magnitude of health risks associated with exposure to en-
vironmental pollution through studies of humans in their
natural environment and to refine methods for conducting
this research. The products of epidemiologic studies pro-
vide data that help to confirm hazards to human health, to
illuminate health hazards not identified experimentally
(e.g., by clinical, animal, or in vitro research), and to
clarify the relative importance of hazards to the popula-
tion. The HERL epidemiology research program takes ad-
vantage of collaborative interactions with investigators in
the field who are associated with universities, public
health departments, and other federal, state, or local agen-
cies.
Epidemiology research has made important contribu-
tions to the regulatory and rule-making activities of the
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Agency, especially relative to drinking water, water
quality, air toxics, and pollutants for which the Agency
has established National Ambient Air Quality Standards.
A major thrust of the HERL program is to integrate infor-
mation from diverse sources (e.g., clinical, in vitro,
animal, and field work) to develop research programs that
effectively address questions of environmental relevance.
In addition, new projects have been initiated to develop
and validate biomarkers of dose and effect that can be
used to study the impact on individuals and populations of
exposure to toxic materials in the environment. Future
directions in environmental epidemiological research can
be highlighted as follows:
Hazard Identification
n Develop and implement techniques to evaluate
public health impacts of drinking water disinfec-
tion
n Develop and validate methods to model and test
study design as field studies are being developed
n Develop estimates of human exposure and dose
that can be used to establish correlations to effects
observed in human populations in epidemiology
studies
Dose-Response Assessment
n Identify populations that are particularly sensitive
(responsive) to ozone in the environment
n Determine the magnitude of risk associated with
exposure to air-borne pollutants in individuals and
populations
D Determine the association between an acute
response to pollutant exposure and the develop-
ment of chronic conditions
Cross-Cutting Issues
a Develop and validate biomarkers of exposure and
effect for use in human populations naturally ex-
posed to toxic materials
2.2 NEUROTOXICOLOGY DIVISION
Most biological manifestations of mammalian ^"in-
cluding muscular movements, the capability to reproduce,
respiration, cardiac function, sensation, perception, and
cognition"are controlled by the nervous system, which is
composed of the brain, spinal cord, and a complex net-
work of nerves and supporting cells. The proper function-
ing of the nervous system is essential for health and
productive life. One of the major functions of the nervous
system is to transmit information or commands from one
component of the body to another; this function is ac-
complished by a complex interaction of various kinds of
nerve cells. Interruption of this process can result in a
variety of subtle effects, such as lowering of IQ scores in
children; indications of neurological dysfunction, such as
paresthesias in the extremities, muscle weakness, or
seizures; or neurodegenerative states resembling naturally
occurring neurological diseases such as Parkinson's and
Alzheimer's diseases.
Figure 2-4 is a simplified representation of the nerv-
ous system. Like other organs, the nervous system serves
to maintain homeostasis under certain input/output condi-
tions. Information concerning the external and internal
environment is relayed to the central nervous system
(CNS) by afferent neurons using chemical messengers
(neurotransmitters). Processing of this information by the
CNS may or may not result in output, which is transmitted
by efferent neurons also using chemical messengers. Out-
put from the CNS is usually seen as a changed motor
function or neurohumoral status. A chemical may inter-
fere with these input/output relationships, either by direct-
ly affecting the level of neuronal organization or by
interacting with another organ and, thus, affecting nervous
system function indirectly. The full range of neurotoxic
changes reported in humans include motor, sensory, cog-
nitive, and autonomic disturbances; and these effects are
studied at the neurobehavioral, neurophysiological,
neurochemical, and neuroanatomical levels of neural or-
ganization.
Neurotoxicants are chemicals, drugs, or other agents
that interfere adversely with the function of the nervous
system. There are many chemicals to assess for
neurotoxicity: EPA receives approximately 1,500 notices
of intent to produce new substances each year and 65,000
chemicals are already listed in the EPA inventory of
chemicals. Estimates concerning how many of these
chemicals are neurotoxicants vary by the class of chemical
in question and the exposure scenario. For example, of
the more than 1,400 active pesticide ingredients registered
by EPA, more than half are considered to be neurotoxic.
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ENVIRONMENTAL
STIMULI
\ 1 /
RECEPTORS
i
Afferent Neuron
' Transmitters
INPUT
HOMEOSTASB
CENTRAL
PROCESSOR
T
COGNITION AND
ASSOCIATION
Efferent Neurons
Transmitters
EFFECTORS
OUTPUT
Fig. 2-4:
Simplified Representation of the Nervous System
Some estimates suggest that only 3-5 percent of industrial
chemicals, excluding pesticides, have neurotoxic potential,
although 28 percent of industrial chemicals for which
there are occupational exposure standards have
demonstrated neurotoxic activity. As a result, estimates of
potential neurotoxicants in the EPA inventory range from
2,600 to over 18,000 chemicals.
Chemicals causing neurotoxicity include simple ele-
ments (e.g., lead, mercury), biological neurotoxins (e.g.,
botulinum toxin, tetrodotoxin), and synthetic compounds
(e.g., DDT and industrial solvents). In humans,
neurotoxic agents can cause a number of adverse effects
on the nervous system, including impairment in muscular
movement, alterations in sensation, deficits in learning and
memory, mood and personality changes, and disruption of
autonomic function.
Perhaps one of the earliest recognized neurotoxicants
is lead, which is contained in industrial emissions, leaded
gasoline, lead-based paints, foods, and beverages. Rela-
tively low levels of lead have been shown to impair cogni-
tive function in children. Another potent neurotoxicant is
mercury, which was implicated in an environmental
catastrophe during the 1950s in Minamata, Japan.
Methylmercury formed from an industrial effluent became
concentrated in fish and shellfish, which were
eventually consumed by local inhabitants. Com-
mon signs of mercury exposure included a lack
of coordination, speech impairment, and visual
problems; functional deficits were eventually as-
sociated with specific neurohistopathological al-
terations. A different type of neurotoxicant is the
mixture of chemicals known as the
polychlorinated biphenyls (PCBs), which are
stable, lipophilic industrial compounds known to
cross the placenta and intoxicate the fetus. Some
children of women exposed to PCBs during
pregnancy have been developmentally impaired
(e.g., hyperreflexia at birth, delays in
psychomotor development, and deficits in visual
accommodation).
Thousands of chemicals are produced for in-
dustrial use, and exposure to some of these
chemicals can produce a chemically induced
cetral-peripheral dying-back axonopathy, includ-
ing muscle weakness, alterations in fine motor
control, and paresthesias (e.g., abnormal prick-
ling, tingling) in the extremities. Solvents are
often used in glues, cements, and paints, and the
possibility of industrial exposure is high. In ad-
dition, toluene-based spray paints, various sol-
vents, and modeling cements are sometimes
abused, resulting in intoxication. Cases have been docu-
mented in which excessive abuse of solvents has led to
permanent neurodegeneration. Another class of chemicals
for which the potential for exposure is high is pesticides,
which include insecticides, rodenticides, and herbicides.
Workers exposed to pesticides display obvious signs of
poisoning, including tremors, weakness, ataxia, visual dis-
turbances, and short-term memory loss. Exposure to some
organophosphate pesticides can result in a delayed
neurotoxicity, including irreversible loss of motor function
and associated neuropathology.
Recently, concern has been expressed over the pos-
sibility that progressive neuro-degenerative diseases such
as Parkinson's disease may be related to pesticide ex-
posure, and that Alzheimer's disease may be related to
aluminum concentrations in drinking water. Even more
recent concerns have focused on possible neurobehavioral
effects in children exposed to pesticides via the diet.
Ample evidence links environmental chemicals with
neurotoxicity in humans; thus, there is a need to identify
and regulate such agents in the environment.
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2.2.1 Divisional Program
The overall objective of the Neurotoxicology
Division (NTD) is to provide the scientific basis and tech-
nological means for predicting, with a minimum of uncer-
tainty, whether an environmental agent will produce
neurotoxicity. The approach taken to this problem is to
model human neurotoxic disease in laboratory animals
and then to use the data collected in animals to make
predictions about possible neurotoxic risks in humans.
2.2.2 Division-Specific Research Needs
2.2.2.7 Hazard Identification
Procedures to assess sensory, motor, autonomic, and
cognitive function in animal models need to be developed,
validated, and refined to provide the basis for extrapola-
tion to human neurotoxicology. NTD also needs proce-
dures that will lead to the quantification and categorization
of developmental neurotoxicity. Little is known about the
potential for chemicals to produce long-term, residual al-
terations in the function and/or structure of the nervous
system following exposure in the early stages of develop-
ment. In addition, NTD needs short-term in vivo or in
vitro tests to identify, cellular and molecular changes that
are indicative of neurotoxicity. At the present time, initial
screening procedures are less than ideal due to a lack of
sensitivity to some neurotoxic effects (i.e., sensory deficits
and cognitive dysfunction), the labor-intensive nature of
the tests, and the relatively large number of animals re-
quired to achieve a statistically reliable answer. Proce-
dures based on biochemical endpoints or using more
recent advances in in vitro technology or molecular
neurobiology need to be developed, validated, and refined.
2.2.2J Dose-Response Assessment
Dose-response data generated in animal models must
be interpreted through acute and repeated dosing scenarios
and measures of sensory, cognitive, and motor function.
The use of homologous models to study chemical-induced
neurotoxicity would greatly improve the risk assessment
process. A better understanding is needed of inter- and in-
traspecies sensitivities, especially as they are related to
differences in metabolism and/or distribution of
neurotoxicants in the body. In addition, sensitivities of
various age groups differ. Research in this area should
also emphasize structure-activity relationships and
mechanism of action to the extent that the information
would be useful in the interpretation of dose-response as-
sessments.
Another area in which research is needed is the
analysis of the neural substrates that underlie some func-
tional endpoints of neurotoxicity. A neurotoxicant-in-
duced change in function implies that the chemical has
interfered with the neural (i.e., neurochemical,
neurophysiological) or anatomical substrate that mediates
or controls the functional endpoinL The magnitude or na-
ture of the functional change may be dependent on the
portion of the neural or anatomical substrate mediating the
functional change. A better understanding of these sub-
strates will lead to a reduction in the uncertainty as-
sociated with risk assessments that use those endpoints.
Specific environmental factors, including temperature
and stress, play a role in the manifestation of
neurotoxicity. Little is known about the importance of
temperature in this process; however, because many
toxicants seem to interfere with thermoregulatory
mechanisms, this endpoint might be a useful index of
toxicant exposure. In terms of stress, NTD is particularly
interested in the possibility that stress produced by ex-
posure to a neurotoxicant or as a result of the neurotoxic
effect can add to or synergize the effects of the
neurotoxicant. In addition, the compensation or adapta-
tion that sometimes occurs following repeated exposure to
neurotoxicants is well documented. Studies that lead to a
better understanding of compensation would be valuable
in the risk assessment process because establishing
thresholds is frequently difficult due to the compensation
that occurs after the first few exposures. Compensation
and adaptation are sometimes confounding variables in the
setting of tolerance limits.
2.2.2J Chemical-Specific Data
NTD needs to quantify the dose-dependent effects of
specific agents that may be of interest to the division and
the program offices. Such chemical-specific studies could
be useful for the continual validation and refinement of
functional and structural endpoints. NTD has in the past
provided data on specific agents, including xylene,
styrene, the acrylates, substituted pyridines, and triphenyl-
phosphite.
2.2.2.4 Biological Markers
NTD recognizes the need to assist in developing and
validating biological markers of exposure and effects in
human populations. These studies should provide biologi-
cal markers that identify possible health hazards, docu-
ment exposures and assess the efficacy of risk reduction
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strategies, and provide a scientific basis for interpretation
of biomarker data submitted to the Agency.
2.2.2.5 Pollutant Mixtures
Risks associated with human exposures to pollutant
mixtures also need to be identified and assessed. Such re-
search would determine the applicability of simplifying
assumptions to risk assessments of chemical mixtures and
compare the potency of various classes of pollutant mix-
tures using several bioassays. Of particular interest is the
development of a strategy for hazard identification with
these chemicals, i.e., one agent at a time, combinations of
selected agents, or the composite material obtained at a
specific site. The ability to assess the potential interactive
nature (i.e., additive or synergistic) of specific agents in
the mixtures is vital for reducing uncertainties in this area.
The mammalian nervous system is composed of
heterogeneous cell types that serve as diverse and unpre-
dictable targets of toxic insult. NTD research efforts will
continue to develop, validate, and refine techniques for
routine neurotoxicological assessments (e.g., those using
novel neurotypic and gliotypic proteins as biochemical in-
dicators of neurotoxicity) that are capable of detecting
damage to diverse cell types. This research will be under-
taken to develop and validate, using known neurotoxicants
and negative controls, a number of neurotypic and
gliotypic assays for detecting cellular and subcellular tar-
gets of neurotoxic attack. Identification of gene products
associated with injury-induced changes in protein markers
should also permit the diagnosis of neurotoxicity and ul-
timately lead to the development of in vitro assays. In ad-
dition, NTD will develop a stategy concerning the use of
in vitro screening data within a larger overall screening ef-
fort.
2.2.3 Research Plan
2.2.3.1 Hazard Identification
NTD has adapted a tiered approach to the study of
chemicals for neurotoxicity. This stepwise progression
from simpler to more specific and sophisticated assess-
ments begins with a basic battery of tests to determine if
further studies are necessary. In NTD, the first-tier in vivo
functional observational battery (FOB) and motor activity
tests have been developed and, as a battery, are currently
being validated. The FOB consists primarily of a
neurological examination for rodents, which is done in a
systematic and sequential fashion. It measures chemical-
related effects on sensory, motor, and autonomic function
and can be used to study dose- and time-dependent effects
in acute or repeated dosing protocols. The resulting
profile of neurological effects is expected to have diagnos-
tic value in the evaluation of unknowns. Research in this
program will continue to validate systematically FOB and
motor activity tests using known neurotoxicants that
produce diverse neurologic syndromes.
NTD will also validate second-tier tests used to con-
firm an agent's effect on the nervous system and to pro-
vide a more precise estimate of neurotoxicity for assessing
risk and setting exposure limits. Known neurotoxicants
will be used to assess the usefulness of the tests, which are
thought to measure specific structural and functional
defects in the nervous system. From the resulting data,
NTD will define which tests should be included in the
second-tier analysis of neurotoxicity.
NTD places a high priority on identifying and charac-
terizing the potential effects of chemicals on the develop-
ing nervous system. Both functional and structural
endpoints of neurotoxicity are of interest. The developing
organism is preferentially vulnerable to some
neurotoxicants, and the influence of developmental ex-
posure on functional endpoints such as learning and
memory must be assessed. Thus, some NTD research ef-
forts are focused on developing and validating a model of
cognitive development in an animal model. This research
is designed to obtain tests of cognitive function in animal
models that are homologous with tests used on humans in-
fants. Then, researchers will validate the sensitivity of the
animal model tests in identifying and characterizing cogni-
tive dysfunction following the animals' developmental ex-
posure to neurotoxicants.
Researchers will also focus on detecting and charac-
terizing toxicant-induced anatomical damage to the
developing nervous system. A number of developmental
issues will be addressed, including the influence of mater-
nal stress on postnatal function and the role of critical
periods of exposure in the manifestation and persistence of
developmental neurotoxicity. This program is based on
the premise that functional deficits resulting from develop-
mental toxic exposure are ultimately linked to alteration in
neural connectivity, and that alterations in neural connec-
tivity result from the disruption of certain developmental
processes that are particularly vulnerable to toxic ex-
posure. NTD will develop a test battery with known
developmental neurotoxicants for a number of endpoints,
including functional (sensory, motor, and cognitive func-
tion) and neuroanatomical measures. The long-term ob-
jective of this research is to establish the validity of a
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number of anatomical markers that are indicative of ex-
posure to neurotoxic agents in the developing organism.
In addition, this work aims to establish the functional con-
sequences of toxicant-induced alterations in connectivity.
Eventually, this effort will lead to the development and
validation of indicators of neurotoxic exposure in humans.
Although NTD can perform routine assessments of
material for neurotoxicant-induced structural alterations,
such endpoints are generally insensitive to early and subtle
changes. Nor do they address neurological integrity over
the range of sub-neuropathic doses or at earlier time
points, when overt anatomical damage is not present and,
if present, may be reversible. NTD research will focus on
the use of gene probes/products of neural substances as-
sociated with events that are specific to regeneration
and/or degeneration following toxic exposure. Such an
approach should permit a sensitive, quantifiable measure
of the progression of toxicant-induced nerve damage and
could lead to the development of an in vitro screening pro-
cedure for neurotoxicity.
To understand the physicochemical properties that
cause neurotoxicity, NTD will examine the structure-ac-
tivity relationships of selected series of neurotoxic com-
pounds. The procedures used will depend on the chemical
class and type of neurotoxicity produced.
2.2.32 Dose-Response Assessment
A number of NTD research efforts focus on obtaining
dose-response data in acute and repeated dosing
paradigms. FOB/motor activity, reflex modification,
visual evoked potentials, and schedule-controlled behavior
procedures have been developed. These procedures have
been validated to the extent that dose-response data could
be used to evaluate the applicability of various risk assess-
ment scenarios, including the standard no-effect-level ap-
proach, and effective dose values could be extrapolated
from dose-response curves. NTD will place a high
priority on the generation of complete dose-response cur-
ves for several different functional measures for a select
population of prototypic neurotoxicants in order to com-
pare the effectiveness of the various risk assessment
scenarios. A number of procedures developed in NTD
will also be used to evaluate structure-activity relation-
ships routinely, including the FOB/motor activity first-tier
screen, visual evoked potentials, reflex modification tech-
niques, schedule-controlled behavior, and neurotypic/
gliotypic protein markers. The information derived from
such studies will lead to more accurate assessment of risk
associated with exposure to chemicals.
Research in NTD uses sensory evoked potentials to
measure stimulus-elicited changes in the electroen-
cephalogram recorded from sensory areas of the brain.
Because visual changes are the most frequently reported
sensory effects, NTD research will focus initially on the
use of visual evoked potentials to study dose-related
neurotoxicity. The overall objective of this research effort
is to use tests of visual function having known relation-
ships to visual perception and to structural and functional
segments of the visual system. Research in NTD will also
focus on the brainstem evoked response (BAER) to study
auditory dysfunction in rats. BAER is used widely in
neurology, otology, and audiology to detect and diagnose
auditory and, in some cases, more general central nervous
system dysfunction. This research is intended to develop
a validated test of auditory dysfunction in rats.
Another research emphasis is the use of behavioral
procedures to study chemical-induced sensory dysfunc-
tion. The reflex modification procedure will be validated
in laboratory rats for the auditory reflex using known
ototoxicants, and the results will be compared to data
gathered on other neurotoxicants having no ototoxicity.
Once the procedure has been validated for auditory func-
tion, a multisensory procedure will be used to determine
the usefulness of the technique in measuring
neurotoxicant-induced changes in other sensory modali-
ties.
The central nervous system is essential for directing
attention to important environmental events and for ac-
quisition (learning) and retention (memory) of such infor-
mation. At present, no data suggest that either a
functional observational battery or tests of motor activity
used in a first-tier screen will provide information on
whether a compound alters cognitive function. NTD re-
search will compare simple, rapid tests of learning and
memory to identify potential screening tests for learning
and memory. The effectiveness of these procedures will
be assessed by evaluating a number of positive and nega-
tive control compounds in terms of relative sensitivity and
selectivity of effects. These studies will determine dose-
related effects of the chemicals given under repeated, as
well as acute, dosing regimens. The intended result of this
work is a fully validated behavioral battery of tests for
toxicant-induced cognitive dysfunction that can be used at
the first tier of testing.
Methods are needed to characterize dose-related ef-
fects of neurotoxicants on cognitive function. Using
known neurotoxicants, research in this area will develop
and validate tests in rodents for the quantification of learn-
ing (automaintained reversal, repeated acquisition in the
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radial maze), memory (spatial delayed response, nonspa-
tial delayed response) and attention (signal detection).
This effort is directed to reaching a better understanding of
the neurobiology of cognitive behavior that will facilitate
extrapolation of data from animal models to humans.
Homologous models and/or fully validated models of cog-
nitive function will provide the necessary basis for estab-
lishing safety factors in estimations of risk to human
health.
The limbic system plays a role in mediating convul-
sions and seizure disorders, which are two common
responses following acute exposure to higher levels of
toxicants. Limbic structures show a predilection for the
initiation and spread of epileptiform activity. It is possible
that at lower levels of exposure to some chemicals, epilep-
tiform activity occurs in the brain at doses below those re-
quired to elicit more severe behavioral manifestation of
seizures. Thus, repeated low-level exposure to convul-
sants may lower seizure thresholds, sensitizing the brain
and promoting the initiation and spread of seizures from
the limbic system to other regions of the brain. Research
in this area seeks to examine the selectivity of the brain
site and nature of the neurotoxicant-induced disruption of
limbic forebrain function that may underlie ensuing
seizure disorders.
Numerous toxicants are known to produce motor dys-
function in experimental animals and humans. Study of
chemical-induced motor dysfunction is carried out with a
variety of techniques and in a number of testing scenarios,
including the development of dose-effect and time-course
characterization of toxicants, comparisons across
toxicants, and the determination of the mechanisms by
which toxicants affect motor function. Using positive and
negative controls, researchers will validate the parametric
conditions that are important for optimal testing and use
appropriate testing procedures to determine the relation-
ship between acute and repeated exposure effects, par-
ticularly in regard to compensation. Schedule-controlled
responding will be used to study the mechanism of action
of selected neurotoxicant agents, particularly those that
produce a dying-back axonopathy.
One of the most common characteristics of
neurotoxicants that affect motor function is that they can
cause a degeneration of the axon. NTD researchers will
study the effects of neurotoxicants on the transport of
material in the axon by measuring the time-dependent dis-
tribution of radiolabelled materials injected into it. Project
goals include the development of a relatively simple and
noninvasive method to study the retrograde transport of
materials, determination of the effects of various known
neurotoxicants on retrograde transport, and the develop-
ment of a similar procedure for the study of retrograde
transport in the peripheral nerves of humans.
The metabolism of inositol phosphates (IPs) repre-
sents an important and vulnerable step in signal transduc-
tion in nerve cells. Initial studies have shown that IP
turnover in the sciatic nerve of hens treated with or-
ganophosphate compounds is altered within days of ex-
posure, and some of these changes are directly related to
neuropathic effects rather than inhibition of acetyl-
cholinesterase. Therefore, determination of IP metabolism
may be a relatively simple biochemical marker for or-
ganophosphate-induced neuropathy that will be inde-
pendent of cholinesterase inhibition. This work is
intended to develop more sensitive and earlier tests for im-
pending peripheral neuropathies that could augment the
traditional, less sensitive tests now used in regulatory
programs.
Several projects within NTD could ultimately result
in the application of in vitro techniques, including the use
of neurotypic and gliotypic protein markers and gene
probe analysis, to the assessment of dose-response and
structure-activity relationships. Additional in vitro techni-
ques are needed for neurotoxicity testing, including some
variant of currently existing techniques (i.e., primary cul-
tures, cell lines, or cloned cells). Although few studies in
NTD have been performed solely to study mechanism of
action, work in this area could be key to decreasing uncer-
tainty in the risk assessment process.
To perform such studies, the skill mix at NTD must
be changed or augmented. One area of potential interest is
the neurotoxicant-induced changes in ionic fluxes. Many
neurotoxicants adversely affect neuronal function by inter-
fering with membrane biophysics or the normal opening
and closing of ionic gates located in the membrane. Volt-
age or patch clamp techniques would permit the study of
many types of neurotoxicants, both in terms of their
mechanism of action and structure-activity relationships.
Another important research issue concerns the underlying
neural substrate that mediates functional measures of
neurotoxicity. Because information in this area would
facilitate the interpretation of neurotoxicant-induced chan-
ges in these endpoints, work in this area is another means
of reducing the uncertainty in risk assessment. At the
present time, several functional endpoints have been
developed and show sufficient promise, particularly in the
area of learning and memory, evoked potentials, reflex
modification, and brainstem evoked potentials.
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NTD currently has no capability for determining the
levels of parent compound or metabolites in regions of the
brain or periphery. During the next three to five years,
this capability will either be developed within NTD or
through the on-site contractor mechanism. NTD currently
conducts studies concerning responsiveness of different
strains of rats to selected prototypic neurotoxicants in the
FOB and motor activity evaluations. These studies will
provide the basis for designing future studies. Future
work in this area will also be concerned with the develop-
ment of tests or a strategy for testing nonrodent species.
NTD is also interested in the differential susceptibilities of
different populations to neurotoxicants.
Stress and the changes associated with stress in-
fluence the manifestation of neurotoxicity by affecting the
absorption, metabolism, and elimination of chemicals.
Research efforts will examine the influence of nonspecific
stress on dose-response curves to neurotoxicants; this
work could provide useful information concerning high-
to-low-dose extrapolation. Another important issue revol-
ves around the possible adverse effect of adrenotoxic
agents on brain structure and function. Research in this
area will use known neurotoxicant and adrenotoxicants to
damage components of the hypothalamic-pituitary-adrenal
axis and determine the response of the system to toxic in-
jury. This information 'is critical for understanding the in-
teraction between stress and the manifestations of
neurotoxicity"a necessary link for reducing uncertainty in
the risk assessment process.
In terms of the influence of temperature on the
manifestation of neurotoxicity, NTD research will com-
pare the consequences of acute versus repeated exposure
to prototypic neurotoxicant agents at various temperatures
and investigate the mechanism by which selected
neurotoxicants can influence the thermoregulatory system.
This work is directed toward the gathering of data that
permit the extrapolation of thermoregulatory effects of
xenobiotics seen in laboratory animals to humans, thereby
providing a basis for limit setting.
NTD will develop a strategy for studying the compen-
sation and adaptation that accompanies repeated exposure
to some neurotoxicants. Initially, studies will focus on the
use of a functional endpoint having a stable baseline of
responding, such as schedule-controlled behavior, and su-
perimpose repeated dosing to known neurotoxicants on
the baseline. The rate and extent of compensation that oc-
curs will be measured; and strategies to reveal underlying
homeostatic changes, such as the application of phar-
macological drug challenges, will be explored. The over-
all objective of this program is a strategy for studying the
role compensatory processes play in the expression of
neurotoxicity; such information can be used to reduce un-
certainty in the risk assessment process.
2.2.3.3 Chemical-Specific Data
NTD can study specific chemicals at several levels of
neural organization. Routine questions concerning the
capability of an agent to produce neurotoxicity can be
studied using the FOB and motor activity tests; while
more focused questions concerning sensory, motor, or
cognitive deficits can be studied with existing procedures.
The search for new and more efficient fuel products
has accelerated in recent years. Considerable concern has
been raised about the increasing use of methanol as an
energy source. NTD will conduct research on the potential
neurotoxicity of methanol. If other alternative sources or
additives are developed, further research will have to be
conducted to support the hazard identification process and
to characterize the chemicals for potential neurotoxicity.
2.2.3.4 Biological Markers
Using gliotypic and neurotypic protein markers as in-
dices of neurotoxicity may eventually lead to the develop-
ment of a biomarker for neurotoxicant exposure. Other
approaches to the development of such a biomarker show
promise and could be explored, perhaps through coopera-
tive agreements. For example, some neurotoxicant agents
(e.g., hexane or acrylamide) might produce an antigenic
reaction after exposure. Future work could be directed to
determining the nature of the antigenic reaction to these
molecules and developing a simple, rapid test that could
routinely be used in the work place or environment after
exposure has occurred.
2.2.3.5 Pollutant Mixtures
Currently, NTD is not conducting research on chemi-
cal mixtures. There is a clear need to study the principal
components of some chemical mixtures, particularly with
respect to possible additivity or synergism of some of the
components. Because many of the mixtures have large
amounts of known neurotoxicants'Tor example, metals
and organic solvents"this area of research would have a
high priority. A critical need in this area is the develop-
ment of a rapid, sensitive in vitro test, possibly based on
the use of cell cultures, to rapidly screen mixtures and
their components for potential neurotoxicity. The FOB
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and motor activity tests should also be used to study
chemical mixtures.
2.2.4 Emerging Issues
A number of major issues could change the priorities
of the NTD program in the future. These include concerns
regarding microorganisms, environmental agents and
neurodegenerative disease, and alternative fuels.
2.2.4.1 Microorganisms
Research is needed to determine whether a causal
relationship exists between environmental exposures to
microorganisms (i.e., bacteria, viruses, fungi, genetically
engineered and naturally occurring microorganisms) and
human health effects. The central nervous system might
be particularly vulnerable to microorganisms, as shown by
the susceptibility of the brain to the AIDS virus. Research
efforts may be necessary to develop appropriate test
methods for hazard identification and to support a quan-
titative health risk assessment.
effects on sensorimotor function in rodents. In vitro pro-
cedures for routine neurotoxicological assessments will
also be developed and validated. Focusing on the second
tier of lexicological assessments, researchers will then
define which currently available tests are appropriate for
inclusion at this level.
In the area of developmental neurotoxicity, NTD will
develop and validate methods to identify and characterize
developmental toxicity. After developing and validating a
model of cognitive development in an animal model, re-
searchers will also validate the sensitivity of animal model
tests in identifying and characterizing cognitive dysfunc-
tion.
NTD will also develop and validate cellular and
molecular endpoints as indicators and predictors of
neurotoxicity. This research will exploit the natural
response of the central and peripheral nervous system to
injury induced by chemicals. Research efforts will also be
directed to using structure-activity relationships to under-
stand the physicochemical properties of chemicals that
cause neurotoxicity.
2.2.42 Environmental Agents and Neurodegenerative
Disease
Both environmental agents and naturally occurring
substances can affect the nervous system, resulting in
neurodegenerative disorders. Possible diseases include
amyotrophic lateral sclerosis, Parkinsonism, and an
Alzheimer-like dementia syndrome. The last of these has
been linked to the ingestion of products made from the
false sago palm, which contains agents that resemble ex-
citatory amino acids (which are neurotoxic in animal
models). The discovery of this link between a naturally
occurring compound and a neurodegenerative disease has
stimulated the search for other toxic substances; and it is
now known that other foods contain neurotoxic substances
that may be related to the etiology of neurodegenerative
disorders. Future research in this area may prove fruitful
for identifying environmental links between exposure to
chemicals and the development of progressive,
neurodegenerative diseases such as Parkinson's and
Alzheimer's diseases.
2.2.5 Summary
2.2.5.7 Hazard Identification
NTD will continue to systematically validate a first-
tier in vivo screen for the assessment of chemical-related
2.2.52 Dose-Response Assessment
Researchers will study mechanism(s) of action of
neurotoxicants by examining the effects of agents at the
cellular, subcellular, and molecular levels and then cor-
relating those effects with neurotoxic signs measured in
the whole animal. Of particular interest will be the inter-
action of metals or solvents with endpoints such as ionic
fluxes inside and outside the cell.
Work will also emphasize issues associated with
various extrapolations (i.e., route-to-route, high-to-low-
dose, species-to-species). Special emphasis will be placed
on experiments in which animals will inhale chemicals
while they are being tested.
Because the body often compensates for or adapts to
the effects of chemicals, NTD will develop a strategy for
assessing the influence of these factors. Acute and
repeated exposure to a prototypic class of neurotoxicants
will be used to assess the underlying mechanism(s) of ac-
tion. Also, NTD will examine the neurotoxicity as-
sociated with chemical mixtures. Known neurotoxic
compounds will be tested in pairs, and the data compared
to the results of testing individual compounds.
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2.3 GENETIC TOXICOLOGY DIVISION
Mixtures (e.g., analgesics, coal tars/pitches, soots)
Interactions between genetic material and environ-
mental agents can result in mutations. There are two types
of mutations: 1) those that occur in germ cells (germ cell
mutations), which may be passed on to the next genera-
tion, and 2) those that occur in somatic cells (somatic
mutations), which are not passed on to the next generation.
Genetic diseases, such as hemophilia, cystic fibrosis, and
Tay-Sachs disease are examples of health effects as-
sociated with germ cell mutation; an example of a health
effect due to a somatic mutation is cancer.
Germ cell mutations are rare and many, if not most,
result in "carrier" individuals rather than diseased off-
spring; thus, no cases have been documented in humans of
new genetic diseases induced by environmental exposures.
Studies in rodents and other animals, however, clearly in-
dicate that germ cell mutations can be induced by chemi-
cal exposure. It is thus theoretically likely that chemicals
pose a threat both to individual offspring and to the in-
tegrity of the human genome and thus to future genera-
tions. While definitive proof is lacking, little doubt exists
that many chemicals have the potential to induce transmis-
sible human mutations.
By comparison, a large body of data suggests that en-
vironmental exposures can result in somatic mutations
leading to the development of cancer. Various genes that
control cell growth, proliferation, and differentiation have
been implicated in the genesis of cancer. Activated cel-
lular oncogenes have been detected in tumors from
humans and animals, suggesting that cellular oncogenes
are critical targets of carcinogens. In addition, the inac-
tivation of certain tumor suppressor genes (anti-on-
cogenes) is postulated to result in the induction of cancer.
Evidence also has accumulated implicating a class of
agents that can cause cancer, yet do not appear to produce
chemical damage to DNA. These agents have been called
"nongenotoxic carcinogens."
In 1987, the International Agency for Research on
Cancer (IARC) conducted an evaluation of the causal as-
sociations between exposure to carcinogens and human
cancer. Since 1969, IARC has reviewed, assessed, and
classified those agents, processes, occupational exposures,
and industries to which humans are exposed. To date,
IARC has classified 50 sources as carcinogenic to
humans, including:
Single agents (e.g., aromatic amines, cancer
chemotherapeutic drugs)
Processes (e.g., aluminum production, coal gasifica-
tion, iron and steel founding, coke production)
Occupational exposures (e.g., petroleum refining)
Industries (e.g., the rubber industry)
IARC has also classified another 37 agents as
"probably carcinogenic to humans" and a further group of
150 agents as "possibly carcinogenic to humans."
Taken in totality, cancer and other diseases of genetic
origin vitally affect human welfare. These diseases often
result in debilitation, death, and human suffering. By
some estimates, at least 10 percent of all human disease
exhibits a significant genetic component. Exposures to
new mutagenic agents in the environment could be in-
creasing the number of individuals who are "carriers" of
altered genes. Such an increase would lead to an in-
creased incidence of cancer and genetic diseases in future
generations.
2.3.1 Divisional Program
Research conducted by the Genetic Toxicology
Division (GTD) addresses both mutation and cancer. The
program is designed to explore the influences of environ-
mental agents on 1) the processes of genetic change in
somatic and germinal tissues (mutagenesis) and 2) the
conversion of normal cells to neoplastic cells (car-
cinogenesis). All direct and indirect interactions between
chemicals and the genetic material of the test organisms
are of interest if they may lead to adverse health effects.
Research efforts are also focused on the genetic toxicol-
ogy of environmental chemicals and complex mixtures,
including the toxicology of genetically engineered
microorganisms and their metabolites.
The hypothesis that carcinogenesis results from
genetic changes"the somatic mutation theory of cancer"is
the basis for the study of genetic toxicology. Cancer is
thought to progress from carcinogen exposure to tumor
formation through a multistage process, including initia-
tion, promotion, conversion, progression, and metastasis
(see Figure 2-5). Germ cell mutagenesis, on the other
hand, is highly cell-stage specific. A mutation in the germ
line may occur in premeiotic or postmeiotic cell stages
(i.e., before or after reduction division) and may result in
mutated offspring or reduced fertility. Chemicals that
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have the ability to induce germ cell mutations deserve spe-
cial attention from a regulatory and a research perspective.
identification, dose-response assessment, chemical-
specific data, biological markers, and pollutant mixtures.
The somatic mutation theory of cancer also provides
the rationale for the use of short-term tests to detect poten-
tial carcinogens. These tests may also be used to detect
active metabolites of mutagens or carcinogens in body
fluids and tissues, to identify genetically active com-
ponents in complex mixtures, and to investigate possible
mechanisms of carcinogenesis and germ cell mutagenesis.
From a mechanistic perspective, both cancer and heritable
mutation are complex. Studies in whole animals are es-
sential to define the intricacies of chemical carcinogenesis
and mutagenesis"to determine appropriate biological
markers for cancer and genetic disease, to model the shape
of dose-response curves for genotoxic as well as non-
genotoxic carcinogens, and ultimately to refine estimates
of human health risks.
2.3.2.7 Hazard Identification
Short-term tests are efficient means of detecting
DNA-reactive genotoxic chemicals. These tests, however,
vary in the types of genetic damage that can be evaluated
and in the specific classes of mutagens/ carcinogens that
can be detected. Research is needed to define the
capabilities and limitations of test systems. Short-term
test databases and structure-activity relationship (SAR)
methods are needed for specific chemical classes of con-
cern to the program offices to 1) solve data needs and 2)
provide the theoretical and practical bases for correlating
molecular struc- ture with biological activity. In conjunc-
tion with these needs, research is required to identify me-
tabolites in critical toxicologic processes. Depending on
the specific nature of
the data gap or SAR
problem, GTD or
MULTISTAGE CARCINOGENESIS
INITIATION
(mutation)
PROMOTION
(selected clonal
expansion)
CONVERSION
(mutation)
PROGRESSION
(multiple
factors)
METASTASIS
(multiple
factors)
Somatic
tissue
Initiated
cell
I
Preneoplastic
lesion
Malignant
tumor
Clinical
cancer
Metastatic
cancer
HERITABLE MUTAGENESIS
PREMEIOSIS
(mutation)
MEIOSIS
(mutation)
I
SPERMIOGENESIS
OOGENESIS
(mutation)
GerminaL
tissue
Mutated
stem cell
Mutated
germ cell
Fig. 2-5: Multistage Carcinogenesis
2.3.2 Division-Specific Research Needs
The most important questions GTD faces in the next
three to five years are described in this section. These re-
search needs are covered under the research topics hazard
other HERL divisions
may perform research
to generate the re-
quired data.
A major em-
phasis must be placed
on the definitions of
test system capabil-
ities and limitations.
Because certain sub-
stances seem to in-
duce tumors without
appearing to induce
genotoxic effects,
short-term tests are
needed for nongeno-
toxic carcinogens
and specifically, for
chemicals that are not
genetically active in
short-term mutagen-
esis screening assays
but cause cancer at
selected target sites in
rodent bioassays.
Additional effort should be focused on the development of
tests that are relevant to rodent hepatic and renal car-
cinogenesis (and research is required to ascertain the
human relevance of these cancers).
FERTILIZATION
(mutation)
1
Mutated
egg or sperm
Mutated
offspring
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Short-term in vivo and in vitro tests for tumor
promoters need to be evaluated. One GTD project in this
area deals with cell-cell communication as a short-term
bioassay for nongenotoxic carcinogens. In addition, short-
term in vivo tests are needed to detect tumor initiators as
well as promoters. Such methods, based on an assessment
of DNA damage (e.g., gastrointestinal tract nuclear
anomalies or DNA strand breaks), can provide a higher
level of confidence than in vitro approaches because they
can account for factors such as species-specific tis-
sue/organ sensitivity, pharmacokinetics, and phar-
macodynamics.
The EPA Mutagenesis Guidelines address environ-
mentally induced, potentially inheritable genetic damage
and stress the need for improved means of identifying
chemicals that may be hazardous to germ-line cells. The
"standard" germ cell assays now available are relatively
insensitive as chemical screens, and relatively few such
studies of chemical effects on germ cells have been
reported. Extrapolation of test results from somatic cell
assays is limited by the processes/targets that are unique to
germ cells. Hence, new short-term methods are needed to
test for chemical induction of heritable genetic effects.
Other efforts should determine if genetically en-
gineered microorganisms (GEMs) can survive in the gut,
invade other tissues, and induce adverse health effects.
Projects would be designed to identify genotoxic and me-
tabolic changes that may occur as a result of direct ex-
posure to GEMs or as a result of their use for
bioremediation of hazardous spills and waste sites and in
other environmental and sanitary operations.
2.3.22 Dose-Response Assessment
In both mutagenicity and carcinogenicity testing, the
development of dose-response data and mechanistic infor-
mation is critical for the quantitative evaluation of the
biological effects considered in risk assessment models.
Research efforts are needed to explore the extent to which
dose-response investigations in both cell culture and ro-
dent systems are applicable to humans at realistic environ-
mental exposure levels, i.e., appropriate extrapolation
models must be developed in support of the risk assess-
ment process.
Mechanistic research efforts using molecular techni-
ques should be designed to identify specific types of in-
duced genetic damage. Mutation induction is a multistep
process of initial lesion occurrence (perhaps from the for-
mation of a DNA-chemical adduct), the repair or misrepair
of that lesion, and the generation of daughter cells contain-
ing the mutation. Because different cell types and dif-
ferent animal species may repair DNA with different
degrees of fidelity, mechanistic research also must include
interspecies comparisons. Cancer induction involves
many potential mechanisms (e.g., gene or chromosomal
mutation, heritable changes in DNA transcription reflected
in altered gene expression) and multiple stages. A variety
of factors or conditions"including enzyme and hormone
levels, rates of cellular proliferation, target cell specificity,
and genetic predisposition"influence the expression of
neoplasia. Therefore, to properly model the relationship
between exposure to cancer-causing environmental agents
and expression of the disease, researchers must explore the
possible mechanisms of carcinogenesis and the factors
that modulate neoplastic change.
One type of work that is needed is an examination of
the relationship between critical biochemical processes
and/or preneoplastic lesions and the development of can-
cer in experimental animals. These experiments have a
twofold goal: 1) to develop data on mechanistic aspects of
environmental carcinogens (both genotoxic and non-
genotoxic) so that the risk associated with exposure to
these chemicals can be assessed both qualitatively and
quantitatively; and 2) to produce data for use in develop-
ing biologically based models for carcinogenesis (for
quantitatively estimating carcinogen potency), and in
qualitatively determining the appropriateness of particular
oncogenic endpoints (e.g., mouse liver or rat kidney
tumors) for human risk assessment.
2.3.2.3 Chemical-Specific Data
What chemical-specific information program offices
need depends on their legislative mandates; however, con-
siderable overlap exists among these requirements.
GTD's capabilities for addressing chemical-specific infor-
mation needs are generally applicable to all program of-
fices. Specific chemicals of interest to various offices are
as follows:
Drinking water: Products of disinfection and specific
compounds for regulation
Air: Representative or index chemicals for broad
chemical classes
Hazardous waste: Chemicals not represented in the
Integrated Risk Information System (IRIS) or for
which important data gaps exist
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Superfund: Chemicals included in the Superfund
comprehensive risk assessment for sites; data needs
include reference doses, potency factors, and SAR in-
formation
Toxics: Chemicals that represent broad classes or
conceptual assessment criteria
2.3.2.4 Biological Markers
A major source of uncertainty in risk assessment lies
in estimating the extent of exposure. For genotoxic effects,
the dose delivered to the cell DNA is critical. New
methods are needed for measuring the internal exposure of
experimental animals and humans to environmental
genotoxic chemicals using biochemical entities (e.g., DNA
and protein adducts and urinary metabolites) via ultrasen-
sitive methods (e.g., GC-MS and P-postlabeling).
An experimental basis must also be established for
relating dose to the DNA (DNA adducts) to the induced
genetic effects (sister chromatid exchange, chromosome
aberrations, micronuclei formation, and gene mutation)
that can be monitored in humans. Further research is re-
quired to establish the shape of dose-response curves and
to develop biologically based dose-response models, espe-
cially for nongenotoxic carcinogens.
2.3.2.5 Pollutant Mixtures
Methods and approaches are needed to detect
genotoxic agents as complex mixtures in ambient air and
indoor air combustion sources, waste incinerators, en-
vironmental tobacco smoke, drinking water, wastewater,
sludges, and products resulting from bioremediation. New
methods are required to detect potentially carcinogenic
chlorinated hydrocarbons, highly reactive gaseous species
(e.g., PAN), and nongenotoxic carcinogens (e.g., diethyl-
hexylphthalate). Additional effort should be directed to
developing and applying green plant genetic test systems
for on-site monitoring of chemical dump sites and other
areas impacted by pollutant sources. After these tests are
developed, their utility must be defined through in situ as-
sessment and environmental monitoring.
Another area for research is modeling and testing the
interactions of genotoxicants with other pollutants using
artificial mixtures and actual fractions of environmental
mixtures. Environmental substances that should be ex-
amined include drinking water, ambient outdoor and in-
door air, industrial effluents, hazardous waste, and source
emissions.
Major areas for GTD research effort have been
defined as the development of methods/approaches for 1)
determining which emission sources are the major con-
tributors of carcinogens to ambient air (both indoor and
outdoor); 2) estimating human exposure to complex mix-
tures, including biomonitoring source, ambient, microen-
vironmental, and personal samples, as well as in situ
monitoring; and 3) determining the carcinogenic potency
of complex mixtures.
These objectives, taken together, include continued
research to test and further develop the comparative poten-
cy method for cancer risk assessment of combustion emis-
sions.
2.3.3 Research Plan
2.3.3.1 Hazard Identification
Several new directions will be pursued relative to
SAR and database development. Specialized databases
for use in SAR chemical analysis will be assembled in the
areas of genetic and developmental toxicology (with
HERL's Developmental Toxicology Division). In col-
laboration with the Office of Pesticide Programs, data sub-
mitted by registrants will be computerized to provide
similar database and SAR analytical capabilities.. In the
area of computational SAR:
Molecular similarity will be quantitated as it relates to
molecular recognition and biological activity.
Causal molecular models will be developed from
computationally available molecular properties that
predict the distribution, transformation, deposition, or
biological activity of classes of chemicals.
Mechanisms of action of nongenotoxic carcinogens
will be explored by assessing molecular similarity in-
dices.
Conventional short-term tests and test batteries for
identifying potential genotoxic agents will be evaluated
and adapted as necessary to efficiently detect specific clas-
ses of chemicals. To detect chemicals that induce
aneuploidy in mammalian cells, new test methods will be
developed. As new molecular techniques become avail-
able, this technology will be applied to develop assays that
are capable of evaluating specific types of genotoxic
damage. GTD investigators will develop short-term test
methods to improve the detection of nongenotoxic car-
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cinogens. Endpoints of immediate interest include altera-
tions in gap junction proteins, alterations in gene expres-
sion, and morphological cell transformation.
The heritable mutation research program will be
refocused in the next two years to utilize new recombinant
DNA techniques for evaluating mechanisms of mutation
in germ cells. The significance of genetic recombination
and segregation events at meiosis leading to mutation will
be analyzed in yeast, with the objective of extending these
studies to the mouse. The long-range goal in this program
is to achieve a better understanding of germline-specific
targets and mechanisms in the induction of heritable muta-
tions, and to improve analytical capabilities to detect such
events.
Research on GEMs will be designed to evaluate
various mutant and recombinant microorganisms and to
identify any adverse effects they could have on human
health. Efforts will be made to develop new and improve
existing methods for monitoring exposure to engineered
bacterial strains and/or genotoxic metabolites generated by
these organisms. New research will detect alterations in
the activity of biomarker enzymes and the effect of these
changes on the biotransformation of xenobiotics. GTD re-
searchers will give particular attention to changes leading
to increased levels of toxic, mutagenic, or carcinogenic
metabolites particular attention.
2.3.3 2 Dose-Response Assessment
The major issues associated with improving cancer
risk assessment are:
Improving the accuracy of the exposure assessment
Comparing the mechanisms of action of chemicals in
both rodent and human tissues
Improving methods for extrapolating rodent cancer
data to humans
Problems in these areas can contribute significantly to the
potential uncertainty, unavailability of data, and inac-
curacy found in current cancer risk assessment procedures.
Designing and evaluating biological marker/molecular
biological techniques for biological monitoring would
greatly increase the accuracy of exposure assessment. In
addition, understanding the mechanisms of action of
chemicals and the differences in the mechanisms between
rodents and humans will improve the basis for human risk
estimation. Incorporation of the biology of cancer induc-
tion, promotion, progression, and metastasis into the risk
estimation/extrapolation models will also improve the can-
cer risk estimates.
The use of internal or target dose measures such as
DNA adducts or cytogenetic effects will improve the ac-
curacy of dose determinations for interspecies extrapola-
tion and will be useful in species-species and route-route
extrapolations. The development of exposure-, dose-, and
genetic effects relationships for genetically active chemi-
cals will allow a more precise extrapolation of risk from
rodent data to the human population. This will be
evaluated with nonsite-specific carcinogens through sys-
temic exposure. GTD researchers will determine the
dosimetric relationships between exposure and dose to the
target cells and induced genetic effects. As an example,
peripheral blood lymphocytes will serve as target cells in
rodents, and humans exposed to the cancer
chemotherapeutic drug, diazaquone (AZQ), will serve as
target tissues for this drug.
Uncertainty in carcinogenesis risk assessment arises,
in part, from a lack of understanding about the underlying
chemical and biological mechanisms that are responsible
for the development of the cancer cell. Knowledge of
mechanisms of action can assist in the choice of the most
appropriate dose-response models for the extrapolation
process. Mechanisms of action of carcinogens can be
genotoxic or nongenotoxic in nature; therefore, the
similarities and differences in the mechanisms of action of
carcinogens in both rodent and human tissues need to be
defined.
The induction of protein kinase c or other enzymes
associated with nongenotoxic carcinogenesis, alterations
in the expression oncogenes or tumor marker genes, and
alterations in the structure of oncogenes will be measured
by state-of-the-art molecular biological techniques. Al-
terations in gap junction intercellular communication has
become recognized as an important element in responses
to nongenotoxic carcinogen treatment in cells as they
progress to a more tumorigenic state. GTD investigators
will study the functional aspects of gap junction
membrane components in fibroblastic and epithelial cells
at the molecular, organelle, and cellular level using long-
term cell culture systems. In addition, they will assess the
involvement of oncogenes and oncogene products in early
promotional events and intercellular communication. This
work will lead to a better knowledge of mechanisms of ac-
tion of different classes of nongenotoxic chemicals and
will provide a more explicit basis for extrapolation model
development.
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A major advance in cancer research during the last 30
years has been catalyzed by the simultaneous recognition
that:
Carcinogenesis is a complex, multistaged biological
process.
Chemicals can increase the incidence of experimental
cancer via a variety of separate, unrelated biochemical
mechanisms, some of which are irreversible (e.g.,
tumor initiation) and others of which may be revers-
ible. In theory at least, these latter mechanisms have
thresholds for activity so that exposures below the
threshold could result in no, or greatly reduced, harm-
ful consequences.
Along with these advances in understanding has come
the realization that the statistically based cancer risk as-
sessment models (e.g., the linearized multistage model)
now in use are probably not appropriate for all types of
chemical carcinogens. This limitation has, in turn,
prompted the development of biologically based dose-
response (BB-DR) models for cancer risk assessment.
BB-DR models are based on the quantification of certain
preselected biochemical processes that are thought to be
critical to the progression of carcinogenesis.
The recently developed Moolgavkar-Knudson-Ven-
zon (MKV) model is one of the more widely used BB-
DRs. It quantifies the carcinogenesis process in terms of a
series of biological parameters, such as mutation rates in
target cells and the rate of growth of various putative
premalignant cellular lesions. This model has proved use-
ful in explaining the incidence of a number of spontaneous
human cancers. More research is needed to evaluate the
feasibility of using BB-DR models to explain the in-
cidence of chemically induced cancer in experimental
animal models. The successful development and applica-
tion of these models should 1) greatly facilitate extrapola-
tions from experimental cancer data to human risk, and 2)
permit the use of numerous types of biochemical data
(e.g., measurement of DNA synthesis and preneoplastic
lesions) in the cancer risk assessment process.
Several experimental approaches have been
developed to determine interspecies sensitivity; all are
based on comparing responses of humans, animals, or
their tissues to carcinogen exposure. Humans and rodents
are exposed in vivo, in vitro, or by a combination of the
two (parallelogram approach); and the data are then com-
pared. Interspecies sensitivity for selected classes of car-
cinogens can be determined by using the same agent and
similar exposure conditions for both rodents and humans.
When available, results from quantitative epidemiology
will be incorporated to define the potency of a human car-
cinogen. An effort will also be made to define the potency
of a carcinogen in an experimental situation using the
same route of exposure as associated with the human data.
Although the measurement of tumor formation is the most
desirable endpoint, other endpoints that may be related to
tumor formation will be used (e.g., genetic or cytogenetic
damage).
When human exposure and effects information are
not available or cannot be generated, GTD researchers will
use human and animal cells or their tissues in culture (in
vitro) to obtain interspecies sensitivity constants. Al-
though the measurement of tumor formation is not pos-
sible in vitro, other endpoints related to the cancer
process"DNA damage, gene mutation, chromosomal ef-
fects, and morphological cell transformation"can be used.
The researchers will apply new molecular methods to un-
derstand the fundamental interspecies differences between
the responses of rodent and human cells to genotoxic
agents. A systematic evaluation of genotoxic response
(including specific gene loci and differences in DNA
repair) will allow a determination of how genotoxic ef-
fects in rodents extrapolate to similar effects in humans.
GTD investigators will use molecular techniques (inser-
tion of human DNA repair genes and specific target genes)
to define and quantify the specific interspecies differen-
ces'^ process that will reduce the uncertainty in the inter-
pretation of in vitro and in vivo rodent data and allow a
more realistic and accurate assessment of human risk from
particular environmental exposures.
The parallelogram method examines responses from
both in vitro and in vivo situations and combines four con-
stants into a prediction of carcinogenic effects in man.
Each one of these interspecies sensitivity/extrapolation
constants requires a determination of the potency of the
chemical under the treatment conditions. "Chemical
potency" is a complex issue to resolve. GTD researchers
will explore the development of a potency measure using a
combination of dose, tumor multiplicity, tumor incidence,
species, sex, site specificity, malignancy, and metastasis.
Lung cancer, which is associated with exposure to
carcinogens in the lung, is a major cause of death. The
primary target site for these carcinogens is the bronchial
epithelial tissues, which are transformed by a series of
dysplastic, metaplastic, and neoplastic changes. Methods
for obtaining and culturing rodent and human respiratory
tissues are needed to define interspecies sensitivity con-
stants to respiratory carcinogens. After these procedures
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are developed, they can be used to subject tissues to car-
cinogen exposures; and then various parameters of the ex-
pression of the neoplastic phenotype or of genotoxic
injury will be measured in a comparative fashion. With
these endpoints, interspecies extrapolation constants will
be determined. Investigators will also use this research to
assess the correspondence between mechanisms of
neoplasia in humans and rodents, and to quantitate their
similarities.
A major confounding factor in defining interspecies
sensitivity to carcinogens is the large interindividual varia-
tion observed in the human population. In contrast, rela-
tively small interindividual variation is observed in
experimental animals. Human interindividual variation in
response to carcinogens is crucial for defining interspecies
extrapolation in the risk assessment process: The issue is
whether to regulate a carcinogen based on the most sus-
ceptible human population or on the "average" human
response. Interindividual variation will be examined using
explanted tissues from surgical specimens from a variety
of tissues.
Recent advances in molecular biology indicate that
genetic changes are involved in the etiology of cancer.
There should be, therefore, a set of biomarkers that signal
the relevant genetic alterations, and these markers should
be detectable before the emergence of cancer. Delineating
such a set of mechanistically consistent biomarkers could
significantly diminish the time needed to perform assess-
ments of human carcinogenic activity. Furthermore, these
assessments could be performed in the exposed human tar-
get tissues. If used to complement existing animal bioas-
says or lengthy in vitro transformation bioassays,
biomarkers could significantly shorten the time and cost of
such bioassays.
2.3.3.3 Chemical-Specific Data
GTD has the capacity to fully evaluate the mutagenic
and oncogenic potential of agents of environmental con-
cern, including pure chemicals and complex environmen-
tal mixtures. The process of evaluating potential
environmental genotoxicants is frequently approached
through the step-wise application of bioassay
methodologies involving short-term screening tests, con-
firmatory short-term bioassays, and, eventually, estab-
lished whole-animal mutagenesis and carcinogenesis
bioassays.
Working collaboratively, scientists from HERL and
the Office of Toxic Substances (OTS) have identified
several chemical classes for SAR research. Chemical
classes were selected according to the perceived need for
understanding the chemical properties and structures
responsible for the observed biological activity. The re-
search team has evaluated three classes of chemicals
(acrylates/methacrylates, anthraquinone dyes, and azo
dyes) to pinpoint the structural properties responsible for
their biological activity. These properties, which are ap-
plicable to other chemicals as well, will be used to
evaluate the SAR approach to studying chemicals'
biological activity.
GTD will also conduct research on the genotoxic
potential of complex mixtures and on specific chemicals
in drinking water, such as chlorinated organic pesticides;
high-volume organic chlorinated hydrocarbon solvents;
chlorinated phenols; chlorinated acids; disinfection
byproducts of ozonation, chloramination, and a combina-
tion of the two; and complex mixtures of wastes and
drinking water concentrates containing chlorinated or-
ganics. Such research is necessary to fill the Office of
Drinking Water's data gaps and to develop the concept of
surrogate measures for estimating the toxicity of complex
mixtures.
2.3.3.4 Biological Markers
'1'j
Analytical methods ( P-postlabeling and cytogen-
etic) will be improved for increased sensitivity and resolu-
tion, and these will be validated for both biomonitoring
and site evaluation. Exposure, dose, and cytogenetic ef-
fects relationships in blood lymphocytes of rodents and
humans exposed to chemicals will be determined. These
methods and calibration studies will provide the ex-
perimental background for measuring DNA adducts and
the cytogenetic effects induced in humans environmental-
ly exposed to these substances.
Biomarkers that can distinguish preneoplastic or
neoplastic cells in human or experimental animal car-
cinogenesis will be developed and validated. The occur-
rence and frequency of alterations of genes implicated in
the cancer process will be assessed in tissues and cell
types that are relevant to environmental cancer etiology.
In particular, carcinogen-exposed cells from humans and
from rodents will be compared for changes in expression
of specific cellular oncogenes (the ras and myc family), al-
terations in gene copy number, and the presence of
specific DNA sequence alterations. In addition, other
markers of cell differentiation or functions, such as recep-
tors that code for essential nutrients or factors, will be
measured in human tissues that have been cultured or by
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culture of animal tissues. Analysis of DNA sequence al-
terations will be performed, and alterations in genes or
their expression will be analyzed. This research involves
studies on the genetic effects induced by environmental
carcinogens at specific sites in target genes using a shuttle
vector system. Relationships between identified biomark-
ers, genetic effects, and carcinogen exposure will be
studied with regard to mechanistic consistency.
GTD has developed the capability to conduct in-
tegrated exposure assessment studies jointly with the
monitoring and engineering laboratories. Future research
in this area will focus on improving methods to conduct
total human exposure assessments that employ new
genetic bioassays for biomonitoring external exposure and
biomarkers for internal exposure assessment (DNA and
protein adducts and metabolites). In studies jointly con-
ducted with the Human Studies Division, human tissues
and body fluids will be used to assess the relationships be-
tween internal exposure, biologically effective dose (target
tissue dose), and genotoxic and nongenotoxic effects (e.g.,
cytogenetic effects).
The current risk assessment guidelines that employ
additivity are most widely used in conjunction with Super-
fund site evaluations and in the assessment of waste in-
cineration because very little toxicology data are available
for the complex waste mixtures to which humans are ex-
posed. As more hazardous municipal and hospital waste
is incinerated rather than buried, toxicology data on the
complex mixture of incineration emissions will become
increasingly critical; and GTD research efforts will con-
tinue to be directed to developing the methods and
database necessary for the cancer risk assessment of these
emissions. Evaluating the gaseous emissions will become
more important in the future, and GTD researchers will
develop and employ new direct bioassay methods for this
task that analyze the emissions both before and after at-
mospheric transformation. Incineration emission research,
as well as the additivity work mentioned above, will focus
on chemical class interactions. Potential interactions be-
tween chlorinated dioxins, metals, PAH, and substituted
PAH are important subjects for these efforts.
2.3.4 Emerging Issues
2.3.3.5 Pollutant Mixtures
Genetic bioassay methods will be developed in con-
junction with personal and microenvironmental sampling
methods in order to use bioassays in the biomonitoring of
exposure to complex mixtures. Research in the water and
waste areas will focus on developing alternative bioassays
(in vitro, cellular systems or fish and plant systems) for
monitoring industrial effluents, waste sites, and drinking
water.
Complex-mixture risk extrapolation issues are in-
creasingly emerging in all areas of air toxics (e.g., urban
soup, alternative fuels) and in indoor air research. Future
GTD air research will expand the use of new
methodologies for identifying and assessing complex mix-
tures of air pollutants. These methodologies will encom-
pass human and environmental exposure assessment
through genetic bioassays for both external and internal
exposure assessment. New emphasis will be applied to
source apportionment of exposure sources; future research
efforts will apply such methodologies to complex mix-
tures of DNA adducts from human exposures. The com-
parative potency and parallelogram methodologies will be
further expanded and applied to new air risk problems; at
the same time, they will be used to test additivity assump-
tions that may be used in alternative approaches.
2.3.4.1 Nongenotoxic Cancer
Work over the past 30 years has uncovered a variety
of mechanisms of action for carcinogens, including ones
that involve the induction of cancer by direct (genotoxic)
or indirect (nongenotoxic) interactions with the genome.
The incorporation of mechanisms of action into risk as-
sessment is a key element of that process. When positive
correlations are made between the mechanisms respon-
sible for an agent's known biological effects and the cur-
rently understood mechanisms of carcinogenesis,
mechanistic information can be used with more con-
fidence in determining the hazard to man.
Although a good deal of information has been
gathered on the mechanisms of action of genotoxic car-
cinogens, data on nongenotoxic carcinogens are sparse.
Nongenotoxic carcinogens are thought to have a wide
range of mechanisms of action because this class of
chemicals is so heterogeneous (e.g., tumor promoters,
peroxisome proliferators, solid state carcinogens, hor-
mones, and hyperplastic agents). Studies are needed to
identify these mechanisms in experimental animals and in
man through exposure of rodent and human mesenchymal
or epithelial cells. The effects of this exposure to non-
genotoxic chemicals should be measured at the molecular
level in terms of alterations in genes and gene products
thought to be associated with the induction or progression
of cancer.
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2.3.42 Susceptible Populations
The risk assessment process both for cancer and for
heritable gene mutation is based on extrapolations from
available animal data. The quantitated risk factor is
generally calculated for the "average" human even though
the human population consists of a heterogenous mix of
diverse genetic backgrounds. This genetic diversity,
coupled with environmentally induced illness or ill health
(infectious or chemically induced, such as cigarette smok-
ing), creates a stratified population with varying suscep-
tibility to particular environmental exposures. As our
ability to quantitate the risk factor for the "normal" human
improves, the need to determine and evaluate the risk for
susceptible subpopulations will emerge as a major re-
search area.
Identifying populations of individuals with unusual
sensitivity to certain cancers fosters understanding of the
etiology and mechanism of occurrence of those cancers.
A major issue in cancer mechanism concerns the
mechanistic role of oncogene alteration and expression in
human cancer and the oncogene's role as a biomarker.
Work in this area will benefit by significant interaction
with other investigations on "molecular mechanisms of
carcinogenesis." The combined research effort is aimed at
determining the role of environmental chemicals in the ex-
pression of oncogenes and tumor suppressor genes. By
establishing the biological basis for the alteration of these
genes, the research effort can also assess the usefulness of
these genes as biomarkers for preneoplastic and neoplastic
lesions in exposed or susceptible human populations.
2.3.4.3 Microorganisms
The health effects caused by microorganisms will be
an important concern for EPA in the coming years. Sig-
nificant research issues associated with this problem in-
clude:
Potential adverse health effects due to the registration
of engineered organisms
Potential adverse health effects due to the use of
microorganisms for environmental bioremediation
Understanding the role microorganisms play in the
"sick building syndrome"
Potential release of pathogenic organisms, especially
viruses, during the incineration of medical/pathologi-
cal waste
The first two of these issues are addressed to some
extent in the Biotechnology Health Research Strategy
Plan. For example, a BSAC subcommittee has recom-
mended that EPA examine the interaction of pol-
lutants"especially metals"and the co-selection of antibiotic
resistant microorganisms. Due to increased recirculation
of air within buildings and lower air exchange rates due to
insulation, an increasing number of "sick building"
syndromes are being caused by organisms that are not
often seen in clinical settings (e.g., aspergillus). To
separate chemically introduced causes of adverse health
effects from those that are induced by microorganisms or
interactive, EPA should establish a program that encom-
passes health work associated with such organisms as
fungi, soil bacteria, and spirochetes.
Finally, most medical facilities are now turning to in-
cineration (municipal and facility) to dispose of biological,
medical/pathological waste. Some incinerator conditions
(e.g., malfunctions that cause bursts of unburnt com-
ponents to be emitted) allow pathogenic organisms to be
encountered in either the airborne or ash waste streams.
2.3.5 Summary
2.3.5.1 Hazard Identification
GTD will increase efforts in computational structure-
activity analysis using quantitative mutagenesis, car-
cinogenesis, and developmental lexicological data. Test
method development efforts will address those chemical
classes (e.g., chlorinated compounds) and kinds of genetic
damage (e.g., aneuploidy) that are not readily detected
using present techniques. Continued mechanistic studies
on mutation in germ cells will lead to the development of
improved analytical capabilities to detect such changes.
Methods to detect chemicals that induce tumors through
nongenotoxic mechanisms also will be given special atten-
tion.
To identify potential environmental health hazards of
special programmatic interest, researchers will focus on
biological markers of dose and effect, in situ bioassay
methods, and microbiological investigations on recom-
binant microorganisms.
2.3.52 Dose-Response Assessment
Mechanistic studies will form the cornerstone of re-
search in carcinogenesis and heritable mutagenesis. The
development and ultimately the usefulness of biologically
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based dose-response models depend on the recognition
that carcinogenesis and heritable mutagenesis are both
complex, multistage^ biological processes. Since genetic
changes are clearly involved in the etiology of cancer, the
development of a set of mechanistically consistent
biomarkers of genetic change may be expected to lead to a
clearer understanding of both mutagenesis and car-
cinogenesis in experimental animals and in man. Par-
ticular attention will be paid to developing models that
improve the ability to extrapolate from rodents to humans.
A mechanistic focus on the biological basis for al-
tered gene expression should yield techniques that will 1)
demonstrate the importance of oncogenes and tumor sup-
pressor genes in carcinogenesis, 2) shorten the time for
analysis of the mutagenic and carcinogenic activity of
chemicals in humans, and 3) identify populations of in-
dividuals with unusual sensitivity to certain genetic dis-
eases and cancers.
GTD's complex mixture research will emphasize im-
proved methodology for the application of bioassay and
molecular genetic methods in the identification of
mutagens and carcinogens. Researchers will make in-
creased effort to interpret and extrapolate the results from
comparative biological evaluations in human, animal, and
in vitro studies of various types of complex mixtures. Re-
search on component interactions and modeling assump-
tions (e.g., additivity, antagonism, synergism) will be
given special attention in order to address complex mix-
ture risk assessment issues.
2.4 ENVIRONMENTAL TOXICOLOGY
DIVISION
Humans are exposed to thousands of toxic chemicals
by inhalation, ingestion, and dermal contact. The surfaces
of the respiratory tract, gastrointestinal (Gl) tract, and skin
function as primary barriers, or portals of entry, that
separate viscera from toxic chemicals; however, chemicals
can cross these physical barriers by both active and pas-
sive processes of absorption and enter the bloodstream.
During absorption, the chemicals can also be transformed.
The major portals of entry for chemicals are the:
Skin
Lung
GI tract
Introduction of the chemicals into the body can result
in cellular damage, disease, changes in metabolic proces-
ses, or activation of protective mechanisms. Moreover,
because the functional integrity of the organs acting as
protective barriers may be compromised by their defensive
roles, susceptible populations"such as children, the elder-
ly, and people with chronic lung or liver disease"can be at
risk of disease with only minor chemical challenge.
Obstructive diseases of the lungs (i.e., emphysema,
asthma, chronic bronchitis) are the third leading cause of
death among the populace. While cigarette smoke ac-
counts for a considerable portion of the 50,000 deaths per
year, deaths due to obstructive lung diseases are increas-
ing. Morbidity, which is more closely associated with the
overall health of the population and the economy, follows
a similar, but more substantial course in terms of person-
years lost. Ozone and smog-related pollutants have been
associated with the respiratory infections that strike 80-
100,000 people per year and kill more than 60,000 per
year. Similarly, exposure to anthropogenic pollutants or
disinfectant-derived de novo pollutants in water has been
associated with neoplasms and cardiovascular diseases
that kill or disable millions each year.
Environmentally linked diseases encompass an ex-
tremely broad range of human illnesses. Examples in-
clude bronchitis and/or emphysema in persons chronically
exposed to fossil fuel-derived air pollution, lung fibrosis
and cancer in individuals exposed to asbestos, heart and
vascular diseases in individuals exposed to carbon
monoxide, and impairment of immune function in men
and women exposed to certain pesticides. In addition, the
possible effects of chronic exposure to cigarette smoke are
well documented, including lung and other cancers, heart
disease, and respiratory diseases. Less severe effects have
been tentatively attributed to passive inhalation of
cigarette smoke in chronically exposed nonsmokers, and
recent evidence suggests that children are a sensitive
group for this pollutant.
The immune system is both a target for and a defense
against environmental insult. For example, suppression of
a delayed hypersensitivity response and increased suscep-
tibility to respiratory infections have been found in
patients who accidentally ingested pesticide-contaminated
rice oil. Although the long-term deleterious consequences
of exposure to polybrominated biphenyls are as yet un-
documented in humans, early data indicate a correlation
between immune alterations and increased tumor in-
cidence. Other pollutants such as nitrogen dioxide may
alter immune function and increase the risk of respiratory
infections, most notably of viruses. Because a large num-
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i!^
her of people die from respiratory infections each year,
even a small effect on immune system components or their
functions can have major consequences on the physical
and economic health of the population.
Pharmacokinetics, which defines the absorption, dis-
tribution, metabolism, and excretion rates of environmen-
tal chemicals, can be used to highlight the relationship
between exposure and effect. Body mechanisms and
processes, such as deposition and clearance of inhaled par-
ticles, affect the amount of the chemical that is absorbed
rather than eliminated and that reaches a target tissue or
active site (e.g., cell membrane, receptor, gene) in the
form of a parent compound, a form that was absorbed or
distributed to that site, or a metabolite. This difference be-
tween exposure and dose concentrations affects the
severity of the health effects that potentially result from
exposure to the chemical and complicates extrapolations
from one set of conditions to another (e.g., route-to-route,
high-to-low-dose, acute-to-chronic-exposure, species-to-
species). Confidence in these extrapolations can be in-
creased by both experimental and modeling dosimetric
studies.
2.4.1 Divisional Program
The Environmental Toxicology Division (ETD) uses
both animal research and in vitro methodologies to study
the potential for human health effects associated with ex-
posure to environmental pollutants. Research efforts will
cover exposure, emphasizing the three primary routes by
which the environmental pollutants enter the body (inhala-
tion, oral, and dermal); dose, in an effort to describe and
predict the concentration of pollutant that reaches the tar-
get organ(s); and effects, focusing on the response in the
respiratory and immune systems, with expertise developed
as needed in the cardiovascular, hepatic, renal, dermal, and
gastrointestinal systems. The division's research efforts in
the three areas of the risk assessment paradigm are
focused on improving human health risk assessment
through quantitative animal-to-human extrapolations (see
Figure 2-6).
ETD's research program is unique among the HERL
divisions in that it comprises many areas of biological ex-
pertise and encompasses a broad spectrum of environmen-
tal health research. The division serves as a primary
technical resource within the Agency for activities requir-
ing expertise in animal inhalation studies and in the health
effects of air pollutants. Because of the diversity of scien-
tific approaches to the research issues, multidisciplinary
teams composed of scientists inside and outside the
division are vital for the success of ETD research efforts.
2.4.2 Division-Specific Research Needs
2.4.2.1 Hazard Identification
One of the most critical research issues facing EPA is
identifying potential health hazards so that the relationship
between environmental exposures and health effects can
be predicted and interpreted. To meet this end, the Agen-
cy needs validated, short-term test methods to screen new
and existing environmental threats. The tests should allow
for timely determination of causality and adversity.
Similarly, animal models of infectious/neoplastic and
allergic disease need to be developed that correlate the ef-
fects of toxic chemicals on immune function tests and thus
improve estimates of the risk of increased disease. As an
example, effects of several toxic chemicals administered
by several routes on natural killer cell activity in mice (a
commonly used immune function test) have been corre-
lated with effects on susceptibility to mouse
cytomegalovirus (a common opportunistic infection).
Hence, chemicals that suppress this particular immune
function test can be predicted to also increase suscep-
tibility to certain types of infection.
2.4.22 Dose-Response Assessment
Dose-response assessment comprises three areas of
research: 1) the relationship between exposure and dose,
2) the relationship between dose and biological response,
and 3) the biological mechanism responsible for the ob-
served effect. ETD can identify specific research needs in
all three areas. To understand the relationship between
exposure and dose to the target tissue, both experimental
and theoretical approaches must be used to define
dosimetric and PB-PK models of inhaled, ingested, and
dermally applied chemicals. Because of the importance of
the lung, skin, and gastrointestinal tract as both target or-
gans and the major portals of entry for pollutants, research
into the uptake and absorption of pollutant gases, solvents,
and particles is critical.
A wide variety of chemical and physical factors,
depending on the portal of entry, determine uptake and ab-
sorption rates. Data are needed, for example, on the fol-
lowing factors:
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Inhalation
Ingestion
Dermal
Absorption
Physiologically
based
pharmacokinetics
Pulmonary
Immunologic
Hepatotoxic
Fig. 2-6:
Environmental Toxicology Division Research Perspective
Determinants of gaseous and particle deposition in
and clearance from the respiratory tract, including
morphology
Experimental measurements of physiological factors
governing chemical disposition
Comparison of in vivo and in vitro absorption and
metabolism systems
Rates of chemical reactions between pollutants and
the liquid linings and tissues of the portals of entry;
determining these requires a detailed knowledge of
the particular biochemical constituents within liquid
linings and tissue
Morphology of the portals of entry at the macroscopic
and microscopic levels
To conduct reliable and cost-effective extrapolation
between animals and humans and between routes of ex-
posure, researchers must account for all of these factors in
the mathematical models they develop.
The next step in dose-response assessment is to ex-
plore the relationship between target organ dose and
biological response by
developing animal models of
human disease states (e.g., car-
diopulmonary disease). Ani-
mal models of various cardio-
pulmonary disease states such
as emphysema, asthma, and
chronic obstructive and restric-
tive lung diseases are critical to
understanding the development
and progression of lung dys-
function and pathology in
humans, especially under con-
ditions of chronic exposure.
For example, the role of en-
dogenous ly released elastase
from inflammatory cells is now
known to be a key element in
the development of em-
physema. Exogenously ad-
ministered elastase to the
animal lung accelerates a
pathogenesis that closely
resembles a state of illness
which takes decades in man.
The development of this
animal model has revealed that al-antitrypsin (al-Pi) also
plays an important role in the pathogenesis of emphysema,
since it normally inhibits secreted elastases. These find-
ings demonstrate that the same disease entity can be
induced by quite different mechanisms"induction of in-
flammation or inhibition of al-Pi. Hence, modeling is
critical for understanding not only the pathogenesis of
disease, but also the impact of environmental stresses that
can induce or enhance its progression.
A critical research need lies in determining deposition
and clearance patterns of inhaled compounds. From a
dosimetric point of view, the lung is currently considered
as a whole. Inhaled particle dose is computed assuming
100 percent deposition efficiency, no clearance, and no
distinction among different regions of the lung. This ap-
proach ignores the dynamics of deposition and clearance
as well as the nonhomogeneity of various regions, both in
structure and function and in response to inhaled pol-
lutants. To better relate response to dose of inhaled par-
ticles, the regional deposition and clearance of particles
must be quantitated in animals and correlated with similar
data in humans.
Another area of needed research is the mechanistic
origin of cardiovascular, pulmonary, liver, and immune
function disease. Work in this area is critical for ex-
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trapolations across species, from acute to chronic ex-
posures, and from high to low doses. Two related issues
are species sensitivity and animal-to-human extrapola-
tions, including route-to-route, acute-to-chronic, and in
vitro-to-in vivo. Research needs associated with these is-
sues center on gathering experimental data across species
that will fill the theoretical data gaps for animal-to-human
extrapolations. Several approaches are possible:
In vivo monitoring of the deposition and clearance of
radiolabeled particles in animals
Investigating the relationship between macrophage
functions and particle clearance
Microscopic analysis of tissue samples to assess
regional dose and effect
2.4.2.3 Chemical-Specific Data
Some of EPA's specific short-term research needs in-
clude information specific to certain chemicals, such as:
High-production chemicals
National Ambient Air Quality Standards (NAAQS)
pollutants (e.g., subchronic and chronic Os, NO2,
methanol, and acid aerosols)
2.4.2.4 Pollutant Mixtures
EPA research needs also include information on:
Specific complex mixtures (e.g., alternative fuels,
urban mixtures, drinking water disinfectants, and in-
door air mixtures)
EPA must develop strategies to handle issues of
potentially chronic noncancer health effects and their
relationship to acute response and interactions of atmos-
pheric mixtures. In conjunction with the Human Studies
Division (HSD), ETD conducts chemical-specific studies
using similar research protocols to improve animal-to-
human extrapolations.
2.4.3 Research Plan
2.4.3.1 Hazard Identification
ETD work under this research topic presently in-
cludes:
Development of immunotoxicologic methods in sup-
port of immunotoxicity guidelines
Validation and refinement of methodologies to im-
prove predictive capabilities in pulmonary toxicology,
immunotoxicology, and other target organ toxicities
Physiologically based pharmacokinetic (PB-PK)
model development based on experimental data
ETD will continue to develop an immunotoxicity test-
ing tier in rats similar to that for mice by 1) developing ap-
propriate host resistance models in rats, and 2) adapting
immune function tests to rats for purposes of supporting
guideline development. In addition to the traditional test-
ing schemes analogous to those applied in mice, methods
for assessing immune responses in the lung and host resis-
tance models that are particularly applicable to the assess-
ment of effects from inhaled compounds will be
developed. A major goal of these research efforts will be
to correlate effects of chemical exposure on immune func-
tion tests with effects on susceptibility to infectious,
neoplastic allergic or autoimmune disease; host resistance
models will be used to study the adversity of alterations in
immune function.
Work on PB-PK models comprises a major com-
ponent of the SAR research effort. Such research is criti-
cal to hazard identification because SAR approaches to
both toxicology and dosimetry may allow estimates of risk
for compounds without the need for detailed toxicity test-
ing or pharmacokinetic determinations. For example, a
compound (so long as it is not highly toxic) may be ex-
cluded as a dermal exposure hazard because its chemical
structure leads to minimal dermal penetration.
2.4.32 Dose-Response Assessment
To perform accurate quantitative animal-to-human
extrapolations, researchers must lessen the uncertainty as-
sociated with extrapolating effects associated with one
route of exposure to those associated with another. To aid
in this task, ETD is investigating, both experimentally and
theoretically, the absorption of toxic chemicals by all three
2-31
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illSHlW
primary routes of exposure. The different processes that
affect the quantity of chemical absorbed through a par-
ticular route (e.g., similarity or dissimilarity of partitioning
and absorption across membrane barriers) are major con-
cerns. These studies require expertise in inhalation en-
gineering, inhalation toxicology, systemic and in vitro
toxicology, dermal absorption, pharmacokinetics, and me-
tabolism.
ETD also addresses issues such as gas and particle
dosimetry and extrapolation accuracy. Research efforts
are directed to developing and validating biomathematical
models to compare intra- and interspecies dosimetry data.
ETD is developing models for the primary routes of ex-
posure that can accurately predict the temporal distribution
of toxicants in various organ systems. Such models are of
particular use in quantitatively extrapolating effective pol-
lutant concentrations between animals and man. Other
work is focused on development of state-of-the-art model-
ing techniques for route-to-route, acute-to-chronic, in
vitro-to-in vivo, and animal-to-man extrapolations.
Physiologically based dosimetry models and structure-ac-
tivity principles form the basis for theoretical constructs to
examine dose-effect relationships.
PB-PK research involves extensive work in both ex-
perimental research and modeling. Once a compound
penetrates beyond the portal of entry, it enters the sys-
temic circulation and can be taken up by the systemic or-
gans, such as the liver and kidney. Predicting the amount
of a compound that will appear in an organ as a function
of time can be addressed through PB-PK modeling. The
data these models require as input include organ size,
blood flow, blood-tissue partition coefficients, and meta-
bolism rates. Because these data have usually not been
gathered for compounds of interest to the Agency, further
experiments are required. Established methodologies can
be used to obtain some of these data, but gathering some
of the input data will require development of new
methodologies. To be useful, PB-PK models must not be
compound- or species-specific; in other words, they must
allow highly accurate predictions of dose with only mini-
mal input. Further knowledge about structure-activity
relationships (SAR) will help achieve this goal.
ETD researchers will also develop and use the
analytical methods that are essential for quantifying the in-
ternal dose of a xenobiotic and its critical metabolites per
unit of sensitive (target) tissues or cells. Such data are
used in biomathematical models for making intra- and in-
terspecies comparisons of dose-response relationships in
animals and humans in terms of tissue levels of key
toxicologic substances. This conversion is dependent on a
thorough knowledge of the metabolism and phar-
macokinetics of the parent chemical in the species of inter-
est Experimental efforts include both in vitro and in vivo
methodologies for the determination of metabolic rate
constants and the validation of extrapolation from one
database to another. Methods for the isolation and iden-
tification of chemicals and metabolites in tissues and
biological fluids are being developed and applied in ex-
perimental dosimetry research programs that address
various types of extrapolation. ETD's pharmacokinetic
and toxicodynamic research addresses problems relevant
to all decision units that provide resources to HERL.
In addition, ETD investigates the toxic health effects
of environmental pollutants using laboratory animals. So
that this research can be used more directly in regulatory
processes, a major effort using animal models of potential-
ly impaired humans is underway to determine whether
certain segments of the population are more susceptible to
air pollutants. This work includes the use of conventional
techniques as well as the development of new methods to
evaluate the effects of pollutants on the lung, skin, liver,
immune, host defense, and cardiovascular systems. Al-
though pulmonary effects and route of exposure comprise
a major focus for much of the animal research, additional
emphasis will be directed toward the dermal and oral
routes in order to better assess the health consequences of
environmental exposures.
To help identify adverse health effects, ETD re-
searchers will work on correlating structural, biochemical,
and functional changes in animal organ systems, principal-
ly the lung and liver. Such correlation is inherently com-
plex, requiring a research team with specialists in
biostatistics, biochemistry, pharmacology, engineering,
immunology, physiology, physics, toxicology, and
chemistry. As pharmacokinetic profiles are developed for
xenobiotics, ETD will continue to study the biochemical,
physiologic, and pathologic effects of these substances to
ascertain mechanisms of toxic action. Specific health ef-
fects issues being addressed include animal-to-man
relationships in toxic response, mechanisms of injury,
animal models of disease, chronic lung disease, and
species sensitivity.
ETD studies on the chronic effects of environmental
pollutants cross a variety of scientific disciplines, includ-
ing inhalation engineering, biostatistics, cardiopulmonary
physiology, biochemistry, immunology, morphometry,
and histopathology. A primary focus of ETD research has
been the lung; thus, the design, fabrication, and operation
of state-of-the-art inhalation facilities by the ETD en-
gineering staff is essential. Such engineering-based re-
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search and development that meets the exposure requests
for a spectrum of gases, particles, and mixtures is an ongo-
ing function of a broadly skilled team experienced with
both acute and chronic exposures of laboratory animals to
a wide variety of gases and particles.
Given the myriad of chemicals in air, it is critical that
ETD investigate the effects of inhalation exposure to these
pollutants on pulmonary and cardiovascular function.
Hazard identification is the first step; but, as data from the
primary air pollutants show, certain subpopulations are
likely to be more sensitive than others to the adverse ef-
fects of toxic inhalants. Hence, emphasis must be placed
on the development and use of animal models that
resemble human disease states such as emphysema,
asthma, hypertension, congestive heart failure, and pul-
monary fibrosis. Assessment of the relative sensitivity of
these subgroups as compared to the normal healthy state
would also provide basic susceptibility data as well as in-
formation on the mechanisms of response that come into
play in the responses seen in the normal, less susceptible
state.
At present, most animals used in experimental studies
are highly inbred strains. While these animals provide
good control of that experimental variable, perturbing the
animals' health through selective/specific empirical tech-
niques is useful for inducing potential risk factors to the
target organ of interest"the lung, for example. Given suf-
ficient knowledge of a particular disease state or the
mechanisms by which the risk factor is induced, a disease
state can be induced. No intervention (chemical, surgical,
or otherwise) is totally specific; thus, this imposed factor
must be incorporated into all data interpretation.
This disadvantage may soon be ameliorated by new
research in genetic manipulation. Human genes can now
be transplanted into the embryos of newly developed,
genetically altered rodent strains to "create" so-called syn-
geneic animals; this work has opened a new avenue for
this type of research. The advantages of exploring the
genetic basis for a disease"e.g., specificity, control"may
greatly aid in health interpretations. Using these new syn-
geneic animals as well as refined, (typically) chemically
induced models of disease, ETD researchers will elucidate
alterations in pathogenesis, overall health sensitivities, and
mechanisms of action. Studies such as these should pro-
vide great insight into those abnormal or deficient states of
health that are most likely to be worsened by exposure to
environmental pollutants, or that could subject the whole
organism to these effects.
ETD investigation of the effects of environmental
chemicals on immune functions and on a variety of dis-
ease models will have three major goals:
Developing methods for assessing immune responses
in the lung to improve the assessment of the im-
munotoxic effects of inhaled compounds; these
methods could then be used along with host resistance
models to provide information on chemicals regulated
by NAAQS and other air toxic compounds for
regulatory purposes
Correlating effects of toxic chemicals on immune
function tests with effects on host resistance and dis-
ease models to improve the assessment of risk of in-
creased debility based on effects of immune function
tests
Improving predictions of the effects on humans based
on animal research
A key aspect of ETD's research program is its ability
to relate exposure to dose to effect in a coordinated man-
ner. Dosimetric models of inhaled compounds require in-
puts of measurable physical and chemical parameters.
Once models are developed, they will be validated ex-
perimentally and further refined. A coordinated research
approach involving modelers and experimentalists is es-
sential for successful development, validation, and refine-
ment.
Once exposure concentration of a pollutant can be re-
lated to tissue dose through dosimetric techniques, re-
search on relative tissue sensitivity among species can be
conducted. Hence, in concert with dosimetry research,
animal toxicology research will focus on the issues of
homology and species sensitivity of experimental
laboratory animals relative to humans. Identification of
acute responses to inhaled toxicants and the responsible
underlying mechanisms over relevant dose ranges will be
key to understanding the relevance of the particular animal
model to humans. For example, a more thorough under-
standing of inflammatory processes in the lung and the
events leading to tissue injury or disease will enable a bet-
ter comparison with the fragmented human data and estab-
lish a base from which an assessment of chronic health
risks can be derived (only animals studies can provide
controlled chronic exposure studies to ascertain long-term
effects). Identification of factors that lead to disease or
otherwise contribute to susceptibility of the exposed sub-
populations is an important step in evaluating the major
air toxicants in the urban environment The collective ef-
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forts from a multidisciplined group of investigators"from
ETD, HSD, and collaborating universities"will develop
better study models and potentially useful markers or in-
dicators of risk for use in human populations.
The appropriateness of any animal model used in the
study of a lexicological event is most clear when the
response of the animal can be correlated, at least qualita-
tively, with the human response (i.e., a homology of effect
exists). If homology exists, then determination of relative
dosimetry and sensitivity will provide a straightforward
picture of the phenomenon that can be directly incor-
porated into the risk assessment process. Unfortunately,
because this information scenario is not typical, the
mechanism of injury must be elucidated for lexicological
data to be used confidently (i.e., with minimal uncertainty)
in risk assessment. By such a process, the type and degree
of injury can be better related to the human siluation.
ETD investigations in this area will amplify understanding
of the mechanisms of toxicily; additionally, they will en-
hance interpretations of the chronic toxicity data for which
there is virtually no human exposure correlation.
The importance of the various routes of exposure is
clearly associated with the physical form of the pollutant
in the environment, although all exposures occur by a
mixture of routes. Water pollutants enter the body
primarily through ingestion; ambienl air pollulanls (bolh
gases and particles) enter primarily through the lung,
though dermal absorption is important for exposure to
compounds such as pesticides. Despite such distinctions,
a potentially considerable overlap exists between exposure
routes. The portal of entry by which a pollutanl enters the
body can have a significant impact on the dose to the
eventual target organ"especially if the portal of entry is
also the primary target organ.
When a pollutant, or class of pollutants, can enter the
body by multiple routes, it is both costly and impractical
to conduct toxicity testing by all routes of exposure. Be-
cause the portal of entry can influence eventual organ
dose, however, the resulls of a toxicity lesl by one ex-
posure route musl be extrapolated to predict the outcome
of exposure by another route. The level of research effort
into portal-of-entry questions is determined 1) by the com-
plexity of the organ serving as the portal of entry, and 2)
by the organ's accessibility to direct experimentation. Re-
search into gas and particle uptake by the respiratory tract
requires a high level of effort because of the complex
physical interactions mat occur, and because of the wide
diversity of cellular and biochemical components in this
organ system. These problems are further complicated by
the inaccessibility of me lung to direct observation in in-
lacl animals.
In contrast, dermal absorption of compounds can be
measured directly, because the skin is a readily accessible
organ to experimentation. Studying gastrointestinal ab-
sorption of compounds is a problem midway in difficulty
between work on the lung and skin. The GI tract lends il-
self to more simple compartmenial modeling approaches
than the lung, but it still requires more coordinated ex-
perimental and modeling approaches lhan the skin.
Many proteins thai are important to cell functioning
are lipophilic and resident in the cell membrane. The lipid
bilayer that forms the membrane provides a matrix in
which the proteins may move relative to one another.
Some functions are dependent on two proteins being able
to diffuse into proximity with one another. The rate at
which a protein can move in ihe lipid matrix is limited by
the fluidity of the membrane, a characteristic that is the in-
verse of viscosity. The functions of several membrane
proteins are known to be affected by the fluidity of the
membrane. In particular, fluidity affects the binding of
neurotransmitters for some receptors, e.g., the serotonin
and the beta-adrenergic receptors.
Mosl organic xenobiotics are lipophilic and rapidly
partition into cell membranes, with a steady-slate con-
centration in the cell being reached with continuous ex-
posure. Accumulation of the compound in the membrane
will affect its fluidity and consequently the function of Ihe
membrane proteins lhal are sensitive lo fluidity. Many
anesthetics work in this manner, although the detailed
mechanism is nol well understood. Xenobiotics that are
not metabolized or only slowly metabolized could also act
in this manner, and may result in toxicity at the cellular
level. This response will be investigated by examining
receptor binding as a function of fluidity (i.e., concentra-
tion of chemical) induced by potentially toxic chemicals
representing different struclural classes. This research ef-
fort is designed to examine the role of membrane proper-
ties in toxicity, explain the basis for toxic effecls, and
identify heretofore unsuspected toxicants.
The ral has become a primary animal model used in
lexicological research; however, this rodenl species may
not be the most appropriate system in which to study all
types of responses. Differences in melabolism or an-
tioxidanl levels between ihe rodenl and human, for ex-
ample, could result in divergent responses that may affect
either ihe homology or sensitivity of effect. The mouse
offers another rodent model with extensive genetic charac-
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terization. In fact, the mouse is frequently the species of
choice for certain mechanistic studies. The animal model
should be chosen in light of the specific questions under
study. Various responses should be studied in at least one
other species; such data, when used in conjunction with
the limited human data, would allow researchers to piece
the puzzle of response together with relevant dosimetry to
determine ultimate human risk. This task is not simple,
but well-conceived studies of response in multiple species
and dosimetry will yield more useful health assessments.
For example, guinea pigs have a very sensitive
bronchoconstrictive reflex to irritants such as sulfuric acid,
and rats do not. On the other hand, long-term exposures
to acid in rats induces a pathology resembling bronchitis
in man, but similar exposures do not appear to affect
guinea pigs to the same degree. Such a disparity is dif-
ficult to interpret: perhaps dosimetric as well as species-
specific factors such as mucus fluid neutralization
capacities are involved. Such differences in response
should guide investigators in selecting appropriate species
as well as in interpreting data for comparison to humans.
Clearance of inhaled nonaqueous particles from the
respiratory tract of animals is another focus of ETD re-
search. The database on clearance of particles from the
conducting airways and pulmonary regions of animal
lungs is very sparse. Yet, clearance kinetics is an impor-
tant component, along with deposition, of particle
dosimetry models. ETD investigations, conducted in con-
cert with HSD, will focus on whole lung kinetics as well
as on the cellular processes that govern clearance kinetics.
Toxicokinetics of environmental chemicals will be-
come a major focus of ETD research. The absorption of
compounds by the oral, dermal, and inhalation routes of
exposure will be studied both in vivo and with in vitro
methodology. Researchers will examine in vivo the dis-
tribution of the absorbed compound and develop models
to predict such transport. They will also analyze the rate
of metabolism in the liver, portals of entry, and target tis-
sue in the animal as well as in tissue extracts. Excretion of
both the parent and metabolite compounds will be studied,
with emphasis on pulmonary elimination of volatiles,
hepatobiliary elimination of large molecules, and renal
elimination of the majority of chemicals. In vitro
methodologies will be developed and used where ap-
propriate, so that researchers can measure the integrated
dose of toxic chemical to the target tissue for repre-
sentatives of a given class of chemicals. Physiological
and experimental data will be used as input for phar-
macokinetic models, which will be designed to allow
dose, route, and species comparisons.
2.4.3.3 Chemical-Specific Data
ETD must continue to work on the toxicities of
specific toxicants to assist the program offices in develop-
ing emission and exposure standards for the myriad of
chemicals in the environment. At present, these chemicals
are controlled individually; but so little is known about
most of them that developing control strategies for interac-
tions is impossible without sufficient databases. ETD re-
search is progressing on several levels:
Identifying health effects
Assessing the chemicals' dose-response behaviors
Testing the worthiness of dosimetric and PB-PK
models in predicting observations (so that data can be
extrapolated across species to humans)
Interactions that may include metabolism, while
under investigation as single and mixed toxicants, will
provide a new dimension to this effort; and, as a result of
this work, the ever-evolving tools for risk assessment
should be improved in their capacity to estimate potential
human hazards. ETD research is designed to integrate
components from outdoor and indoor issues because these
comprise the entire exposure scenario. Much is unknown
about the effects of encountered chemicals in their par-
ticular exposure scenario"for example, volatile organic
compounds (VOCs) found indoors or alternative fuels ad-
ding to urban "soup."
The health issues facing experimental toxicology are
growing more complex and urgent. Current regulations
based on acute alterations in lung function in exposed
humans have little relevance for the potential threat of
chronic lung disease or impairment. Moreover, single-
contaminant studies clearly oversimplify even the classic
atmospheres of cities like Los Angeles.
Recent decisions to replace conventional fuels with
alternative, more combustible fuels are intended to reduce
tropospheric ozone as well as national dependence on
foreign oil. The enhanced volatility of these alternative
fuels and their combustion products, however, may lead to
additional atmospheric exposures. Possible health effects
associated with the switch require examination.
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2.4.3.4 Biological Markers
In collaboration with HSD, ETD will conduct re-
search to develop and evaluate quantitative biochemical
markers for toxic chemical exposure, and to integrate
these markers into current models of toxicity, adaptation,
and repair. In this effort, relationships between tissue dose
(binding or absorption of toxicant molecules) and toxicity
will be examined, as well as between species, exposure
scenarios, and altered states of susceptibility. The
linkages developed through this research should address
EPA's need to extrapolate data from animals to humans,
from acute to chronic situations, and from resistant to sus-
ceptible individuals. Questions involving the adversity of
biochemical changes will be addressed through correlation
of these changes with morphological and physiological ef-
fects. Research efforts will also include development of
immunologic assays as biomarkers for allergenic reactions
to inhaled compounds and establishment of methods for
testing the allergenic potential of inhaled compounds.
2.4.3.5 Pollutant Mixtures
Much ETD research on complex mixtures is covered
under Section 2.4.3.3, Chemical-Specific Data. In addi-
tion, ETD plans to develop bioassays to evaluate the
toxicity of complex mixtures.
2.4.4 Emerging Issues
2.4.4.1 Biologicals/Microorganisms
In spite of our substantial knowledge of microbiol-
ogy, the role of microorganisms and their products as one
component of the environmental milieu is not well ap-
preciated. Clearly, Legionnaire's disease is a salient ex-
ample of the insidious influence of microorganisms on
health, and the polio epidemic of the 1940s and 50s stands
out as a waterborne problem. Today, we are just begin-
ning to consider the many microorganisms that populate
our homes and work places; and data concerning cases of
sick building syndrome suggest that these life forms, their
spores, and gaseous emanations may contribute to im-
paired health"from malaise to respiratory stress.
Destruction of biological wastes, particularly from
medical applications, is another cause of concern. In-
cineration is probably the most efficient, but expensive,
means of destruction and decontamination. If these in-
cinerators are poorly operated, the contaminated materials
may not be totally destroyed and thus the smoke from the
incinerators may be contaminated. The resolution seems
easy enough, but the problem is of growing concern due to
lack of knowledge and enforcement.
2.4.42 Natural Versus Synthetic Fibers
The health effects associated with asbestos, a natural-
ly occurring fiber, are well known. As manmade fibers
are developed, questions arise as to their potential for
similar health consequences. Research into the dosimetry
and the potential for a relationship between fiber
dosimetry and toxicity needs to be explored.
2.4.4.3 Ultraviolet Radiation
Sunlight contains an ultraviolet light of short wave
length known as the "B form" that appears to have not
only genetic implications (skin carcinogenesis), but may
penetrate enough to affect systemic immune function.
Too little is known at this time to do anything but specu-
late on the problem. If UV-B has immune suppressive
capabilities, however, any increases in this type of radia-
tion due to ozone layer degradation could represent a
problem of potentially great significance.
2.4.4.4 Bioavailability of Chemicals from Soils or
Other Matrices
Many potentially toxic compounds are found in the
soil and may pose a hazard to humans by all three routes
of exposure. Because these compounds may be bound up
in the soil matrix, their potential health consequences are
likely to be a function of the ease with which they can be
released from the soil after it has entered the body through
the gut or respiratory tract, or after it is applied to the skin.
Research into this issue is likely to be important for future
assessment of hazards from toxic waste sites.
2.4.5 Summary
2.4.5.1 Hazard Identification
Because more cellular and molecular approaches, in
addition to pulmonary function tests, are needed to iden-
tify hazards, researchers will develop in vitro
methodologies for evaluating pulmonary toxicity.
Mechanistic studies on respiratory injury will focus on the
pathogenesis of toxicant-induced lung disorders, with em-
phasis on the development of animal models for pul-
monary disease and populations at special risk (e.g.,
children, the elderly). In all cases, studies will be con-
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Early Embryonic
Development
\
Gamete
Function
Organogenesis
Ovarian
Function
Testicular
Function
Neonatal
Development
Fig. 2-7: Lifespan Reproductive Events
By focusing on the life cycle, the division has been
able to divide its research activities into a continuum of
smaller, well-defined processes. Relative sensitivities or
progression of toxicities can then be followed through all
the important stages of reproduction. Within the area of
gonadal function, for example, concerns include endocrine
and paracrine regulation of oogenesis and sper-
matogenesis; the production, release, transport, and
maturation of the gametes; and the development of
biomarkers of function that can be readily extrapolated to
humans. If functional gametes reach the site of fertiliza-
tion, the events of fertilization, cleavage and embryo
transport, maternal hormonal milieu, and uterine recep-
tivity associated with implantation become the focus of
the DTD effort.
Once pregnancy has been initiated, a new period of
special susceptibility is encoun-tered: embryogenesis.
Here, DTD research efforts are concentrated on under-
standing the mechanisms of abnormal development; deter-
mining the significance of "minor" versus "major"
malformations for extrapolation purposes; determining the
relationships between pharmacokinetic and phar-
macodynamic parameters; and assessing the significance
of confounding factors such as maternal toxicity or poten-
tial reversibility of effects. To understand alterations in
developmental processes, investigations of the physiology
and biochemistry of postnatal
animals is sometimes neces-
sary because many of the
organ systems (e.g., lungs) do
not become operational until
after birth. The neonate may
also be at risk to toxicant ex-
posure during the period when
physiological functions are still
developing into the adult
capabilities.
The last major develop-
mental hurdle is puberty, or the
transition phase that cul-
minates in full reproductive
competence. This stage is
used as a launching point to as-
sess the overall effects of
xenobiotics on reproductive
processes, which might include
a diminution in numbers or
health of future generations.
Finally, aging is the ultimate
developmental process. Avail-
able data suggest that aging of
the reproductive tract can be
modified by toxicant exposures during critical preceding
developmental periods. This lifestage may also pose sig-
nificant new risks to individuals: their homeostatic
processes may have diminished to the extent that they are
less capable of withstanding previously innocuous
xenobiotic exposures.
Human health effects caused by biotechnology agents
can result from direct action of a microbe as well as
through interactive mechanisms. With natural agents,
direct effects such as infection, pathogenicity, toxicity,
and oncogenic cell transformation are the basis for con-
cern. In contrast, concerns with genetically engineered
agents relate to more indirect mechanisms and center
around the genetic instability that may result from disper-
sal of genes into other strains or species. Potential health
consequences or inappropriate genetic interactions leading
to the disruption of coordinated regulation of gene activity
are therefore important considerations in deciding whether
to allow their environmental release.
2.5.2 Division-Specific Research Needs
The importance of understanding and characterizing
the human developmental and reproductive lifecycle
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catalyzes a number of research needs in these areas in
hazard identification, dose-response assessment, and
chemical-specific information. These needs are separately
categorized in the discussion below under reproductive
and developmental toxicology and microbial pesticides.
2.5.2.1 Hazard Identification
Reproductive Toxicology. The relatively new dis-
cipline of reproductive toxicology presents four major
challenges. First, evaluation of reproductive function in
animals and humans requires a multifaceted approach,
taking into consideration the marked physiological dif-
ferences 1) between males and females, 2) among
reproductively immature, mature, and senescent in-
dividuals of both sexes, and 3) among diverse but inter-
dependent target organs (e.g., brain, pituitary, and
gonads). Second, due to the relatively high background
rates of infertility and spontaneous abortions in the general
population, detecting and quantitating the potential of en-
vironmental agents to initiate adverse reproductive out-
comes in humans is difficult. Factors responsible for the
high background incidence are generally unknown. Third,
outcomes resulting from acute or chronic exposures to
reproductive toxicants are likely to differ with regard to
their chronology, perseverance, and severity. These dif-
ferences need to be defined and factored into the risk as-
sessment process. Finally, reproductive toxicology must
interface with the related disciplines of developmental
toxicology (to meld fertility and adverse pregnancy out-
come) and germ cell mutagenesis (to integrate the genetic
and morphological integrity of the gametes to support nor-
mal development).
Reproductive toxicology has traditionally focused
primarily on the male, with emphasis on direct testicular
toxicity and the resultant decrease in sperm production.
Current research issues revolve around defining endpoint
relationships with respect to each other and to fertility,
determining the significance of low-dose measures of ef-
fects, and evaluating the duration and reversibility of ef-
fects. However, an increased emphasis on the female is
forecast as a result of recent reports of significant (about
25 percent) early, peri-implantation pregnancy loss in
humans. In both sexes, the predictiveness of hormonal
endpoints for evaluating loss of fertility must be deter-
mined, especially in the early stages of loss of reproduc-
tive competence.
Reproductive toxicity in animal models is presently
evaluated through multigeneration or continuous breeding
protocols: adult animals of both sexes are treated over
periods of time that encompass at least one spermatogenic
cycle in the male and two or more generations in the
female. These protocols have serious shortcomings due to
their reliance on fertility as their main endpoint. As
pointed out in the proposed EPA reproductive risk assess-
ment guidelines, fertility is an inadequate measurement on
which to base assessment of reproductive dysfunction in
rodents as many components of the reproductive axis can
be compromised without affecting fertility. In addition,
the present tests generally do not provide information on
the affected sex, the likely target tissue of toxicant
damage, or the mechanism of toxicity. Thus, a major need
in reproductive toxicology is the improvement of testing
protocols. Ideally, a flexible decision-tree approach should
be developed for assembling an appropriate protocol in ac-
cordance with compound-specific information available at
the time.
Also, a more comprehensive set of reproductive
endpoints is needed in both the male and female to pro-
vide essential information about the affected sex and rela-
tive end-organ sensitivities. In the male, technological
advances on semen analysis have been accomplished, in-
cluding computer-assisted analysis of sperm motility; but
these need to be rigorously evaluated in toxicologic and
epidemiologic settings. In the female, methods to identify
early pregnancy loss in animals are needed so that animal
models can be developed for experimental toxicologic
studies.
Developmental Toxicology. Current testing guide-
lines established by the principal regulatory offices are
generally considered adequate for assessing the classical
manifestation of developmental toxicity"embryonic death,
gross structural malformations, and intrauterine growth
retardation. However, for other major areas of concern, no
acceptable detection methods are available. For example,
there are currently no acceptable procedures for evaluating
postnatal manifestations of developmental toxicity (in-
cluding, but not limited to, effects on the central nervous
system). This deficit exists despite the recognized in-
ability of standard assays to evaluate alterations in organs
and organ systems, such as the thyroid, adrenal, pul-
monary, renal and immune, which remain quiescent until
the postpartum period. Other key needs under hazard
identification are techniques for developing structure-ac-
tivity relationships, assessing the potential special suscep-
tibilities of the lactating female and the pre-adolescent,
and examining the relative strengths of in vitro test sys-
tems as primary screening versus mechanistic tools.
Finally, the issue of transplacental carcinogenesis presents
additional challenges to the risk assessment process.
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Microbial Pesticides. In the past, microbial pesticide
research efforts sought to improve the guidelines for
safety assessments and registration standards of naturally
occurring microorganisms. The present pesticide assess-
ment guidelines for microbial pesticides appear to be ade-
quate for most of the natural agents and simple genetic
alterations. However, future efforts in genetic engineering
will produce increasingly complex and heterogeneous
lifeforms. An exotic array of agents possessing unique
characteristics and novel modes of action is anticipated.
The suitability of present tests for such agents must there-
fore be reassessed. Additionally, safety evaluation needs
for nonpesticidal, genetically altered agents (e.g. bio-
remediation organisms) have yet to be clarified.
At present, the predominant uncertainties in assessing
the risk associated with genetically altered microbial
agents lie in hazard identification. Hazards associated
with natural progenitor microbial agents and potential
dangers associated with novel, genetically engineered
strains have to be identified and characterized; and then
tests must be developed to assess their potential to
produce detrimental health effects. Such tests, when
standardized and shown to be of general utility, can be
used by the Agency for regulatory purposes, such as in
subdivision M of the Pesticide Assessment Guidelines.
2.5.22 Dose-Response Assessment
Reproductive Toxicology. A clear need exists for re-
search directed at the identification of toxic mechanisms,
sites of action, cell/organ targets, and critical periods of
vulnerability within the life cycle. This type of informa-
tion is essential in establishing a biological basis for ex-
trapolation of animal data to humans. Protocols using
short-term exposures are particularly suitable for deter-
mining biologically based dose-responses using multiple
endpoints, for evaluating reversibility, and for identifying
effects during critical time periods (e.g., early pregnancy)
or in specific populations (e.g., young versus old in-
dividuals).
Another major area of concern and research need is
the identification of susceptible populations. For example,
more information is needed about the potential of early
lifetime exposures, especially to hormonally active agents
at critical developmental stages, to affect the normal
progression of puberty and sexual differentiation.
Likewise, little is known about the relative sensitivity of
aging individuals to reproductive effects, or about the
potential of xenobiotics to accelerate the onset of
reproductive senescence.
Developmental Toxicology. Clearly, the area of
greatest need within the Agency for developmental
toxicity data is the improved understanding and use of in-
formation generated in standardized test protocols. Short-
term needs involve research to support the modification of
these protocols to reflect greater understanding of the
biological significance(s) of the various manifestations of
developmental toxicity (e.g., skeletal variation, dilated
kidneys, intrauterine growth retardation). This informa-
tion would be viewed in terms of the risk equivalence and
comparative expression of the four primary manifestations
of developmental toxicity (death, structural modifications,
altered growth, and functional alterations). Another major
concern is determining the relationship(s), if any, between
maternal toxicity and developmental toxicity. Can better
indicators of each be developed and implemented? Are
some forms of maternal toxicity casually involved in ab-
normal development? Answers to such questions will
support greater confidence in the use of safety factors
based on the most sensitive effect in the most sensitive
species.
Longer-term needs entail the development of more
comprehensive determinations of exposure and response
variables in animal models, including incorporation of
pharmacokinetic, pharmacodynamic, and quantitative
dose-response models. Efforts must be directed at iden-
tifying and quantifying species-specific parameters that
can be applied in mathematical modeling efforts, deter-
mining the relationship and time dependency between
delivered dose and biological outcome, and characterizing
the biochemical and molecular events that precede disrup-
tions in morphological development. Ultimately, quantita-
tive dose-response models linked with mechanistic
information and physiologically based pharmacokinetic
models will allow risk assessors to scale the developmen-
tal toxicity in accordance with known species-specific fac-
tors. Conceptual frameworks for this revolutionary
approach to risk assessment need to be formalized,
analyzed as to their underlying assumptions, and empiri-
cally validated.
Microbial Pesticides. Because inappropriate genetic
interaction of genetically altered agents with other
microbes that are either in the environment or endogenous
in human cells, tissues, or organs could lead to detrimental
health effects, the mechanism involved in such processes
must be characterized. Interactive mechanisms could lead
to the spread or acquisition of undesirable traits or en-
hance mechanisms that potentiate the occurrence of
detrimental health effects. At present, few genetically en-
gineered commercial products have reached the
marketplace. Needs pertaining to dose-response assess-
2-41
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ments for these products are presently being discussed
within the Agency.
2.5.2.3 Chemical- or Agent-Specific Data
Reproductive and Developmental Toxicology.
Defining research needs for chemical-specific information
is difficult. Some program offices (e.g., Office of Drink-
ing Water) may be able to lay out long-term regulatory
agendas, and others (e.g., Office of Air Quality Planning
and Standards) may be able to identify emerging research
issues, but others require information on an ad hoc basis.
Thus, OPP may be faced with a immediate need for data
on a particular pesticide to make decisions about imminent
hazard, or some national environmental disaster may dic-
tate development of a critical database. DTD has tradi-
tionally been involved in both defined and unanticipated
studies; and it has the staff and capabilities to meet such
needs as they arise.
Methanol is currendy a chemical of interest because
its use as a clean fuel is expected to increase greatly.
Evidence available to date indicates that methanol may
represent a developmental and reproductive hazard.
Therefore, a comprehensive evaluation of the effects of
this compound is a current research need.
Microbial Pesticides. Because many of the geneti-
cally altered strains and natural progenitors used in
microbial pesticides and other biotechnology products are
frequently obscure agents, information such as specific
strain identification and detection are required. Specific
strain identification becomes critical for bacteria, since
their major properties are frequently determined by dis-
sociable extrachromosomal genetic elements (i.e., plas-
mids).
2.5.3 Research Plan
DTD's staff has traditionally been closely involved in
risk assessment activities within the Agency. DTD scien-
tists have served as members on committees drafting the
risk assessment guidelines for developmental and
reproductive toxicology, and have planned and par-
ticipated in workshops and conferences to formulate
recommendations for designing and interpreting studies
that address major unresolved issues. In fact, they have
often produced the key research papers that have formed
the basis for many of these events and are routinely sought
to participate in them.
Significant interactions"including the transfer of
scientific and technical advice as well as research sup-
port"have occurred between DTD staff and the program
offices. Not only do the risk assessment concerns of the
program offices influence the research direction of the
division, but the division's research outputs have had sig-
nificant impact on both specific and generic elements of
the risk assessment process, and thus on regulations
governing various chemicals. In fact, the first decision to
label a pesticide with information regarding potential ad-
verse developmental effects, and the first decision to
remove a herbicide from the U.S. market on the basis of
developmental effects, were made on the basis of research
performed by the staff.
Thus, through close interactions with other elements
of ORD and the Agency, DTD maintains a high degree of
programmatic relevance while advancing the state-of-the-
science in developmental and reproductive toxicology.
The research plan for each discipline has been crafted in
light of the long-term needs of the Agency for improved
detection, interpretation, and extrapolation of developmen-
tal and reproductive toxicities.
The specific goals in the area of microbial pesticides
are to identify, characterize, and develop test mediods for
the assessment of health hazards associated with natural
and genetically altered microbial pesticides and products
of modern biotechnology. Because health effects research
with microbial agents generally involves macromolecule
and concerns center around genetic issues, a biomolecular
approach is required.
2.5.3.1 Hazard Identification
Reproductive Toxicology. To address the need for
improved reproductive tests, alternative reproductive
toxicity test (ART) strategies are under development and
validation. ART protocols may eventually replace or sup-
plement the current multigeneration tests. DTD re-
searchers will assess known reproductive toxicants using a
protocol that begins with prepubertal animals and extends
through sexual maturation, pregnancy, and the first
generation. This protocol detects developmental effects
during critical periods of sexual maturation, and at the
same time evaluates the entire reproductive life cycle. It
incorporates multiple endpoint measures, including cel-
lular and endocrine measures, which will be evaluated for
specificity and sensitivity. DTD's tiered approach incor-
porates the use of novel and sometimes more specialized
endpoints when appropriate, particularly in follow-up,
mechanistic studies. As research proceeds during the next
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Illliillillliiliiiilliiilli
two to three years, the protocol will be refined and recom-
mendations made for its incorporation into EPA risk as-
sessment and/or testing guidelines.
To increase the number and usefulness of male and
female reproductive endpoints, research efforts will im-
prove methods for quantitative evaluation of gamete
production and quality. Current work is focused on com-
puter-assisted image analysis of rat sperm motility, and fu-
ture work will extend this technology for morphometric
analysis of sperm cells as well as testicular and ovarian
histology. In the endocrine arena, a battery of hormone
assays has been developed for use in rodent toxicology
studies. Information provided by endocrine profiles will
be compared with more invasive measures of reproductive
dysfunction and their use as biomarkers of effects on the
reproductive axis evaluated. A major advantage of both
the endocrine and semen analyses is that similar measure-
ments could be easily performed in humans with
suspected exposures to environmental agents.
Developmental Toxicology. Within the next year,
the DTD research program evaluating the relationships of
maternal (adult) and developmental toxicity will have at-
tained its major milestones. DTD researchers, however,
will collaborate with scientists from NTD to continue the
investigation of the role of maternal stress in altered
embryonic development. This effort may define alterna-
tive methods of establishing the maximum tolerated dose
levels used in standard developmental toxicity assays.
DTD and NTD scientists will also work together to
evaluate the effect of maternal separation (an experimental
manipulation required for dermal or inhalation exposures
during lactation) on postnatal growth, differentiation, and
behavior. Results from this effort may ultimately feed
into the developmental neurotoxicity testing guidelines
that are currently being drafted.
DTD will also continue work in areas of interpreta-
tion of malformations versus "variations," focusing on the
induction of supernumerary ribs. Research efforts will
determine the mechanism behind this alteration, as well as
how the endpoint is expressed across various laboratory
species. Efforts to characterize SARs for substituted
phenols and branched chain aliphatic acids will continue
in support of the Office of Toxic Substances (OTS).
Finally, DTD will continue to monitor the development
and validation of in vitro teratology screening assays for
potential applications in SAR as well as bioassay-directed
fractionation projects.
Microbial Pesticides. Categorizing research under
the hazard identification and dose-response assessment
topics is more difficult for microbial pesticides than for
chemical agents. Therefore, these types of efforts are ad-
dressed together here.
Some of the proteins contained in spores of the
registered pesticidal agent, Bacillus thuringiensis subsp.
israelensis, are toxic to mammals. DTD is investigating
the precise nature of the physiological effects, the mode of
action, the molecular genetics, and the potential for
mutagenic inactivation of the toxin gene. Aside from
providing data for health effects assessment of the toxin,
the assignment of the mammalian toxic activity to a
specific gene product distinct from the insecticidal com-
ponents will prevent mistaken use of the mammalian toxin
gene in genetic engineering of other bacteria and plants.
Inappropriate interaction between the altered agent
and other microbes inhabiting the environment or human
cells and tissues may lead to detrimental human health ef-
fects. An ongoing investigation is characterizing mecha-
nisms involved in the movement of a marker mammalian
toxin gene between bacterial chromosome and other
genetic elements, as well as other bacteria strains and
species. The results of this work will provide a better un-
derstanding of how an unstable engineered gene could be
transferred (or detrimental genes acquired) by genetically
altered agents to produce agents which then could induce
adverse health effects. Inappropriate genetic interactions
could also occur at the molecular level, resulting in the ac-
tivation of latent endogenous agents. Current research is
investigating the potential for activation of human
cytomegalovirus infections by genetically engineered
baculoviruses.
Finally, DTD is developing more appropriate and sen-
sitive tests for assessing the health effects of microbial
agents used as pesticides. The division is attempting to in-
corporate state-of-the-art technology into preregistration
tests recommended by the Agency for candidate viral pes-
ticides; this effort should increase their reproducibility,
sensitivity, and cost effectiveness. These new tests will be
standardized, and the general efficacy over a range of
human and other nontarget species cell cultures deter-
mined. Special emphasis will be placed on developing
tests to assess the likelihood and potential effect of foreign
genes moving into human cells from engineered or-
ganisms.
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iiiiiiM
2.5.32 Dose-Response Assessment
Reproductive Toxicology. In response to the need
for short-term reproductive assessments, current efforts
are directed at evaluating the pathogenesis of known
reproductive toxicants. A variety of cellular and en-
docrinologic endpoints are being applied and compared
for relative sensitivity and predictive value. Researchers
are periodically evaluating endpoints over a short-term
period to allow identification of the primary site of lesion
(hypothalamus, pituitary, gonad) and localization of cel-
lular targets in the gonad. This approach is being used to
evaluate susceptible populations (e.g., effects in young
versus old animals) and to compare acute and subchronic
effects. The long-range goal of these studies is to make
recommendations regarding choice of endpoints in
reproductive toxicology and extrapolation from acute to
(sub)chronic effects in specific categories of reproductive
toxicants.
The DTD program is also developing and applying in
vitro tests using reproductive cells and tissues to conduct
mechanistic studies and to provide data for interspecies
extrapolation. For example, in vitro production of steroids
by Leydig cells from the testes or granulosa cells from the
ovary are being used to dissect effects on the steroidogenic
pathways in animal models. Likewise, secretion of
proteins from epididymal epithelial cells is being ex-
amined in an attempt to find improved biomarkers of
epididymal function that may one day be applied to
analyze human semen. Other cellular approaches under
evaluation include in vitro tests to detect local changes in
neuroendocrine function (such as hypothalamic and
pituitary perfusion) and to assess gamete function (in vitro
fertilization, sperm microinjection). Hormone receptor as-
says and biochemical analysis of placental tissues during
implantation are also under development.
In response to the need for enhanced efforts in female
reproductive toxicology, research efforts under the Re-
search to Improve Health Risk Assessment (RIHRA) pro-
gram will examine the homology between animal models
and humans regarding ovarian and uterine function.
Short-term tests will continue to be used to assess effects
of chemicals on the luteinizing hormone (LH) surge that
triggers ovulation, on the fertilization process, and on the
initiation and maintenance of pregnancy. Again, multiple
endpoints will be compared for their relative utility, and
recommendations will be made regarding identification of
hazards to early pregnancy in animals and hence to
humans.
Developmental Toxicology. The long-range goal in
this area is to develop procedures for pharmacokinetic and
biologically based dose-response assessments of develop-
mental toxicity. Pharmacokinetic studies will initially
focus on theoretical aspects of placental transport and
embryonic deposition of a series of phenols and aliphatic
acids used in previous SAR studies in the lab. These data
will be used to define the relationships among ad-
ministered dose, delivered dose, and developmental out-
come. Researchers will compare results from both in vivo
and in vitro systems and will evaluate physiologically
based pharmacokinetic models as a tool in the risk assess-
ment process. Other pharmacokinetic studies will
evaluate the inter-litter (maternal), intra-litter (uterine
position), and embryo (genomic) sources of variation in
developmental response. In addition, efforts will continue
in evaluating the role of the vehicle in bioavailability and
in the relative importance of peak versus total exposure es-
timates for the developmental toxicities induced by disin-
fectant byproducts. Through co-cultures of mammalian
embryos and heterologous metabolic activation systems,
the role of pharmacokinetic versus pharmacodynamic fac-
tors in species sensitivities will be approached. Finally,
through techniques of biochemistry, cell biology, and flow
cytometry, early events leading to stage and species-
specific alterations in embryonic development will be
probed to elucidate mechanisms of abnormal development
and to evaluate the existence of thresholds. DTD will as-
semble the information compiled in the various com-
ponents of this research to produce biologically relevant
quantitative dose-response models.
Other work involves better use of available informa-
tion on human developmental toxicants. Examining the
animal literature on these agents highlights the tremen-
dous gaps in knowledge of these toxicants' dose-responses
in standard animal models. Many of these agents have
defined pharmacokinetic and pharmacodynamic profiles
that could be readily compared across species. So-called
retrospective risk assessment can be used to evaluate how
a well-developed animal database predicts known human
risk. Lessons learned from this endeavor will feed back
into improved "prospective" risk assessments for develop-
mental toxicity.
2.5.3.3 Chemical- or Agent-Specific Data
Reproductive and Developmental Toxicology. A
small proportion of DTD's research strategy is directed at
chemical-specific information for developmental toxicol-
ogy. Of that, the largest component has been catalyzed by
the Office of Drinking Water's (ODW's) need to evaluate
health hazards imposed by drinking water disinfectants
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and their byproducts. Given the types of compounds in-
volved, this information should ultimately be useful in
SAR evaluations. The induction of cardiovascular malfor-
mations by several related disinfectant by-products will
form the basis for a broader-scale analysis of the dose-
response characteristics, the nature of the biochemical
lesion, and the potential interactions of simultaneous ex-
posure to multiple disinfectant by-prod-ucts. SAR studies
involving MX-type chemicals are also a possibility.
OPP has sporadic but continuing needs for specific
research efforts on particular pesticides. DTD's involve-
ment is determined by the criticality of the need in terms
of time, the ability to utilize state-of-the-science technol-
ogy to answer a perplexing problem, and/or the need to ar-
bitrate conflicting data. OTS has interests in SAR
evaluations on specific chemical classes, such as the
aliphatic acids for which DTD will be constructing the
necessary databases over the next several years. DTD will
also continue to coordinate efforts to develop Develop-
mental Toxicity Activity Profiles analogous to those
developed for genetic-toxicity bioassays.
In response to the Agency's need for information
regarding the reproductive effects of methanol, short-term
dose-response and route-to-route studies have been in-
itiated using endocrine assessments and testicular/sperm
evaluation in male rats. Effects are related to blood
methanol levels as well as applied dose. The acute effect
of methanol on the LH surge in females is also under in-
vestigation; and, pending results, effects on fertilization
and preimplantation development will also be explored.
In addition, researchers will evaluate the adverse effects
on prenatal development, including alterations in the func-
tion of the nervous system in postnatal animals and on
development of a database on a second rodent test species.
Microbial Pesticides. A major problem for the
Agency in regulating microbial pesticides is definitive
identification and differentiation of bacterial strains. This
problem has been exacerbated by modern biotechnological
methods in which genes are readily moved between strains
by recombinant DNA methods and mobile genetic ele-
ments. One answer to the problem is to obtain a genetic
map of the bacterial chromosome. In conjunction with
studies on the movement of the bacterial mammalian toxin
gene described above, investigations are underway to
identify sites on the bacterial chromosome that can be
used to construct a genomic map. Fine genetic maps
showing unique positions of genes will permit differentia-
tion of closely related strains for regulatory purposes by
the Agency.
2.5.4 Emerging Issues
2.5.4.7 Improvements in Risk Assessment
Over the next several years, the Division will con-
tinue to focus on detecting, interpreting, and extrapolating
test results to the human situation. Advancements in risk
assessment will follow the interactive process in which
improvements in detection lead to questions of interpreta-
tion"which, when answered, clarify the extrapolation
process. Likewise, improved recognition of the weak
points in risk assessment yield research questions ad-
dressed by the detection and interpretation components of
the mission. Research in developmental toxicology will
become increasingly focused on the extrapolation process,
while that in reproductive toxicology will be moving more
into the interpretation and then extrapolation areas.
To accomplish these goals, greater emphasis must be
placed on the development of more integrative approaches
by the various research teams. Greater coordination of the
study of metabolism, distribution, morphology, biochem-
istry, and physiology as related to lexicological mecha-
nisms is required. Additional resources will have to be
placed in both the pharmacokinetic and pharmacodynamic
efforts. Rapid advances now occurring in cell biology and
morphology will have to be incorporated into all research
endeavors, a feat that will require additional scientific
training and staffing in areas such as biochemical toxicol-
ogy, molecular biology, and experimental morphology.
All research programs will need a greater understanding of
the concentrations of the proximate toxicant in their target
tissue. More involved interactions between scientists of
complementary expertise will be required to provide in-
tegrated assessments of all aspects of developmental and
reproductive risk. Methanol may be a prototype chemical
with which to utilize this combined expertise.
2:5.42 The Female as a Susceptible Population
The female as a susceptible population is an emerging
research issue that needs the support of an ovarian, placen-
ta!, or lactational physiologist. In the area of male
reproductive effects, DTD has no expertise in the area of
risk of heritable genetic damage to germ cells. This is an
important aspect of evaluating the total risk to reproduc-
tive and developmental toxicants, and all would benefit by
closer collaboration with scientists in GTD who are ad-
dressing this issue. In developmental toxicology, an
emerging need for research in developmental neuro-
toxicity is clearly indicated. Collaborations with scientists
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in DTD and NTD is needed to situate HERL in a position
to address this issue.
2.5.4.3 NontraditionalExposure Regimens
Another emerging issue relates to nontraditional ex-
posure regimens. "Traditional" in this context refers to
single daily oral exposure to individual agents. The non-
traditional exposures, in contrast, relate to considerations
of dose-rate effects, dermal and inhalation exposures, mul-
tiagent (i.e., complex) exposures, and the use of phar-
macokinetic information to design more appropriate
exposure scenarios or to extrapolate results between
various routes of administration. Factors relating to study
design (e.g., the impact of maternal isolation on neonatal
development during dermal or inhalation exposures) are
included in this concept.
2.5.4.4 Age and Toxicity
As the population ages, interest is increasing regard-
ing the influence of toxicants on the aging process and the
influence of aging on toxicity. Within the existing struc-
ture, DTD has the base capability of establishing an aging
animal colony for use by HERL staff in such disciplines as
pharmacokinetics, neurotoxicity, cardiovascular, im-
munology, and pulmonary function. The effects of aging
in terms of reproductive senescence will continue to be an
integral component of DTD research.
2.5.4.5 Biotechnology
In the area of biotechnology, the division maintains
expertise in the areas of viral and bacterial pesticides but
lacks a specialist in fungal biology. Representatives from
this phyla are likely to be developed as biological pes-
ticides in the coming years, and DTD will be able to ad-
vise OPP on health issues related to fungi, as is already
done for other types of biological agents.
2.5.5 Summary
2.5.5.1 Hazard Identification
In reproductive toxicology, DTD will improve the
techniques used to detect and characterize impairments in
fertility and fecundity. Better detection procedures are
available in developmental toxicology; thus, scientists in
this area will focus on improving the extrapolation of
toxicologic data. To accomplish these objectives, the
division will shift resources away from describing
lexicological manifestations toward examining the
proximate events in the induction of abnormal mor-
phological and functional development as well as
reproductive dysfunction. Blends of in vivo and in vitro
methods and biochemical and morphological approaches
will be used to develop comprehensive pictures of the full
spectrum of developmental and reproductive alterations.
2.5.52 Dose-Response Assessment
In close conjunction with efforts to biologically char-
acterize toxicity, scientists will use radioisotopic labelling,
autoradiographic techniques, and analytical chemistry to
analyze target tissue disposition of xenobiotics. Where
possible, the biological and pharmacokinetic efforts will
concentrate their joint activities within chemical classes
(e.g., phenols, aliphatic acids) so that structure-dosimetry-
activity relationships can also be derived.
DTD will continue to assess the postnatal functional
consequences of prenatal insults, though at a slightly
reduced level of effort. Researchers will complete the
analysis of effects of maternal deprivation on postnatal
growth and maturation, and these data will be used to es-
tablish testing guidelines for dermal and inhalation ex-
posures over the perinatal period.
In reproductive toxicity, research effort to identify
more sensitive and comprehensive endpoints for assessing
reproductive dysfunction will culminate in improvements
to the Agency's testing and risk assessment guidelines.
Emphasis will be placed on developing biomarkers of
reproductive function for both the male and the female.
To support the program offices, DTD will assess a
wide variety of drinking water disinfectants, the reproduc-
tive and developmental effects of existing and alternative
fuels, and the appropriateness of the additivity assumption
for mixtures.
To improve risk assessments, researchers will use
data gathered in various DTD efforts as input for empirical
and biologically based quantitative dose-response models.
Various complex issues associated with the use and
development of these models will be addressed.
DTD is also developing and validating methods to
screen and quantify the potential adverse health effects as-
sociated with microbial pest control agents (MPCAs). Re-
searchers will examine the mechanism of mammalian
toxicity that results from exposure to components of a
bacterial pesticide. This information will then be used to
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develop specific assays for their identification. These
same methods can be applied to genetically engineered or-
ganisms. DTD will assess the potential for interaction at
the genetic level and for transfer of engineered genes of
viral biotechnology and pesticidal agents that enter human
and other mammalian cells. Researchers will determine
whether genes present in human or other mammalian cells
can be activated by biotechnological agents and, if so, to
what level. To assess the importance of gene transfer as a
route by which genetically engineered agents can cause
health effects, DTD will also investigate the movement of
genes for mammalian toxins between genetic components
of a bacterial pesticidal agent.
2.6 HUMAN STUDIES DIVISION
2.6.1 Divisional Program
Investigators in the Human Studies Division (HSD)
study people's responses to exposure to pollutants and
mixtures of pollutants commonly found in the environ-
ment. Human research conducted in HSD has immediate,
direct, and significant application to the regulatory and
rule-making activities of the Agency.
HSD's research program encompasses all aspects of
health research"exposure, dose, and effects. Determining
exposure is particularly important in HSD's epidemiologic
and field studies, which are generally conducted in
natural, real-world settings. (In the division's clinical and
in vitro studies, on the other hand, exposure levels are
known and carefully controlled.) In an effort to interpret
mechanisms of action and to extrapolate data from one set
of conditions to another, HSD is also exploring the
relationship between exposure concentrations and the
amount of pollutants or their metabolite(s) that reach tar-
get tissues and cells in humans (i.e., the dose). With these
data, HSD can work to characterize the risk associated
with exposure to a given pollutant by establishing relation-
ships among exposure, target dose, and adverse health
consequences. Improved risk characterization results in
better risk assessments and in more accurate monitoring of
the effectiveness of risk reduction strategies.
In HSD clinical studies, human volunteers are ex-
posed to common environmental pollutants in laboratory
"chambers" under carefully controlled and monitored con-
ditions. The pollutant levels are comparable to or less
than ambient exposures in many urban areas and are ex-
pected to cause only reversible, transient effects such as
symptoms, decrements in pulmonary function, and
biochemical changes. HSD scientists note the acute ef-
fects experienced by the volunteers as a result of the pol-
lutant exposures and assess whether longer-term or
higher-level exposures to the same pollutants could cause
disease. They also conduct dosimetric studies on the ab-
sorption, delivery to target sites, and elimination of en-
vironmental materials.
Sample tissues, cells, and lung fluids are collected for
in vitro studies (e.g., by nasal or bronchial washing or by
epithelial scraping) during clinical studies. In vitro
studies, which are conducted with tissues, cells, and cell
lines obtained from volunteers, enable HSD investigators
to obtain very specific data on cell function; these data
contribute to our understanding of the immunotoxic and
cytotoxic mechanisms of effect that pollutants have on
human health. Such studies are especially important for
gathering exposure data on pollutants to which humans
cannot be exposed experimentally. Both clinical and in
vitro exposure data are used to identify biomarkers of ex-
posure, which can then be tested in field studies.
In epidemiologic and related studies, HSD inves-
tigators look for patterns of biological effects (pre-clinical
and clinical), disease, and mortality in groups of people
who have been exposed, over both long and short terms, to
pollutants in their living or working environment Such
studies are a vital means of collecting effects data on pol-
lutants to which humans cannot be exposed experimental-
ly. Epidemiologic work is also used to monitor the
effectiveness of risk reduction strategies, i.e., to determine
whether reduced pollutant levels have benefited human
health. Field studies can address these same issues, as
well as provide a means to quantify the incidence of dis-
ease and death in humans. Combining clinical, in vitro,
and epidemiologic data allows HSD researchers to identify
potential human health effects associated with exposure to
pollutants and verify their occurrence in real-world set-
tings. Together, these three types of studies give HSD the
opportunity to study human responses to a wide variety of
exposures "ranging from uncontrolled natural exposures to
highly controlled laboratory exposures"and to study a
wide range of effects at different levels of biological or-
ganization (whole body, systemic, cellular, and biochemi-
cal). With such an integrated approach, investigators can
better understand the dose of pollutants delivered to the in-
dividual and to the target site under a variety of exposure
conditions, as well as the effects of the exposure and the
mechanism of the effects in man. In addition, human re-
search offers the opportunity to validate postulates
developed in animal models and to evaluate many risk as-
sessments that are based on theoretical and experimental
efforts.
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HSD research has helped serve as the basis for several
important regulatory decisions by the Agency, including
the current NAAQS for ozone (based on both clinical and
epidemiologic data); the 10-micron standard for panicu-
late matter (epidemiologic data) and the recent recommen-
dation by the Science Advisory Board that a NAAQS be
set for acid aerosols (based on epidemiologic data). HSD
epidemiologic data have also demonstrated that improved
ambient air quality resulting from clean air regulations has
resulted in an improvement in human health.
2.6.2 Division-Specific Research Needs
2.6.2.7 Hazard Identification
One issue of major importance to risk assessment is
how much of the pollutants to which we are exposed
reaches target organs. Improved and novel dosimetry
methods are needed to answer this question. Data from
dosimetry studies is being used to evaluate the potential
human health effects of exposure to inhaled environmental
pollutants and to develop mathematical models of how the
human respiratory tract handles such inhaled pollutants.
This research needs to be carefully integrated with the
animal dosimetry program conducted in the ETD.
Environmental research requires innovative ap-
proaches and a wide variety of methods to test for dif-
ferent endpoints and to understand the effects associated
with low levels of exposure to pollutant(s) on a variety of
biological and health endpoints. Improved methods are
also needed to measure a wider range of biological, pre-
clinical, and clinical endpoints more accurately. New tests
that are more sensitive to subtle changes and that focus on
specific areas known to be susceptible to injury need to be
developed and validated. For example, small airways,
which constitute a large portion of the surface area in the
lungs, are thought to be particularly vulnerable.
Dosimetry and modeling studies indicate that small air-
ways are the site of greatest deposition for many airborne
pollutants. Epidemiologic and animal data have shown
that pulmonary responses and morphological changes
occur in the small airways of the lung in response to pol-
lutant exposures; however, detecting such effects in
humans in clinical settings has proved particularly dif-
ficult. More sensitive methods are clearly needed for
detecting effects on the small airways, as well as on other
tissues and systems.
Research is also needed to increase the sensitivity and
specificity of the exposure and disease endpoints used in
epidemiologic research. In addition, HSD epidemiologists
need:
Methods for determining the best available exposed
populations and study opportunities
Modeling approaches for studying the feasibility and
optimal design of proposed studies
Information on the extent to which modern com-
puterization of health records (such as registries and
hospital admissions) can facilitate cost-effective
monitoring of health effects associated with pollution
Methods for subclassifying diseases into components
that are related and unrelated to specific environmen-
tal agents
Methods (measurements) that effectively estimate
population or person exposures in epidemiologic
studies
2.6.2.2 Dose-Response Assessment
Much HSD research has focused on establishing
dose-response relationships for a variety of inhaled pol-
lutants, particularly NAAQS pollutants. This research has
identified a range of acute effects found in different
human populations or subpopulations following short-
term exposure to the pollutants"including bronchocon-
striction in asthmatics after exposure to sulfur dioxide,
loss of pulmonary function in individuals who exercise in
ozone, or decreased vigilance due to carbon monoxide ex-
posure. Such acute responses are considered adverse be-
cause they could interfere with the normal activities of
daily living or significantly impair human performance
capabilities.
A key question that needs to be addressed through
mechanistic studies is the relationship between exposure
and delivered dose to the target organ. Historically, HSD
research has emphasized uptake of gases and particles by
the lung. Physiologically based pharmacokinetic studies
are needed to investigate the absorption and fate of pol-
lutants in the lungs and other systems, such as the skin and
the gastrointestinal tract.
Research is also needed to understand the
mechanisms of response to pollutants. For example, when
a responsive individual is repeatedly exposed to ozone on
consecutive days, an initial increase in pulmonary function
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response (on day two) is followed by a progressive and
remarkable attenuation in response: By day four or five,
no measurable functional response to the exposure can be
measured. Does this attenuation represent a positive adap-
tation, or does it mean that the epithelial cells have been
damaged and are no longer able to respond to the chal-
lenge of inhaled pollutants? Will repeated exposures to
ozone also attenuate the immune and inflammatory
responses? The answers to these questions have major
implications for the regulation of air pollutants.
Some individuals or populations (e.g., the elderly,
children, immunocompromised individuals, and patients
with chronic obstructive lung disease or ischemic heart
disease) are more responsive to a given pollutant than are
others. This increased responsiveness to pollutant stimuli
can be caused by factors such as preexisting disease, other
current exposures, individual sensitivity, or perhaps
genetic predisposition. These populations or individuals
may be particularly sensitive to pollutants such as ozone,
sulfur dioxide, carbon monoxide, and volatile organic
hydrocarbons. To improve risk assessments and thus
regulatory decision-making, research is needed to identify
sensitive populations, determine the reasons for their sen-
sitivity, and characterize their responses to various levels
of exposure. The Clean Air Act of 1970, as amended
(1977), for example, mandated that protection from air-
borne pollution be extended not only to normal, but also to
the most sensitive, individuals in the population. In addi-
tion, work with sensitive individuals may shed light on the
mechanisms causing air pollution effects.
One particularly sensitive population to certain air
pollutants is asthmatics. Asthma hospital admissions are
increasing in the United States, and little is known regard-
ing the causes"though some theories point to air pollution.
Asthma, which is a disease characterized by hyper-reac-
tive airways, can be life threatening. Epidemiologic
studies have shown that asthmatic subjects are at added
risk during periods of increased air pollution, though con-
trolled laboratory studies indicate that asthmatics are par-
ticularly responsive to some"but not alTpollutants.
Important questions remain regarding the response of
asthmatic individuals to air pollutants and the role of air
pollutants in the development or exacerbation of asthma.
Due to the limitations on conducting experiments
with human subjects, most data used to assess risk to
human health from exposure to various pollutants are
gathered from studies on laboratory animals. Currently,
however, much uncertainty surrounds the extrapolation of
data from animals to humans and from the high doses
commonly used in the laboratory to the low doses to
which humans are usually exposed. To determine which
animal models can most accurately predict human health
effects, scientists must compare the pollutants'
mechanisms of action in humans with those in various
laboratory animals. Such research contributes substantial-
ly to improvements in health risk assessment.
Epidemiologic and animal research has demonstrated
changes in host defenses associated with exposure to en-
vironmental pollutants: For certain individuals, exposure
appears to increase susceptibility to respiratory infections.
Additional epidemiologic research evaluating the associa-
tions between pollution episodes and illness (perhaps
using hospital records) is needed to assess these risks, hi
addition, clinical research across a broad range of host
defense mechanisms, including deposition and clearance
of inhaled materials, is needed for this task.
Understanding the relationship between acute respon-
ses and the development of chronic disease is also of criti-
cal importance in risk assessment. For example, the
immunotoxicological and inflammatory response that has
been observed in human lungs following exposure to en-
vironmental pollutants is a potential stage in the develop-
ment of chronic changes in the lung. Research is needed
to explore the connection between the acute responses ob-
served in clinical studies and later chronic effects.
2.6.2.3 Chemical-Specific Data
Toxics and Superfund. Many chemicals (e.g.,
PCBs, TCDD, phosphene, lead) known to be toxic or
found on Superfund sites raise public health concerns.
Studying the health effects associated with exposure to
each one of this large number of chemicals would be im-
possible. Therefore, research efforts must be directed to
understanding principles of action so that risk associated
with exposure to various compounds or toxic chemicals
can be extrapolated from available data on other chemicals
or compounds. Investigations with human tissues will in-
volve primarily immune host defense studies in vitro.
Acid Aerosols. Acid aerosols are another group of
chemicals for which additional health effects data are
needed. Cumulative evidence from animal, clinical, and
epidemiologic studies clearly suggests that health effects
can be associated with exposure to acid particles. Based
on this evidence, the Science Advisory Board's Clean Air
Science Advisory Committee recommended to the Ad-
ministrator in October 1988 that acid particles be con-
sidered for listing under Section 108 of the Clean Air Act.
Within 12 months of a listing decision, the Agency would
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have to issue air quality criteria and propose standards.
Because the database for acid aerosols is incomplete, how-
ever, research is needed to determine whether the listing is
justified and, if so, to provide a basis for developing
criteria.
UV-B. Environmental pollution is thought to be
depleting the ozone layer in the earth's stratosphere, thus
increasing human exposure to ultraviolet (UV) radiation
from sunlight. Available animal data suggest that such ex-
posure may have both local and systemic immunosuppres-
sive effects. Suppression of the immune system increases
the risk of disease, especially infectious disease, and may
also contribute to higher rates of cancer. Research is
needed to investigate whether the correlation between im-
munosuppressive effects that has been shown in
laboratory animal models is also valid for human health
effects.
Indoor Air. Relatively little is known about human
physiological responses to indoor air pollution, which ac-
counts for much lost work and productivity (the "sick
building" syndrome). Symptoms associated with the
syndrome suggest irritation of the upper airways, but more
research is needed to pinpoint mechanisms of response to
the pervasive and ubiquitous compounds found in indoor
air.
Exposure to radon is thought to be responsible for
most lung cancers occurring among nonsmokers, and it
probably also contributes to smoking-related lung cancers.
In some areas of the United States (e.g., Maine), drinking
water contains very high levels of naturally occurring
radon. Volatilization of radon during water use (e.g.,
showering) can be the major contributor to radon in indoor
air. Research is needed to determine whether this source
of radon results in significant exposure.
Ozone. Data from both humans and animals strongly
suggest that repeated and long-term exposure to ozone can
be associated with the development of chronic illness.
This connection poses a risk for individuals living in areas
where ozone level requirements have not been attained (an
estimated 100 million people in the continental U.S.
alone). Epidemiologic and clinical studies are needed to
assess this risk for people who live in both urban and rural
areas and who are exposed repeatedly and chronically to
ozone and associated pollutants.
Drinking Water. Chlorination of drinking water has
reduced the waterborne transmission of infectious disease.
Recently, however, a variety of organic contaminants,
both volatile and nonvolatile, have been identified as
resulting from the reaction of the chlorine with naturally
occurring humic substances. With in vitro tests, re-
searchers in other divisions of HERL are identifying the
various chlorination byproducts, assessing the com-
pounds' toxicity to animals, and examining the contam-
inants' mutagenicity. Epidemiologic studies are needed to
determine the cancer and cardiovascular risks associated
with water chlorination.
A number of epidemiologic studies have reported an
association between certain cancers and chlorinated water,
and analytical epidemiologic studies have shown a
moderate increase in risk of bladder and colon cancer in
populations with a relatively long duration of use of
chlorinated drinking water. Establishing any causal as-
sociations between water chlorination and these cancers,
however, must await the completion of additional
epidemiologic studies as well as other research.
2.62.4 Biological Markers
One important area for additional research is the
development of biomarkers of exposure and response.
Biomarkers of exposure are any biologic measurements
(e.g., in tumors or blood specimens) that can be used to
detect responses and quantify exposure to pollutants,
either systemically or in critical target tissues. Biomarkers
of effect are biologic responses that occur in conjunction
with or prior to the development of adverse health effects.
They are important tools in detecting and predicting ef-
fects. Clinical, in vitro, and epidemiologic research all
contribute to the development of these biomarkers.
2.62.5 Pollutant Mixtures
Most pollutants are found in the environment in mix-
tures of multiple pollutants rather than in pure form. Both
epidemiologic and clinical studies are needed to evaluate
how exposure to pollutant mixtures is different from ex-
posure to single chemicals. For example, HSD inves-
tigators need to examine whether pre-exposure to one
pollutant potentiates (or blunts) the response to exposure
to a second pollutant and whether combinations of pol-
lutants act synergistically.
2.6.3 Research Plan
HSD's research program is divided between two of
the division's branches: the Clinical Research Branch and
the Epidemiology Branch. Considerable effort, however,
has been devoted to integrating these research activities
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across branches. For example, new tests developed and
tested in the clinical facility may be applied in
epidemiologic studies conducted primarily in the field.
Similarly, clinical research lends itself to identifying
responsive or sensitive populations, who HSD epidem-
iologists can then use as target populations, and vice-
versa.
2.6.3.1 Hazard Identification
Dosimetry. Work in this area will be directed to bet-
ter understanding the relationship between exposure con-
centrations of air pollutants and the dose of those
pollutants or their metabolite(s) that reaches target organs.
The total deposition of gaseous pollutants at various points
in the human respiratory tract will be measured ex-
perimentally. In addition, HSD investigators will evaluate
the locations in the respiratory tract where the compounds
are deposited and the rates at which they are cleared.
The size of an inhaled particle influences its trajec-
tory, which in turn influences where it is deposited. Many
inhaled pollutants grow in size due to moisture acquisition
as they travel through the respiratory tract. Therefore,
techniques are being developed to measure particle growth
in the human respiratory tract to facilitate understanding
of patterns of particle deposition and to develop models of
the transport process.
Development of Sensitive Pulmonary Function
Tests. Tests of pulmonary function are commonly used to
measure responses to inhaled air pollutants in humans.
Tests believed to be sensitive to small airway dysfunction
are being adapted and evaluated for use in human environ-
mental research. Such tools should enable measurement
of subtle effects much earlier than classical pulmonary
function tests presently allow"an improvement that could
make clinical studies more sensitive in detecting these ef-
fects.
These tests, as well as other laboratory test methods,
are being adapted for use in the field. For example, re-
searchers are examining the validity of using heart rate as
an indicator of ventilation rate in subjects who exercise
outdoors. Pollutant dose rate (calculated using the ventila-
tion estimates from the heart rate and the measured am-
bient pollutant concentration) can be correlated with
measured changes in pulmonary function. HSD re-
searchers use portable monitors to measure ambient pol-
lutant levels and calculate pollutant dose rate"mea-
surements that can then be correlated to changes in pul-
monary function. This approach could provide a relatively
simple way to field validate some of the effects data that
are obtained from clinical studies.
With nasal and bronchoalveolar lavage techniques,
HSD investigators can sample tissues, cells, and fluids
from either the upper or lower airways in humans. The
samples are gathered either from naive (unexposed) sub-
jects for use in in vitro studies or from volunteers pre-
viously exposed to environmental pollutants in order to
examine the effects of inhaled pollutants on respiratory
cell biology and immunology. Though the bronchoal-
veolar lavage technique must be conducted by medically
qualified personnel and demands the highest operational
standards for application in this context, the resulting data
are crucial to an understanding of the mechanisms of
response and health effects at the cellular and molecular
levels. The nasal lavage procedure, on the other hand, is
relatively simple and noninvasive. Because of its
simplicity, the nasal lavage procedure may also have use-
ful applications in field studies. The application of novel
molecular techniques to these types of study will improve
our ability to detect changes in cells exposed to pollutants
and provide new endpoints that could be used as
biomarkers of exposure.
Other methods are currently being developed to
analyze whether and how pollutants affect human resis-
tance to infection. In one effort, HSD investigators are
developing techniques to grow human epithelial cells and
cell lines in culture. The objective of these studies is to
determine the susceptibility of these cells to viral infec-
tions, and their contribution (e.g., mediators and
cytokines) to the inflammatory response following ex-
posure to pollutants. Epithelial cells are the first target for
exposure in the upper respiratory tract, and their ability to
remain functional and anatomically intact is important in
maintaining normal lung homeostasis.
According to available animal data, some behavioral
responses can result from exposure to a number of pol-
lutants. HSD researchers are developing and adapting
new tests of neurobehavioral function and validating them
for use with volunteers. These tests, which are more sen-
sitive and specific than those currently available, will
allow behavioral responses to be measured directly in
humans. For example, research will be directed to deter-
mining whether changes in the visual evoked potential
(the measure of electrical brain activity in response to a
visual stimulus) can be used as a predictor of visual dis-
function. If evoked potential is a biomarker of effect, it
will be an important tool for future studies of the
neurobehavioral effects of individual pollutants.
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In addition, more objective measures for behavioral
responses are needed to study areas of potential effects,
such as the sick building syndrome, that are now identified
largely by anecdotal data. A computerized battery of
neurobehavioral tests is being validated and tested that
should permit analysis of larger groups of people. This
computerized test battery may have important applications
in field studies, where larger numbers of individuals from
exposed populations need to be tested.
Most of the tests described above have potential ap-
plication in both clinical exposure studies and in
epidemiologic (field) studies.
Drinking Water. HSD epidemiologists will continue
to support local public health departments and other
federal agencies in determining whether specific outbreaks
of infectious diseases resulted from faulty filtration or dis-
infection of drinking water. These outbreaks provide the
opportunity to refine the epidemiologic methods used for
answering this question.
2.6.3.2 Dose-Response Assessment
Dosimetry. Ongoing studies on the dosimetry of
gases and particles in humans will continue in order to in-
crease the accuracy of animal-to-human extrapolations of
data. These studies examine the dose to human target tis-
sues from exposure to air pollutants as well as the
mechanisms of delivery of these materials, including loca-
tion of distribution and quantity deposited. By closely
coordinating HSD's dosimetry efforts with those in ETD,
HERL researchers can compare human and animal data to
determine which animal models most closely mimic the
human deposition pattern for particular pollutants.
Mechanisms of Response. A major focus of human
research is the mechanisms of response to air pollution.
This research is coordinated with animal studies develop-
ing parallel models and projects. A concerted effort is
also being made to integrate epidemiology and clinical re-
search"particularly in the development of biomarkers and
in understanding associations between acute responses ob-
served under conditions of acute exposure (such as in ex-
perimental laboratory [clinical] or field studies) and the
chronic changes that might be produced following long-
term daily exposures (such as in many urban or rural areas
in the United States).
Both for scientific and regulatory purposes, the Agen-
cy needs to define the mechanisms of response to environ-
mental pollutants; and HSD is pursuing several important
lines of investigation in this direction. One effect of pol-
lutant exposure can be the development of an acute in-
flammatory response evidenced by an influx of
neutrophils and inflammatory mediators into the lungs.
Understanding the significance of this response may allow
researchers to answer the question: Do repeated bouts of
inflammation lead to chronic damage in the lungs, as
might be expected because of the presence of materials as-
sociated with both destructive and fibrotic processes; or is
the lung capable of resolving these acute incidents as they
occur?
Another important mechanistic question possibly as-
sociated with the inflammatory response is whether
repeated intermittent and/or daily exposures over a period
of many years leads to or contributes to chronic problems
in individuals or groups of individuals. A multiyear and
multidisciplinary study to investigate how the acute
responses measured in clinical and field studies relate to
the development of chronic respiratory disease and
decrease in pulmonary function is being evaluated. This
study would require 2-3 years of pilot research and then
sustained funding for 10-12 years. Defining an associa-
tion between the acute response to pollutants experienced
by some individuals (e.g., to ozone) and the ultimate
development of chronic pulmonary disease would have a
profound effect on the scientific and regulatory com-
munity.
Another important issue is the mechanism by which
the physiological response of many individuals to repeated
sequential exposures is attenuated. An understanding of
this phenomenon requires research efforts on the pul-
monary function (airways), inflammatory, and im-
munologic responses in humans.
Sensitive Individuals. HSD has studied a large num-
ber of subjects at 0.18 ppm ozone to better define the dis-
tribution of responsiveness to this pollutant in a normal
population. Some of these subjects, when re-exposed one
or two times, were found to have reproducible responses1^
finding that indicates that some individuals are consistent-
ly more responsive than others. Although this effort has
provided useful data for risk assessment, much additional
work is required to identify the determinants of this
variability in responsiveness.
In addition, numerous studies have provided an exten-
sive database concerning the response of asthmatics to sul-
fur dioxide exposure. In most of these studies, young,
generally unmedicated, mild asthmatics have undergone
short-term sulfur dioxide exposure at increased ventilatory
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rates (exercise or eucapnic hyperventilation) with a variety
of coexisting variables. Although asthmatics appear not to
be hyper-responsive to ozone, as measured by symptoms
or pulmonary function, their responses to other provoca-
tive agents (e.g., antigen) and bronchoconstrictors may be
intensified following ozone exposure; and these responses
should be investigated.
In addition, because epidemiologic evidence suggests
that air pollution may increase the incidence of asthmatic
attacks, longer-term exposures (i.e., extended exposures
on several consecutive days) will be analyzed to determine
if such exposures contribute to the development or exacer-
bation of asthmatic attacks. Such studies are also needed
to evaluate whether the incidence of asthma increases with
elevated ambient pollutant concentrations and increased
exposure levels.
HSD is also examining the responses to pollutant ex-
posures shown by people with compromised immunity,
such as people:
With genetic immunodeficiencies (e.g., severe com-
bined immunodeficiency [SCID])
Undergoing cancer chemotherapy
Receiving immunosuppressive drugs (e.g., cyclo-
sporin A) after transplants
Receiving corticosteroid treatment for various dis-
eases
With immune-suppressing infections (e.g., acquired
immune deficiency syndrome)
Studies with these patients could provide insights into
the questions of sensitivity and mechanisms of pollutant
action.
People with angina"pain induced by cardiac insuf-
ficiency, and the typical symptom of heart disease"are sen-
sitive to carbon monoxide, which affects the time of onset
and duration of angina. In the United States, approximate-
ly 50 percent of sudden cardiovascular deaths occur in
people who have had no previous indication of heart
trouble. Many of these people had silent ischemia, i.e., is-
chemic heart disease without angina. HSD will develop
techniques to identify and study individuals with silent is-
chemia to determine whether they also represent a par-
ticularly sensitive population to carbon monoxide. The
results of this work will contribute to an understanding of
the mechanism of effect of carbon monoxide.
Environmental Epidemiology. Epidemiologic re-
search is conducted primarily on a collaborative or
cooperative basis with investigators in different univer-
sities, agencies, public health departments, and foreign
governments. This provides HSD with the opportunity to
enlist investigators with specialized interests, experience,
skills, or opportunities to participate in research projects of
special interest to the Agency. In addition, because
epidemiologic research is very expensive, conducting
projects that are jointly sponsored often provides two (or
more) organizations the opportunity to conduct research
that would have been impossible to a single group.
HSD is co-sponsoring several projects evaluating in-
dividual and population responses to air pollution ex-
posures (e.g., the Harvard Six and Twenty-four City
Studies, children in summer camps, bladder cancer and
chlorinated drinking water) and is very influential in the
development of a major research plan to evaluate health
effects of chronic exposures to ozone.
International Studies. For several years, HSD in-
vestigators have been involved in cooperative studies of
lung cancer in relation to indoor coal burning in China.
They have also been cooperating with the Chinese EPA in
a pilot study of respiratory health (primarily the growth of
children's lung function) in several Chinese cities exhibit-
ing a wide gradient of exposure to particulates, sulfur
oxides, and acid aerosols. These studies offer the oppor-
tunity to determine human health effects across a very
wide range of exposures to both indoor and outdoor air
pollution. In addition, work has also begun with
Thailand's Office of the National Environment Board to
launch pilot studies of short-term and long-term human
health effects of pesticide exposure and automotive and
industrial air pollution exposure in that country. Other
studies evaluating the health effects of inhaled paniculate
matter, particularly acid aerosols, are now possible in
Eastern Europe, countries where poor industrial develop-
ment has resulted in high levels of exposure for the
general population. These efforts are expected to provide
a rare natural experiment in pesticide use as well as useful
information for risk assessment of air pollution.
2.6.3.3 Chemical-Specific Data
Acid Aerosols. To address the need for health effects
data on acid aerosols, HSD will conduct chamber studies
using controlled exposures to different concentrations of
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acid aerosols and measure health effects in the exposed
subjects. In addition to pulmonary function, researchers
will study the clearance of inhaled insoluble particles be-
cause both animal data and some human data indicate that
changes occur in the mucociliary clearance rate, depend-
ing on the exposure levels. The response of the small air-
ways to acid aerosols will also be tested. Other efforts
will investigate the combined effects of acid aerosols and
ozone. This clinical research will be complemented by
acid aerosol-related measurements, which will be taken in
several on-going epidemiologic studies.
UV-B. Because animal data point to UV-B-induced
immunosuppression, HSD is evaluating the response of
volunteers exposed to UV-B light under controlled condi-
tions. The research focuses on tests for cellular immunity,
both at the site of exposure and at a site that was not ex-
posed (systemic immunosuppression). Future work might
include efforts to determine whether the degree of skin
pigmentation influences the immunosuppressive effect,
and whether exposure to UV light diminishes the effec-
tiveness of vaccinations in protecting against infectious
disease.
Ozone. HSD is evaluating a major multiyear
epidemiologic project to reduce the uncertainty surround-
ing chronic respiratory effects of recurrent ambient ozone
exposure. The study would assess the relationship be-
tween ambient oxidant exposure and markers of chronic
pulmonary disease, and it might also assess the relation-
ship of ozone exposure to the incidence of respiratory in-
fection and asthma attacks. The results would be exceed-
ingly valuable in reconsidering the NAAQS for ozone.
Drinking Water. To examine the association be-
tween drinking water and heart disease, HSD is analyzing
the feasibility of conducting an epidemiologic study to in-
vestigate the effect of disinfectant parameters and water
hardness on serum cholesterol. Other research efforts in
drinking water involve comparing the relative importance
of different routes of exposure for specific pollutants. Re-
search on indoor air pollutants has shown that, for various
volatile organic chemicals such as chloroform and TCE,
inhalation is a more important route of exposure than in-
gestion. HSD is currently studying the extent of volatili-
zation of these chemicals from household use of water. In
addition, researchers are conducting a pilot study to deter-
mine if human breath analysis can be used to measure
radon exposure from the ingestion of drinking water. In
the area of disinfection, little is known about the potential
human health effects of alternative disinfectants (e.g.,
ozone, chlorine dioxide, and chloramines) and their by-
products. HSD is evaluating the potential for cancer and
reproductive effects.
Clusters of adverse reproductive effects have been
identified in contaminated water supplies, and animal
studies suggest that some compounds found in drinking
water may cause these effects at high doses. Epidemi-
ologic research will examine whether contaminated drink-
ing water has reproductive effects.
Alternative Fuels. A major legislative effort has
been mounted in the United States to switch from gasoline
to alternative fuels such as methanol. The State of
California, for example, has already mandated this switch
in commercial fleet vehicles. Methanol is a leading can-
didate as an alternative fuel because the technology for
methanol vehicles is well developed and methanol-fueled
vehicles are expected to emit lower levels of criteria pol-
lutants than gasoline or diesel vehicles. While the
decrease in pollutant levels will very likely benefit public
health, very little is known about the potential health ef-
fects of methanol, possible additives, and combustion by-
products, including formaldehyde. Nearly all the available
information on methanol toxicity in humans is related to
acute, uncontrolled, high-level exposures, largely by in-
gestion. Data on human exposure to low levels of
methanol vapors are very limited. Clinical and
epidemiologic research is needed to determine whether
any adverse human health effects are associated with
methanol fuel use.
2.6.3.4 Biological Markers
Asbestos and silica are known to cause disease in
humans. The function of cells (primarily alveolar macro-
phages) that have been exposed to these paniculate sub-
stances in vitro is being compared with the function of
cells from people who have been environmentally or oc-
cupationally exposed to these pollutants. This research is
aimed at developing biomarkers to point to the occurrence
of potentially harmful exposures before disease develops.
Such a tool could contribute to the prevention of environ-
mental diseases.
Other studies will develop and validate both effects
and exposure biomarkers. For example, of the almost
1,000 different proteins found in alveolar macrophages
obtained by bronchoalveolar lavage, over 100 are consis-
tently and predictably changed following exposure to cer-
tain pollutants. This "fingerprint" of response seems to be
unique to individual pollutants in exposed individuals.
Such protein fingerprints could potentially be used as
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biomarkers to indicate that a particular exposure has taken
place and also as a tool to find individuals who might be
particularly sensitive to a given pollutant.
HSD is also developing tools for detecting potential
molecular biomarkers of effect. Researchers are continu-
ing to adapt a molecular biological technique for gene
amplification (the polymerize chain reaction technique) to
look at messenger RNA in cells of subjects exposed to
particular pollutants. This approach, if successful, should
enable measurement of the effects of past pollutant ex-
posures on processes and analysis of factors such as gene
regulation, growth factors, coagulation factors, im-
munological and inflammatory mediators, and cytokines.
Cotinine is a metabolic by-product of nicotine meta-
bolism. HSD is developing a kinetic model to facilitate
the use of cotinine in urine as a biomarker for measuring
children's exposure to secondhand cigarette smoke. The
research effort is obtaining data on how rapidly cotinine in
urine is cleared in children, so that models for relating
cotinine measurements back to levels of cigarette smoke
exposure can be developed.
HSD epidemiologists are developing methods for
detecting genetic damage in human specimens analyzed in
field studies. They are applying assays for detecting
modifications at each of the three levels of organization of
genetic materiaTchromosomal, gene- or locus- specific,
and chemical (DNA nucleotide). Initially, this work will
examine individuals who have relatively high levels of ex-
posure. At a later point, the assays' sensitivity for detect-
ing genetic damage in people with lower, environmental
exposure levels will be determined.
Researchers are using molecular assays in a protocol
involving occupational exposures. In this study, serial
blood samples will be taken from individuals who have
experienced relatively high occupational exposures, and
their genetic material will be examined through time to
determine whether the exposures resulted in biological
changes. Positive results would suggest similar studies
should be conducted in populations with lower, more en-
vironmentally relevant exposures, to determine whether
these biomarkers of exposure are also valid at these lower
concentrations.
In addition, HSD is measuring the chemical modifica-
tion of DNA (DNA adducts) in human tissues, such as
blood samples, bronchoalveolar lavage, and autopsy tis-
sues, to determine if a relationship exists between the
DNA adducts in these tissues and cancer in different or-
gans. Initially, smokers will be selected as the study
population because of their relatively high exposure to
certain pollutants. These studies will help determine the
potential of DNA adducts to serve as markers of biologi-
cally significant exposures and effects. In a related study,
investigations will determine the relationship between
modifications in the DNA adducts and other endpoints for
genetic damage, e.g., mutation or chromosomal changes.
This work is initially being undertaken in subjects with
precisely determined exposures to chemotherapeutic
drugs.
Growing concern is focusing on the potential
reproductive effects of environmental chemicals. Fre-
quently, members of communities that have been exposed
to some form of pollution claim that this exposure has
caused an elevated spontaneous abortion rate. Reproduc-
tive epidemiology is a relatively new area of research that
can help determine whether pollutant exposures are as-
sociated with effects such as infertility, pregnancy loss
(spontaneous abortions), congenital defects, prematurity,
low birth weight, and altered sex ratio.
Many generic issues need to be addressed as
reproductive epidemiology matures. For example,
measuring the rate of spontaneous abortions with any ac-
curacy is currently difficult. Much research in this area
has focused on developing more refined ways of measur-
ing fetal loss, particularly very early fetal loss that occurs
before clinical recognition of pregnancy. The develop-
ment of sophisticated techniques for measuring urine
human chorionic gonadotropin"a biomarker for pregnan-
cy"has aided in better defining the baseline rates of spon-
taneous abortions. Similar research is needed for other
reproductive endpoints in order to increase the accuracy
and uniformity of these studies.
2.6.3.5 Pollutant Mixtures
Much of the clinical research to date has been related
to the effects and mechanisms of response to individual
NAAQS pollutants, even though most real-world ex-
posures are to mixtures of pollutants. While some re-
search has been done on pollutant combinations"
including sulfur dioxide, nitrogen dioxide, ozone, and
several acid aerosols"little is known about the human
response to these mixtures. Research is needed to assess
whether pollutant combinations cause more severe effects
than individual pollutants. One prime candidate for study
is ozone in combination with other pollutants: Animal
data clearly show that ozone synergizes with other pol-
lutants. A finding of synergism could be very useful in
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determining the air quality standards for the chemicals in-
volved.
Epidemiologic studies of the health effects of pol-
lutant mixtures in ambient air are also needed to provide
perspective on the significance and health effects of am-
bient air pollutants. Several industrial areas in the United
States have high levels of chemical complexes, involving
hundreds of different pollutants. To support this type of
research, refinements in epidemiologic methodology, test
methods, and biomarkers are needed to determine the im-
portant exposures in chemical mixtures and how to
measure them.
2.6.4 Emerging Issues
2.6.4.1 Indoor Air
Historically, the focus of much environmental health
research has been effects of pollutants found outdoors. In
recent years, scientists have increasingly realized that the
quality of indoor air plays a major role in human health.
Many people spend far more time indoors than outdoors,
and pollutants can accumulate to relatively high levels in
indoor environments, especially those without adequate
ventilation. An ever-increasing number of indoor air pol-
lutants are being identified:
Combustion products from stoves and fireplaces
Emissions from carpets, furniture, and building
materials
Microbial organisms that grow in air conditioning
systems
Environmental tobacco smoke
Radon
Organics in drinking water that volatilize during
household use
Little is known about the health effects of exposure to
many of these pollutants. Clinical and epidemiologic
studies are needed to determine whether indoor air pol-
lutants pose a significant health risk.
2.6.4.2 Immunotoxicology
Novel and exciting challenges are developing in the
field of immunotoxicology as safety assessment issues
emerge regarding the use of species-specific recombinant
biologicals, biological and biochemical pesticides (e.g.,
recombinant microbes), and monoclonal antibody reagents
designed as drug delivery or detoxification vehicles. Ex-
amining these agents requires the thoughtful application of
flexible lexicological protocols designed to reveal whether
they are immunotoxic, immunopharmacologically active,
or immunogenic. The design of such immunotoxicologic
studies will be based on an understanding of the potential
targets within the immune system and the interactions of
chemicals with immunocompetent cells. This approach
will rely on new methods that will be developed to ex-
amine functional impairment or toxicity in a variety of tar-
get organ systems, and on cell and molecular biological
techniques that will be more rigorously applied for defin-
ing the underlying mechanisms of toxicity. Research is
needed to determine whether exposure is occurring and
whether any health effects can be measured. This is an
area of research of particular relevance to ETD and HSD
because inhalation is likely to be the primary route of ex-
posure, with pulmonary immunologic effects as the
primary health result.
2.6.5 Summary
2.6.5.1 Hazard Identification
HSD investigators are addressing several tasks that
primarily fall under the hazard identification research
topic but that also support ongoing dose-response assess-
ment research. For example, HSD epidemiologists
routinely and continuously evaluate approaches and
develop techniques to estimate exposure and dose to in-
dividuals and populations for pollutants of interest In ad-
dition, they play a unique role in exposure assessment by
providing design and statistical support to the models used
to determine human exposure.
HSD researchers are also developing and evaluating
computerized models for use in study design; with these
models, they can evaluate the power and specificity of ex-
perimental approaches in hypothesis testing.
The development of biomarkers to determine ex-
posure and effects will be facilitated by an integrated ap-
proach that incorporates both human and collaborative
animal research. This knowledge will be applied in
epidemiologic and field studies, which will be used to
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evaluate the health impact of exposure to potential en-
vironmental contaminants in both water and air. HSD will
use in vitro techniques to study toxic materials with
human tissues and cells to identify potential hazards and
to validate animal models. A multidisciplinary approach
that addresses endpoints in immunotoxicology, cell biol-
ogy, and molecular biology will aid in developing and
validating both in vivo and in vitro methods for hazard
identification. Techniques for evaluating the public health
impacts of drinking water disinfection is one of the press-
ing research issues facing HSD.
identification of sensitive populations, the magnitude of
the risk to compromised individuals, determination of the
cumulative dose to target organs and sites under differing
conditions and durations of exposure, interactions of pol-
lutant combinations, development and validation of new
and sensitive tests of response, the mechanism of pollutant
action in individuals, and the association between an acute
response to pollutant exposure and development of a
chronic condition.
Clinical investigators are developing new techniques
to measure dosimetry in humans following inhalation, der-
mal exposure, or ingestion of a variety of materials, in-
cluding volatile organic compounds and oxidant gases.
HSD is also developing new techniques to measure the
growth of inhaled hygroscopic particles in humans. To
extend the research to include more sensitive measure-
ments of human health effects, new tests of small airways
function are being developed, validated, and incorporated
into ongoing studies in which they complement more clas-
sical measurements of pulmonary function.
The need to study the human effects of exposure to
toxic compounds is pressing, yet exposing volunteers to
these substances would be inappropriate. Thus, in vitro
techniques are being developed that use human tissues and
cells. As these approaches mature, resources will be
shifted to support their development.
2.6.5.2 Dose-Response Assessment
A major research initiative has been proposed to
develop a series of epidemiologic and clinical studies for
testing the hypothesis that repeated and continued ex-
posure to ozone, such as occurs in many U.S. urban areas,
causes chronic respiratory effects. Because responsive in-
dividuals can presently only be identified in clinical ex-
posure studies and long-term exposure to ozone has been
shown to produce chronic changes in the lungs of animals,
HSD epidemiologists must carefully refer to and integrate
clinical and animal studies. As new resources are com-
mitted, these investigations will take on increasing
prominence in the HSD research program.
HSD has a strong program in dose-response assess-
ment, particularly for NAAQS pollutants. Epidemiologic
and clinical research has been used to establish and/or sup-
port the NAAQS for ozone, lead, paniculate matter, sulfur
dioxide, and carbon monoxide. Many important issues
remain to be studied for NAAQS pollutants, including
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SECTION THREE
CURRENT AND FUTURE DIRECTIONS IN
HEALTH RESEARCH
BY REGULATORY PROGRAM
In this section, current and future research efforts
under the EPA health program are categorized first by pro-
gram office and program, and then by research topic (the x
and y axes in Figure 1-2). Sections 3.1-3.6 outline needs
and research strategies under the Office of Air and Radia-
tion, Office of Water (Drinking Water), Office of Water
(Water Quality), Office of Pesticides and Toxic Substan-
ces, Office of Emergency and Remedial Response, and
Office of Solid Waste. Program data needs cutting across
hazardous identification and dose-response assessment
(e.g., biomarkers) are listed
under exposure or dose-
response assessment. This dis-
cussion presents the regulatory
point of view on the Agency's
current and future health re-
search needs.
This discussion of research needs of the Office of Air
and Radiation (OAR) first describes the programs sup-
ported by the office, outlines each program's issues and
research needs relative to the research topics listed in Sec-
tion One (i.e., hazard identification, dose-response assess-
ment, and exposure), and highlights the health research
plans that respond to these program needs. The
OHR/HERL research program in support of the Office of
Air and Radiation is designed to fill those needs (see
Table 3-1).
Table 3-1:
NAAQS, AIR TOXICS, MOBILE SOURCES, INDOOR AIR
Issue
Priority Needs
Research
Hazard ID
med
3.1 OFFICE OF
AIR AND
RADIATION
The Clean Air Act (CAA)
gives EPA authority to set na-
tional standards for ambient air
quality and to regulate sources
of pollution. The Act explicitly
states that the basis for regula-
tion is protection of public
health and welfare. In addition
to setting standards, the Agen-
cy has sought to protect public
health from the adverse effects
of air pollution by other
methods, such as public educa-
tion, support of state and local
regulatory programs, and the
banning of products that pol-
lute the air. The foundation of
these activities is also the
protection of public health or
welfare risks.
Dose-Response hi
Exposure
hi
Identification of
chemicals and endpoints
Estimation of human
population risks
Identification of lowest
concentration of concern
Identify emission sources
Identify problem chemicals
Demonstration of cause/
Effect of emissions
Methods development
SAR
Clinical methods
Short-term noncancer
endpoints esp. neurotox,
repro/devel, immunotox,
pulmonary, genetox/cancer
In vitro to in vivo extra-
polation methods
Dosimetry/pharmacokinetics
Extrapolation
Species-to-species
Route-to-route
High-to-low-dose
Sensitive subpopulations
Mixtures
Extrapolation across
various exposure scenarios
Biologic characterization of
atmospheric transformation
Biologic characterization
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3.1.1 Office Programs
OAR supports six statutory and programmatic ac-
tivities with specific health research needs that are ad-
dressed by the OHR/HERL research program:
Ambient Air Quality
Air Toxics
Mobile Sources
Indoor Air
Global Atmospheric
Radiation
3.1.1.1 Ambient Air Quality Program
EPA has established National Ambient Air Quality
Standards (NAAQS) for six pollutants or classes of pol-
lutants identified in the Clean Air Act: carbon monoxide,
fine paniculate matter, lead, nitrogen dioxide, ozone, and
sulfur dioxides. These pollutants were singled out be-
cause they are found ubiquitously and present a substantial
danger to public health and welfare. Significant progress
has been made in controlling emissions of these pol-
lutants. Great increases, however, in the number of
vehicles in recent years have overwhelmed these improve-
ments in many areas. As a consequence, these pollutants
remain a major public health issue. State governments,
with federal assistance, develop and enforce plans to meet
these standards. EPA also establishes standards to restrict
emission from new facilities or from facilities undergoing
major modifications (New Source Performance Stand-
ards). The Agency is required to periodically review the
NAAQS standards and to work with state and local air
pollution control agencies to achieve progress in comply-
ing with these standards.
3.1.1.2 Air Toxics Program
This program has responsibility for all pollutants,
other than the six covered by NAAQS, that are potentially
damaging to public health or welfare, OAR's strategy for
controlling air toxics is three-pronged:
Use the authority provided by federal regulation of
emissions from stationary sources. These National
Emission Standards for Hazardous Air Pollutant
(NESHAPs) control emissions of pollutants that cause
"serious irreversible or incapacitating reversible ill-
ness." Under this program, regulations have been es-
tablished for seven pollutants"asbestos, beryllium,
mercury, benzene, vinyl chloride, radionuclides, ar-
senic"and proposed for coke ovens. The Agency has
also announced an intent to list nine other chemicals
as hazardous air pollutants. Pollutants can also be
regulated using New Source Performance Standards
(NSPS), which are used to restrict emissions of both
NAAQS and specially "designated" pollutants from
new industrial facilities or existing facilities undergo-
ing major modifications or reconstruction.
Under proposed amendments to the CAA, 191 poten-
tially regulated pollutants are identified. Plants emit-
ting greater than 10 tons of any individual pollutant or
25 tons of a mixture of pollutants are subject to Maxi-
mum Achievable Control Technology unless the plant
can demonstrate it poses a negligible public health
risk. It will be up to state agencies to decide whether
the risk is negligible, but EPA must provide guidance
on how to make this determination. To do this, EPA
will identify concentrations or "health benchmarks"
that present a negligible health risk for both cancer
and for non-cancer endpoints. The Agency must also
assess sources of the 191 pollutants for excessive
post-control or "residual" risks. Assessments of
health hazards will be needed to add or delete chemi-
cals from the list of 191 pollutants.
Assess toxic urban air pollution. Although urban air
is known to contain toxic substances, it is difficult to
determine which pollutants are hazardous, the effect
of exposure to mixtures and atmospherically trans-
formed chemicals on public health, the sources of
these emissions, and which chemicals and sources
should be regulated. The Office of Air Quality Plan-
ning and Standards (OAQPS) has efforts directed at
characterizing this problem and determining future
regulatory efforts.
Provide state/local assistance and high-risk sources
programs. EPA also provides technical assistance to
state and local governments. The Agency provides
technical support to state and local agencies and
EPA's regional offices in implementing air pollution
control programs and assists in developing control
strategies for toxic emissions, implementing state
plans, developing regulations, evaluating operation
3-2
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and maintenance problems, reviewing new sources to
determine appropriate control technology, and
evaluating source emission and control strategies. To
facilitate these efforts, the Agency has established a
technology transfer center. The Control Technology
Center (CTC) is a joint effort of the Air and Energy
Engineering Research Laboratory in Research Tri-
angle Park, North Carolina; the Center for Environ-
mental Research Information (CERI) in Cincinnati,
Ohio; and OAQPS in Research Triangle Park, North
Carolina.
An additional technology transfer center is EPA's Air
Risk Information Support Center (Air RISC). This
joint effort of the Office of Health and Environmental
Assessment (OHEA), CERI, and OAQPS assists state
and local air pollution control agencies and EPA
regional offices with technical matters concerning
health, exposure, and risk assessments for toxic air
pollutants. Air RISC's primary goal is to serve as a
focal point for obtaining information and to provide
assistance in the review and interpretation of that in-
formation. It provides health and risk assessment in-
formation for chemicals being evaluated in the permit
review process, assists with on-site risk assessments,
and provides guidance on current methods available
to conduct health risk analyses.
3.1.1.3 Mobile Sources Program
The CAA requires the Agency to ensure that mobile
source emissions from new vehicles, engines, fuels, and
fuel additives do not adversely impact health. The Act
mandates control of hydrocarbons, carbon monoxide, and
nitrogen oxides for light-duty vehicles such as
automobiles. In addition, the Agency has developed or is
developing emission standards for other types of vehicles.
EPA enforces its mobile source standards by an extensive
vehicle testing and certification program. The Agency has
also promoted the development of state programs for
prevention of vehicle tampering and fuel switching. High
ozone and carbon monoxide areas are required to have
vehicle inspection and maintenance programs. In addi-
tion, the CAA authorizes the Agency to regulate motor
vehicle fuels and fuel additives in order to protect air
quality. The Energy Policy and Conservation Act (1974)
and the Alternative Fuels Act (1988) were passed to move
the country toward alternative fuel use in order to decrease
dependence on foreign oil and facilitate attainment of
NAAQS standards in some nonattainment areas of the
country. This shift towards the use of alternative fuels has
significant implications for health research conducted for
the Mobile Sources Program. In addition to the research
conducted at HERL, a significant part of the health effects
research on mobile source emissions is conducted through
the Congressionally mandated Health Effects Institute
(HEI). HEI is jointly funded by EPA and the automobile
industry and performs research on the health effects of
pollutants related to mobile sources.
3.1.1.4 Indoor Air Program
For a number of years, EPA has been addressing the
complex issues associated with indoor air pollution via a
program of public education and information dissemina-
tion. In 1987 Congress passed, as part of a Superfund bill,
the Radon Gas and Indoor Air Quality Research Act This
legislation established an indoor air quality research pro-
gram; directed EPA to coordinate with federal, state, local,
and private efforts to improve indoor air quality; and en-
couraged continued work in information dissemination.
The program focuses on the identification, charac-
terization, and monitoring of the sources of indoor air pol-
lutants, human health effects, mitigation measures, and
dissemination of information to ensure the public
availability of these findings. The Agency has no
regulatory authority and, therefore, cannot set enforceable
standards for indoor air quality. EPA advisory informa-
tion, however, may recommend safe levels for specific
contaminants. A variety of indoor air pollutants such as
tobacco smoke, fumes from combustion appliances,
biological contaminants (e.g., molds, mildew, fungi), and
volatile organic compounds from building materials are
known to have significant adverse health effects and are
encompassed in EPA's efforts.
3.1.1.5 Global Atmospheric Program
EPA is directing research into the potential for air
pollution to create climatic, ecological, and health
problems of global significance. Complex chemical inter-
actions in the earth's atmosphere are now suspected of in-
creasing average temperatures (global warming), changing
climatic patterns, depleting stratospheric ozone, and con-
tributing to acid rain. To correct or reverse these trends,
EPA is studying the policy options that could stabilize the
amount of gases in the atmosphere and control future
warming. EPA is also studying the health and environ-
mental effects of potential warming trends. HERL is cur-
rently researching the association between the depletion of
stratospheric ozone and immune system effects.
Under a Congressional mandate embodied in the
Global Climate Protection Act of 1987, EPA is helping to
coordinate a national policy on global wanning. EPA has
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also directed large-scale research into the atmospheric
chemistry and long-range atmospheric transport problems
of acid rain formation, as well as into methods for restor-
ing lake habitats. EPA also assisted in the development of
the Montreal Protocol, an international agreement to
reduce consumption of chlorofluorocarbons, halons, and
other chemicals that reduce atmospheric ozone. EPA's
position was based partly on the results of a 1987 risk as-
sessment of the projected health and environmental effects
of reductions in atmospheric ozone. The Montreal
Protocol was signed in September 1987 and went into ef-
fect January 1, 1989. EPA promulgated regulations (pub-
lished in the Federal Register, August 12, 1988) to
implement the phaseout of chlorofluorocarbons, halons,
and other chemicals in accordance with the Protocol.
3.1.1.6 Radiation Program
Although radioactive materials fall within the term
"air pollutants" in the CAA, the courts found in favor of
companies that challenged Minnesota when it used the
CAA in an attempt to regulate radioactive materials more
stringently than the Atomic Energy Commission. To
remedy this situation, an amendment was added to the
CAA in 1977, Section 122, that mandated that the EPA
Administrator had to review all "relevant information and
determine whether or not emissions of radioactive pol-
lutants (including source material, special nuclear
material, and byproduct material), cadmium, arsenic, and
polycyclic organic matter into the ambient air, ... may
reasonably be anticipated to endanger public health." If
so, the Administrator must regulate the pollutant under
Sections 108, 111, 112, or any combination of these sec-
tions.
EPA currently implements a number of programs to
protect the public from the health hazards of radiation con-
tamination. While the Department of Energy (DOE) and
the Nuclear Regulatory Commission (NRC) have jurisdic-
tion over many facilities that handle radioactive materials,
EPA regulates the exposure of the general public to radia-
tion. Virtually all of these regulatory, guidance, and
analytical programs are based to some degree on the
health effects of radiation exposure.
3.1.2 Office-Specific Research Needs
3.1.2.1 Ambient Air Quality Program
Two factors shape the risk assessment and research
needs for the NAAQS program: 1) the limited number of
identified pollutants that are regulated by this program;
and 2) the widespread exposure to these pollutants, along
with the tremendous public health, societal, and economic
implications of that exposure. In light of these factors, re-
search needs, by risk assessment step, are as follows.
Hazard Identification. Relative to other programs in
the Agency, little work is required to identify new pol-
lutants. Needs in the hazard identification area include
confirming associations between specific health effects
and identified pollutants, and developing evidence that
these health effects are likely to occur in humans. Re-
search efforts developing methods and protocols to ad-
dress these concerns are ongoing. The focus of this
research is generally on very specific questions, such as:
What are the pulmonary and immune system effects
of chronic low-level ozone and/or acid aerosol ex-
posures?
What are the public health implications of subtle pol-
lutant-related effects observed in animals, such as in-
creased lung membrane permeability ("leaky lung")
or increased collagen build-up?
Dose-Response Assessment. As noted above, the
pollutants addressed by the NAAQS are relatively unusual
in terms of extent of exposure and associated public health
risks. As a consequence, risk assessment questions must
be answered much more precisely and conclusively than is
common for other environmental problems. A central
question for NAAQS pollutants is, what percentage of the
population, including sensitive subpopulations, is likely to
respond at various exposure concentrations? Research ef-
forts to address this question must emphasize human data,
animal-to-human extrapolations, and an understanding of
mechanisms of action to facilitate interpretation of subtle
effects occurring at very low concentrations.
Exposure Assessment. The exposure issues of con-
cern are related to the hazard identification and dose-
response questions noted above; for example:
Can more sensitive biologic indicators of human ex-
posure and resultant effects be developed?
What are the chronic effects of repeated acute ex-
posures?
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3.1.2.2 Air Toxics Program
Two issues significantly shape risk assessment and
research needs for this program: the large number of pol-
lutants and sources, and the lack of a substantial database
for most pollutants. As a consequence, efforts are focused
on improving rapid screening capabilities, as well as
developing approaches and assumptions that are valid for
many pollutants and mixtures. Although complex issues
such as impacts on sensitive human subpopulations are of
concern to the Air Toxics Program, research in these areas
must generally await resolution of more basic questions.
Research needs in this area, by risk assessment step,
are as follows.
Hazard Identification. Regulatory responsibility for
many pollutants makes hazard identification one of the
most difficult charges of this program. Two of the most
pressing questions are:
What are the chemicals of greatest concern for public
health?
Are the health endpoints associated with these chemi-
cals likely to occur in exposed people?
Dose-Response Assessment. The Air Toxics Pro-
gram has historically required two types of dose-response
assessments: 1) relatively simplistic assessments for
preliminary federal regulatory assessments, support of
state and local programs, and assessment of urban toxics;
and 2) detailed assessments, generally concerning
widespread exposures, used in the development of nation-
al emission standards (e.g., for benzene cancer risks). The
relatively simplistic assessments depend heavily on
generic risk assessment procedures and extrapolation as-
sumptions that use pre-existing data. The detailed assess-
ment requires more sophisticated analysis and detailed
data, similar to that required for the NAAQS pollutants;
new data and methodologies must often be generated to
support the assessment Use of these two levels of assess-
ment will likely continue in making negligible risk deter-
minations and residual risk determinations.
Important questions for both simplistic and detailed
assessments include:
How can risks be assessed for large numbers of pol-
lutants based on relatively little data?
How should risks from multipollutant sources be as-
sessed?
To answer these questions, research into rapid screen-
ing and evaluation techniques and improved route-to-
route, species-to-species, and low-dose extrapolation
methods"as well as into the underlying issues of target
dose and mechanisms of action"must be performed.
Equally critical is discerning adverse critical effects for
each target system, the interactions between toxic events
at different organs, and the progression of toxicity, par-
ticularly for toxics where multiple noncancer events are
possible. HERL's role is to develop data to support these
generic approaches and to develop effective assessment
tools, e.g., quantitative methods for noncancer health ef-
fects.
Exposure Assessment. The need to characterize
emissions from a variety of sources for many chemicals
results in several questions for research efforts:
What are the most significant sources of pollution?
Can resulting exposures be demonstrated to be causal-
ly related to adverse health effects?
Are nontoxic chemicals atmospherically transformed
into toxic products?
3.1.2.3 Mobile Sources Program
Historically, the statutory and programmatic issues
that shape mobile source-related research were very
similar to NAAQS: a limited number of classes of pol-
lutants of concern and enormous potential public health
impacts. However, the needs of this program have
broadened over time. Research is now needed to develop
methods and data to identify pollutants of concern and as-
sess the health effects of currently unregulated pollutants
and complex mixtures associated with mobile sources.
Needs center on identifying the health effects of certain
pollutants or mixtures"for example, diesel exhausf'and on
gathering detailed information on dose-response relation-
ships, especially on extrapolation to humans, including
sensitive human subpopulations. The Agency is com-
mitted to analyzing the relative risks of alternative fuels to
enable development of a national alternative fuels
strategy. It is anticipated that the research requirements
for such a comparative assessment, which are discussed in
the Agency's Alternative Fuels Research Strategy, will
receive considerable attention.
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3.1.2.4 Indoor Air Program
3.1.2.6 Radiation Program
The risk assessment questions for indoor air are
similar to those for air toxics, probably due to the
programs' similar responsibilities for many pollutants and
a variety of sources. In some areas, however, the Indoor
Air Program has some special interests:
What are the sensitive indicators of toxicity in readily
identifiable subpopulations"for example, the
"ill" "who are likely to spend all of their time indoors
and are thus at greater risk from indoor pollutants?
What pollutants are harmful and at what concentra-
tions?
Can chemically sensitive or hypersensitive popula-
tions be characterized relative to indoor air pollution
problems?
3.1.2.5 Global Atmospheric Program
Global atmospheric issues are still emerging. The
most pressing health issue identified so far is associated
with ultraviolet (UV) radiation. Due to the potential for
tremendous public health impacts, research on the health
effects resulting from increased UV-B exposure and as-
sociated dose-response relationships has been given a high
priority by HERL. UV appears to have complex effects
on the immune system; at least in mice, these effects ap-
pear to induce the generation of T-lymphocytes that sup-
press immune responses in a long-lasting and specific
manner. Whether increasing UV radiation at the earth's
surface will cause immune suppression, thus leading to in-
creased population susceptibility to infectious agents,
remains a distinct possibility. No studies in humans has
yet addressed whether and how UV exposure alters human
immune response. Questions of concern to the program
office include:
Does UV light exposure in humans result in systemic
immunotoxic effects?
If so, are the effects long-lasting and specific?
Can the effects be demonstrated using in vitro assays?
What are the mechanisms of the effects?
Research is also needed to elucidate the mechanisms
and dose-response relationships associated with exposure
to radiation. An area of emerging interest is electromag-
netic radiation (EMR). Effects have been reported on the
brain, the reproductive system, and the immune system.
EPA is currently developing a research strategy in this
area.
3.1.3 Research Plan
Proposed air research will address the questions of
concern to the program offices. The research areas iden-
tified here are critical to support risk assessment across
programs. Key research areas common to each program's
needs include clinical and animal inhalation toxicology
and epidemiology. Work under the research topic of
hazard identification will focus on method developments
that will enable identification of agents and endpoints of
concern. Efforts will be included to determine the
relevance of these endpoints to the human population.
Dose-response work will focus on dosimetry, phar-
macokinetics, and mechanisms of action as key areas in
understanding target dose, and a variety of extrapolation
questions. The aim of this research is the improvement of
the ability to estimate human population responses to ex-
posure, based on animal and human data for a variety of
exposure scenarios (e.g., intermittent vs. chronic ex-
posures). Exposure research will use biologic endpoints
to characterize exposure and to link exposure to effects.
This work will result in more effective identification of
sources and agents of concern as well as strengthen cause-
and-effect relationships between emissions and human
health effects. Taken together, these research efforts will
ensure the credible and effective use of risk assessment in
Agency decisions.
3.1.3.1 Ambient Air Quality Program
Hazard Identification. Research will focus on the
development of more sensitive and specific bioassays to
identify more subtle indices of damage for known effects
and facilitating the interpretation of the significance of
these effects for public health. Understanding of
mechanisms of action of toxic agents and of disease
processes is an important part of this effort. Two areas of
this research effort are particularly noteworthy:
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Understanding the impacts of chemical exposure
on the immune system. Available data suggest that
substantial impacts on the immune response may
occur as a result of low-level pollutant exposure and
that immunotoxic effects are one of the most sensitive
indicators of toxicity. Currently, the nature of the im-
mune response is characterized using responses in
lung and host-resistance models. A major goal of this
research is to correlate the effects of chemical ex-
posure on immune function test with effects that are
seen in the human population such as susceptibility to
infections, allergic reactions, and autoimmune dis-
ease.
Identifying subtle indices of pulmonary damage
that are believed to be the early hallmarks of
serious respiratory disease. For example, several
lines of animal and epidemiologic evidence indicate
that many airborne pollutants can damage small air-
ways. This damage cannot be detected clinically.
Sensitive clinical techniques need to be developed to
allow acquisition of human data in this critical area.
As noted above, work in this area is designed to
develop sensitive test methods that will allow com-
parisons between human and animal as well as be-
tween in vivo and in vitro data.
Dose-Response Assessment. Research under this
topic will allow risk assessors to accurately estimate
population responses to pollutant exposures. Efforts will
fall in three important areas: understanding the mechanism
of response and its quantitative relationship to dose, im-
proving extrapolation from animal to human response, and
identifying sensitive subpopulations. A key aspect of this
research is joint efforts in human and animal toxicology"a
research design that is uniquely available at HERL.
Animal models of cardiopulmonary and immune system
diseases are being developed in animals and validated
with both in vivo and in vitro clinical data and
epidemiologic data from humans. Refinement of
dosimetric and pharmacokinetic dose-response models is
also essential to this research effort. These models enable
the accurate estimates of critical dose to target organs that
is necessary for risk assessment and extrapolation across
dose, species, and route of exposure. Also, dosimetrics
and pharmacokinetics, coupled with differences in tissue
sensitivity, are the factors that are most likely responsible
for differences in population sensitivity. Both human
(clinical and epidemiologic) and animal dose-response
studies, as well as mathematical modeling, will be given
special attention to determine the deposition, clearance,
and pulmonary function effects of particles, alone and in
combination with ozone, NCh, and SCh.
Exposure Assessment. Sensitive biological
measures of pollutant damage identified as part of hazard
identification and dose-response work will be used in ad-
dressing exposure-related issues. Animal and clinical
studies examining the effects of repeated acute exposures
will be coupled to epidemiologic and/or mechanistic work
that will address chronic implications for human health.
The use of biomarkers is expected to greatly improve both
clinical and epidemiologic estimates of exposure and ef-
fects. These biomarkers, in conjunction with animal ex-
periments, are expected to play a critical role in answering
questions about the effects of ozone, nitrogen dioxide, and
acid aerosol mixtures.
3.1.3.2 Air Toxics Program
Hazard Identification. As an aid in identifying
numerous chemicals and endpoints of concern, HERL is
developing methods for a tiered approach to screening and
characterizing pollutants. Current tests are often expen-
sive and time-consuming and do not identify all important
effects. Work in this area will be directed at developing
more rapid and accurate assessments.
The initial toxicity screening could be performed
based on structure activity and in vitro test data. Sub-
sequent work will validate health concerns identified in
the initial screening assessments as well as their relevance
to humans. Researchers will focus on expanding techni-
ques that have been developed to assess cancer to a wider
variety of chemicals and chemical classes and to develop
new tests for noncancer endpoints. Examples of tests that
have been developed at HERL and are currently being
validated or improved are a gene-toxic screen for reproduc-
tive hazards; bioassay-directed fractionation; a structure-
activity computer program; the Functional Observational
Battery, which measures chemical-related effects on sen-
sory, motor, and autonomic nervous system function; and
the Chemoff/Kavlock screen for developmental effects.
In addition, the research program to support the urban
toxic program involves considerable effort to identify
complex mixtures of concern, including those created by
atmospheric transformation.
Dose-Response Assessment. Of equivalent concern
to the Air Toxics Program is dose-response assessment to
evaluate levels of risks. Research under this topic will im-
prove understanding of dosimetry, pharmacokinetics, and
mechanisms of action and, as a consequence, the dose-
response relationship and species-to-species, route-to-
route, high-to-low-dose extrapolation issues. The major
goal of this research program is to provide better methods
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for extrapolating animal data to human effects. Both
human and animal experiments will provide data on the
functional, morphological, and biochemical changes that
occur following exposures to air pollutants; provide ex-
trapolation techniques to predict human responses; and
provide data to determine the extent to which air pol-
lutants cause or exacerbate the development of chronic
disease. Biological endpoints to be examined include
development of cardiovascular or pulmonary disease, im-
pairment of immune function, reproductive and develop-
mental endpoints, and neurotoxicity, as well as liver and
kidney dysfunction and cancer.
dicators of human exposure, is planned for the near
future. Research into the biologic link between these
markers and actual cancer risk is also needed. Suc-
cessful results will make possible direct estimates of
human exposure to carcinogens and will provide
relatively rapid and inexpensive alternatives to tradi-
tional epidemiologic approaches. Significant im-
provements in such areas as urban cancer risk and
improved cause-and-effect evidence for NESHAPs
candidate pollutants will also be possible.
Biomarkers for pulmonary, immune, and reproductive
system effects are under development.
This information will significantly improve or permit
risk assessments of greater certainty for a much larger
number of chemicals and exposure situations than is cur-
rently possible. Some of the more immediate benefits to
the program are likely to be in estimating risks associated
with doses exceeding the reference dose, reduction or
elimination of some uncertainty factors, and gathering in-
formation concerning appropriate methods to use when as-
sessing the risk associated with chemical mixtures. The
health effects data from this research program will be in-
corporated into EPA documents to support regulations, to
define problems, to assist state and local agencies, and to
evaluate high-risk point sources.
Exposure Assessment. Sensitive biologic measures
of pollutant exposure that will be developed as part of the
hazard identification and dose response work should
greatly improve estimates of exposure. Work in this area
is focused on three issues:
Identification of major sources of air pollution
using biologic screening. This use of mutagen as-
says to screen for significant sources of emissions
was developed at HERL. Future work will develop
methods for more accurately characterizing and iden-
tifying new sources of pollution and evaluation of
noncancer as well as cancer health concerns.
Identification of relevant atmospheric transforma-
tion products. The development and improvement of
similar biologic methods to characterize atmospheric
transformation products is also ongoing. Data being
developed may be critical in regulation of chemicals
that in themselves are innocuous but are transformed
in the atmosphere to potent carcinogens.
Use of biomarkers of exposure. Animal testing of
DNA adducts as biomarkers of exposure is currently
ongoing. Field testing of these biomarkers, as in-
3.1.3.3 Mobile Sources Program
Hazard Identification, Dose-Response, Exposure.
Research is currently ongoing on the mutagenicity and
carcinogenicity of existing mobile source-related emis-
sions. Studies conducted by ORD have shown that motor
vehicles are a major source of risk in urban areas. Re-
search in this area is focused on developing and improving
methods for screening and characterizing carcinogenic
potential using short-term bioassays. Researchers will
also begin to assess noncancer endpoints, particularly
developmental and reproductive toxicity, immunotoxicity,
hepatic toxicity, respiratory toxicity, and neurotoxicity.
Components of mobile source emissions will be identified
and the relationship of these assays to relative mutagenic
and carcinogenic potencies determined. Dose-response
assessment for endpoints of concern will also be evaluated
through animal-to-man extrapolation and estimates of sen-
sitive subpopulations. The program also needs human
data on ozone, oxides of nitrogen, sulfur dioxide, carbon
dioxide, methanol, and aldehydes.
In addition, the health research program plans to col-
lect the data needed to perform a comparative risk assess-
ment of alternative fuels. To do this, the mobile source
program needs to establish bioassay and animal testing
protocols (as mandated in Section 211 of the CAA) so that
it can evaluate the health impacts of new fuels and fuel ad-
ditives. In addition, the mobile source program needs sub-
stantial health research information on methanol and other
alternative fuels. For each alternative fuel, data will be
gathered to speciate emissions, identify and quantify com-
ponents, determine variance with the weather and car per-
formance characteristics, and evaluate possible public
health consequences. Since much of this information is
not available for gasoline, additional research may be un-
dertaken for this fuel as well.
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3.1.3.4 Indoor Air Program
Hazard Identification. Research will continue to
identify agents common in indoor air that produce adverse
health effects. Identifying and interpreting subtle human
health effects associated with identified indoor air pol-
lutants will be a key research area. Characterizing sensi-
tive human subpopulations and their responses to indoor
pollutants is a major part of this effort. To perform this
task, the development of new methods and approaches to
the types of effects caused by indoor pollutants will be
necessary.
Dose-Response and Exposure Assessment. The re-
search in this area will be directed to the indoor agents of
greatest concern, i.e., volatile organic compound (VOC)
mixtures and environmental tobacco smoke. Human ef-
fects and dose-response of noncarcinogenic VOCs will be
determined in relation to the sick building syndrome.
Cancer, impaired immune function, impaired lung func-
tion, and neurotoxicity are the current focus of this pro-
gram. Future efforts will expand into other areas, such as
reproductive/developmental risks. Bioassay of organics
from indoor sources will be conducted in chambers, test
homes, and field studies to provide comparative estimates
of potential health risks from various sources. Biomarkers
of exposure will also be validated to enable demonstration
of cause-and-effect relationships in epidemiologic studies.
3.1.3.5 Global Atmospheric Program
Hazard Identification. Research will determine
whether immunotoxic effects observed in mice in
response to low-level UV-B radiation also occur in
humans. In vitro methods to study these effects will also
be developed.
Dose-Response Assessment. Initial efforts will focus
on emulating the UV dosage schedule that is observed to
cause immunotoxic effects in mice and is appropriate for
human use. Both local and systemic immune responses
will be observed. The time course of responses will also
be determined. Work will emphasize analysis of the
mechanisms of these changes to enable understanding of
the potential impacts on human populations, given
worldwide exposure.
3.1.3.6 Radiation Program
As already noted, EPA is developing a research
strategy in this area. Projects have been proposed to study
the connection between immunosuppression from UV ir-
radiation and pathogenesis of infections diseases; the
changes in innate and acquired defense mechanisms in
mice exposed to radiation; the effects of UV-B on
pathogenic and protective immune responses in murine
cutaneous leishmaniasis; the influence of UV-B irradiation
on the effectiveness of immunization; and the effects of
UV-B radiation on susceptibility to murine
cytomegalovirus and influenza virus using experimental
mouse models.
3.2 OFFICE OF WATER (DRINKING
WATER)
The Safe Drinking Water Act of 1974 (SOWA), as
amended in 1986, mandates that the EPA ensure a high
quality for the nation's drinking water. The focus of the
law, and of the regulations developed by the Office of
Drinking Water (ODW), is the protection of human health.
Public water supplies must be disinfected to prevent pes-
tilence from waterborne infectious disease. Controls are
required on the type and amount of disinfectants and other
water treatment chemicals in drinking water because these
chemicals form by-products after reaction with naturally
occurring chemical substances in water. These by-
products have biologic activity and the potential to cause
adverse human health effects. Thus, regulatory decisions
about water treatment requirements involve balancing the
risks from infectious disease in raw water and the risks
from chemical toxicity in disinfected water. This balanc-
ing is achieved through two levels of drinking water
standards. The primary drinking water standards limit the
amount of microbes and certain chemicals allowed in
drinking water. The secondary drinking water standards,
which regulate parameters related to the aesthetics of
drinking water quality, are not covered here.
The Agency must set Maximum Contaminant Level
Goals (MCLGs) and Maximum Contaminant Levels
(MCLs) for microbes and each chemical or chemical class
to be regulated. MCLGs establish levels in drinking water
that are not expected to result in any adverse human health
effect over a lifetime of exposure. Based on health effects
concerns only, they are not enforceable standards but rep-
resent goals that water treatment operators should strive to
reach. MCLs are enforceable standards based on health
effects concerns, but they also take practical considera-
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tions into account (e.g., the technological feasibility of
control, availability of analytical techniques and their
detection limits, and the economic impact of regulating the
contaminant).
MCLs and MCLGs are based on cancer risk assess-
ments or noncancer risk reference dose calculations
described in Drinking Water Quality Criteria documents.
These documents outline the available scientific database
and the risk assessment logic used to derive the numbers.
For chemicals that are not ubiquitous in U.S. water sup-
plies but that may enter drinking water by accident, the
Office of Drinking Water prepares Health Advisory Docu-
ments (HADs). While shorter than the criteria documents,
HADs contain similar risk assessment information; they
derive One-day, Ten-day, Longer-term, and Lifetime
Health Advisories (HAs), i.e., concentrations of a con-
taminant in drinking water that are not expected to cause
any adverse noncarcinogenic human health effects after
exposures varying from one day to a lifetime.
The 1986 SDWA Amendments establish a time frame
>for completing primary drinking water regulations on 83
substances identified by Congress
(provisions were also made for adding
25 substances every three years after-
wards). The Amendments' require dis-
infection of all public water supplies.
EPA is currently evaluating which
treatment combinations are the most ef-
fective for microbiological control and
produce the least noxious chemicals.
Regulatory options include:
Placing substances on the list for regulation
Setting MCLGs and HAs
Writing Criteria and Health Advisory Documents
Establishing the basis for
n Treatment technologies
D Monitoring
n Public notification
n Evaluating efficacy of control technologies
D Determining risk reduction achieved by regulation
The OHR/HERL research program in support of
ODW is designed to fill those needs (see Table 3-2).
Table 3-2:
SUMMARY OF DRINKING WATER HEALTH RESEARCH
Topic
Disinfectants
and
Disinfectant
Source Water By-Products
Nondisinfectant
Additives and
Distribution
System
Contaminants
Hazard ID
Requiring removal of organic
materials from raw water prior to
disinfection to eliminate the pre-
cursors of disinfection by-products
Weighing the various primary and
residual disinfection possibilities
Filtrating
Setting requirements for other
modifiers of water characteristics
(e.g., pH, alkalinity)
Health research is needed to sup-
port ODW's decisions in these areas:
Dose-Response
Chemical-Specific
Species-to-species/
organ-to-organ
extrapolation;
mechanistic
studies to develop
alternatives to
RfD
As part of other
studies with focus
on metals
Exposure
Databases and
SAR; methods
development
(e.g., bioassays,
reproductive tests)
Pollutant mixtures',
species-to-species
extrapolation;
PB-PK studies
Human and experi-
mental animal
studies of major
disinfectants and
their alternates
Uptake and dis-
tribution of
disinfectants
PB-PK studies on
metals, route-
to-route
extrapolation
As part of other
studies with focus
on metals
Uptake and dis-
tribution of
metals
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3.2.1 Office-Specific Research Needs
The key issues that face ODW in its mission to ensure
a safe, high-quality drinking water supply are:
Microbiological and chemical quality of source
waters
Safety of disinfectant processes
Impact of nondisinfectant chemical additives (e.g.,
clarifying agents) on human health
Safety of corrosion by-products and chemicals that
might leach from the water distribution system prior
to arrival at the tap
These issues are discussed in this section under three topic
areas: 1) source water; 2) water disinfection and disinfec-
tion by-products; and 3) nondisinfectant additives and dis-
tribution system contaminants. These topics are not
mutually exclusive. Points that apply to more than one
area will be made under one topic only.
32.1.1 Source Water
Drinking water comes from either surface (e.g.,
rivers, lakes) or ground waters (aquifers). The water from
these different sources varies greatly in pH, hardness and
other mineral characteristics, microbiological and chemi-
cal contamination, and organic material (e.g., leaf mold).
Ground waters are generally of better quality than surface
waters, with fewer microbes, little organic material, and
fewer pollutants. These waters, however, may contain
higher concentrations of chemical substances, such as pes-
ticides in agricultural areas or solvents from underground
injection of wastes.
These characteristics"which greatly impact the quality
of the treated water"must be considered in choosing treat-
ment technologies. For instance, raising pH lowers the
leaching of lead from pipes and solder in the distribution
system and also lowers the formation of choloroacetic
acids in chlorinated water; at the same time, however, it
increases the formation of trihalomethanes, which include
known human toxicants like chloroform. In addition,
removal of organic material lessens the formation of haz-
ardous disinfectant by-products in treated water, and
reducing water hardness lowers the corrosivity of the
water reaching distribution pipes; but both these options
add a significant cost to finished water and neither is ab-
solutely achievable.
ODW and water suppliers must ask:
What chemicals are present in a given source of
water?
Which have adverse health effects?
Of these, what is the basis for deciding to remove the
substance from drinking water or reduce its amount in
water?
What detection and monitoring techniques are avail-
able?
What is the cost of monitoring and control options?
What is the minimum amount causing an effect?
How should health effects concerns be balanced with
monitoring and control technology to determine how
drinking water contaminants should be regulated?
Research in this area should focus on providing the
scientific basis for setting MCLs and developing HAs.
For most of the metals (e.g., nickel, chromium) and or-
ganic pollutants (e.g., pesticides, solvents) present in
source water, data are available concerning hazard poten-
tial. These data are often generated to meet the mandate
of other environmental laws such as the Federal Insec-
ticide, Fungicide, and Rodenticide Act (FIFRA) or the
Toxic Substances Control Act (TSCA). (For information
on organic materials in source water, see Section 3.2.2.1;
on chemicals responsible for water hardness and corrosion
by-products, see Section 3.2.2.3.) To compare the hazard
potential of exposure to these materials in source water
versus through other media, pharmacokinetic and phar-
macodynamic data from the various media must be com-
pared; thus, the prime focus of HERL research for source
water concerns will be on dose-response research.
32.1.2 Water Disinfection and Disinfection
By-Products
Drinking water disinfection has been practiced for
many years"especially disinfection of surface waters,
which are more heavily impacted by human activity than
are ground waters. Disinfectants are strong oxidants,
highly reactive with biologic material. These charac-
3-11
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teristics allow them to sterilize the pathogenic microbes in
source water. The more reactive the disinfectant, the
greater the immediate disinfectant property, but the less
the stability. Because water is not consumed immediately
after disinfection, microbes that escape treatment or that
are introduced into the distribution system after treatment
are a potential health hazard. As a result, secondary disin-
fection is often employed via residual disinfectant to en-
sure the microbial safety of water reaching the tap. ODW
is seeking ways to reduce the formation of disinfection by-
products by offering water suppliers options such as
precursor removal prior to disinfection and various treat-
ment combinations, such as ozonation (which does not
produce chlorinated by-products) followed by chloramina-
tion (to provide a stable residual disinfectant) while still
maintaining adequate water safety in the development of
its regulations. This process is very complex, and there is
much uncertainty surrounding what chemical substances
are formed by water treatment, the nature of their health
effects, and the choice of the best control technologies.
In evaluating the basis for disinfection requirements,
by-product control, and variances, ODW must ask:
What compounds are formed by the various disinfec-
tant treatments?
What is their ability to cause human health effects?
Which results in the lowest chemical hazard potential
with the greatest disinfectant properties?
What treatment technologies are available to achieve
the desirable characteristics?
What detection methods and approaches are available
to monitor the effectiveness of treatment tech-
nologies?
What is the maximum amount(s) of disinfectant(s)
causing an adverse health effect?
How should the health effects concerns be balanced
with monitoring and control technology capabilities
to determine disinfection requirements?
The research focus in this area must be on developing
the science base to support water disinfection decision-
making. Human data are needed to identify or confirm
health hazards associated with alternate disinfectant treat-
ments (e.g., ozone, chloramine, chlorine dioxide) and sus-
ceptible subpopulations (e.g., pregnant women and
glucose-6-phosphate dehydrogenase deficient persons) ex-
posed to strong oxidants. Extrapolation studies that
evaluate high-to-low-dose relationships need to be con-
ducted to evaluate the risks that are likely to occur at the
levels of chemicals encountered in drinking water. These
studies are key to ensuring safety without undue economic
hardship. The disinfectant regulations will cost the water
industry an estimated additional $1-2 billion per year, or
from 10-20 percent of their current annual income.
Work is also needed on the bioavailability and dis-
tribution of disinfectants and their by-products (e.g., ozone
and chloramine by-products) from water versus other
sources of exposure. Research to develop biomarkers for
sensitive endpoints related to disease outcomes (e.g., liver
enzymes and the threshold mechanism for liver car-
cinogenicity of dichloroacetic acid) also is needed. Re-
search efforts should be designed to understand the
artifacts induced at high exposure levels that have no bear-
ing on health effects at doses humans receive.
To support risk assessment, pollutant mixture studies
are needed to identify the biology of disinfectants and by-
products present in both simple and complex combina-
tions. Sample fractionation followed by bioassay will also
be used to identify biologically active chemicals as a basis
for identifying toxic components and for directing control
technologies. Test methods need to be developed for
evaluating the structure-activity relationships (SAR)
within families of by-products (e.g., trihalomethanes,
chloroacetic acids, furanones) to compare potencies be-
tween different endpoints (e.g., genotoxicity/cancer com-
pared to reproductive, neurotoxic, cardiovascular, and
other organ effects). Databases are needed to systemati-
cally store the SAR information for subsequent use in
model development, to address adversity/severity of ef-
fect, and to select the most appropriate test regimens for
untested compounds. The ultimate goal is the develop-
ment of predictive risk assessment models.
32.1.3 Nondisinfectant Additives and Distribution
System Contaminants
In addition to chemical disinfectants, other chemical
substances are added to drinking water at treatment plants.
Clarifying agents such as alum are used to remove particu-
lates suspended in the incoming water, and other substan-
ces may be added to adjust such characteristics as pH and
water hardness. As drinking water leaves the treatment
plant, it passes through distribution mains to connections
with various buildings and finally into internal building
3-12
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plumbing before reaching the tap. As it passes through
the system, copper, lead, and other metals may leach into
if'for example, from the piping or solder"before the water
reaches the tap. Recent regulations limit the lead allow-
able in new pipes and solders, though pipes and solders in
existing structures are not yet regulated. Little is known,
however, about the toxicity of metals (e.g., antimony)
used to replace lead in solders. ODW must make sure that
water entering the distribution system does not contain
harmful additives, and that once in the pipes, it does not
cause corrosion and leaching of toxic metals.
ODW must set MCLGs and develop HAs for drinking
water additives and distribution system contaminants; to
do so, it needs research data characterizing the uptake of
heavy metals (e.g., nickel, aluminum, copper) and their
distribution and fate in the body. Human studies are
needed to evaluate the ability of the corrosion by-products
to cause reproductive effects and other adverse health out-
comes.
3.2.2 Research Plan
For regulatory decision making, information is
needed about the uptake and distribution of xenobiotics
within the human body, and effects likely to occur at the
low concentrations encountered in the environment. Uni-
que ODW research needs result from the regulatory man-
date for the Office to make sure source water, disinfection
treatments, and distribution systems are consistent with
safe, high-quality water.
32.2.1 Source Water
Dose-Response and Exposure Assessment. Most
information about the chemicals present in source water
will be gleaned from the literature or be derived from
work conducted in support of other Agency programs: the
substances of concern in source waters are largely the pes-
ticides and industrial chemicals used in commerce and
metals leaching from the soil through which the water pas-
ses. In the water program, the potential for adverse human
health effects must generally be based on data from high-
dose exposures in whole animals or in vitro tests. Ques-
tions often arise concerning the uptake, bioavailability,
and tissue distribution of materials from source water; thus
pharmacokinetic studies are needed to interpret and
evaluate data, including:
Fate and disposition of metals (e.g., arsenic, cad-
mium, lead) as related to ingestion via drinking water,
and route-to-route extrapolation
Human epidemiologic studies on populations im-
pacted by high metal content (e.g., arsenic) to relate
exposure to outcome
Extrapolation of effects observed at high doses com-
pared to those observed at low doses
Extrapolation of data from animal studies to humans
A variety of endpoints have been used by ODW to set
MCLs in the regulation of drinking water contaminants:
nervous system effects, liver toxicity, tumor formation,
kidney toxicity, circulato^eart/blood effects, lung toxi-
city, reproductive/developmental effects, and gastrointes-
tinal tract effects. To increase knowledge concerning the
shape of the dose-response relationships for these substan-
ces:
Work will be conducted to understand homologous
endpoints between human and experimental animals
for noncancer endpoints, especially for reproduc-
tive/developmental effects and neurotoxicity.
Emphasis will be placed on identifying species-
specific and cross-species parameters to support
physiologically based (PB-PK) model development.
The relationships and time dependency between ex-
posure, delivered dose, and outcome will be studied
to develop biologic markers of dose and early effect.
The markers will be used to understand the shape of
dose-response curves and the data will be transferred
to the Oiiice of Health and Environmental Assess-
ment (OHEA) and other risk assessment groups for
application in risk assessment modeling to supplant
the use of safety factors in setting RfDs.
Chemical-specific data are needed because ODW can-
not require industry to test chemicals. However, many of
these chemicals are of interest to more than one program
office. For instance, ODW and the Office of Solid Waste
and Emergency Response (OSWER) share an interest in
the effects of exposure to metals. HERL will conduct re-
search using chemicals of interest to ODW, especially me-
tals (e.g., arsenic, nickel, chromium, cadmium).
3-13
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3.2.2 2 Water Disinfection and Disinfection
By-Products
In relation to disinfectants and disinfectant by-
products, HERL researchers will address fundamental
questions about the safety of oxidants at the low doses en-
countered in drinking water and will provide data for com-
paring various disinfection treatment options in regulatory
decision making. HERL will give research in this area
high priority because most of the chemicals of concern
(i.e., the disinfectant by-products) are formed inadvertent-
ly and ODW cannot require industry to test them.
Hazard Identification. Thousands of chemical sub-
stances are formed through chemical reactions between
disinfectants and organic material present in source water.
Few data exist concerning biologic activity associated
with exposure to these substances. Because testing them
would be prohibitively expensive, as well as impractical,
substantial efforts are needed to develop SAR as well as to
develop, validate, and refine test methods and testing
strategies for evaluating the hazard potential of
halogenated disinfectants and their by-products, alone and
in mixtures.
These efforts include:
Developing databases
Using the databases in risk assessment and the
development and refinement of SAR models and
techniques
Constructing graphic displays of a variety of
endpoints, enabling comparisons of toxicity by class
of compound, outcome, and potency as a basis for
biologic SAR
Using SAR and profiles for generating hypotheses,
developing testing strategies, and prioritizing research
compounds
Dose-Response and Exposure Assessment. Some
treatments being considered as alternative technologies
that might replace chlorination, such as ozonation and
chloramination, have been used for several decades or
more in certain communities in the United States and
abroad. A major uncertainty surrounding these chemicals
is whether the treatments and their by-products are as haz-
ardous or perhaps more hazardous than those resulting
from chlorination.
Animal studies. HERL researchers will perform
studies on experimental animals to provide a basis for as-
sessing human risk. Most emphasis will be placed on
evaluating the hazard potential for disinfectants other than
chlorine"especially for ozone and chloramine, which have
been identified as potential candidates for alternative dis-
infection. Stoichiometric differences in the half-lives and
by-product formation of these and other disinfectants (e.g.,
chlorine, chlorine dioxide) in the gut will be modeled as a
basis for predicting outcomes from high-dose experimen-
tal to low-dose environmental exposures and for designing
follow-up lexicological studies. To the extent feasible, the
models will be used to predict toxicity and follow-up
pharmacokinetic and pharmacodynamic studies will be
conducted to verify the predictions.
Such studies will form the groundwork for targeting
subsequent research efforts to identify the presence and
nature of artifacts that may be induced by high-dose
regimens but are not an issue with low-dose environmen-
tal exposures. The work will also be used in developing
biomarkers of organ dose and early biologic effect to
define the shape of the dose-response curves for various
adverse outcomes. This information will be useful in
developing the quantitative risk assessment models that
will eventually replace RfDs.
These efforts will focus on developmental effects,
cancer, and liver toxicity. The carcinogenicity studies will
emphasize the development of parameters for the Mool-
gavkar-Knudson-Venzon model and the elaboration of
dose-response curves for nongenotoxic carcinogens as a
basis for exploring the possibility of threshold
mechanisms of cancer induction. Developmental effects
studies will employ the techniques of cell biology and
flow cytometry to elucidate early events leading to stage-
and species-specific alterations in embryonic develop-
ment. Similarly, studies on liver metabolism will focus on
early biochemical changes of organ function for use in
elucidating the dose-response. To the extent possible,
these studies will be conducted in parallel to provide com-
parative toxicity information.
Human studies. Human data will also be collected to
evaluate increases in morbidity and mortality linked to the
consumption of ozonated, chloraminated, and ozonated/
chloraminated water compared to the consumption of
chlorinated water. As appropriate, similar comparisons
will also be made for persons consuming water treated
with chlorine dioxide and other possible alternative disin-
fectants, if suitable populations can be found. Studies on
persons consuming ozonated water will probably be con-
ducted in Europe, which has the longest history of use.
3-14
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However, care will have to be taken to select communities
that employ treatment processes similar to those likely to
be used in the United States. Studies of populations con-
suming chloraminated water will probably be conducted
in New Orleans, Louisiana.
Studies of mixtures. Because drinking water is a com-
plex mixture of chemical substances, the health research
program will focus on evaluating the effects of disinfec-
tants and their by-products after combined rather than
single exposures. The two thrusts of this effort are:
dose and to compare uptake and response from different
routes of exposure.
Studies of nonconventional, lifetime exposure (e.g.,
effects over two and three pregnancies), and susceptible
life stages (e.g., very young, very old, pregnant) will be
conducted using experimental animals to approximate
human conditions not modeled by conventional assays.
Researchers will also perform human studies to refine the
results of the animal studies for risk assessment use.
To understand principles and develop databases for
risk assessment
To identify the toxic species within mixtures so that
control technologies can be developed to eliminate or
reduce them from potable water
The current emphasis in these studies is on the ap-
plication of short-term tests for genotoxicity in combina-
tion with chemical fractionation. While this approach has
proven successful in identifying genotoxic compounds in
drinking water"for instance, in the identification of
hydroxyfuranones (e.g., MX) as potent genotoxicants"the
relationship between a chemical's potency as a
genotoxicant and its potency for other toxic effects is un-
known. Short-term test methods will be developed for
reproductive and developmental effects, liver, and, if pos-
sible, neurotoxic outcomes; these will be applied to model
compounds, simple mixtures, and then to drinking water
samples. The data will be used to assess the toxicity of
water treated with a variety of disinfectants.
33.2.3 Nondisinfectant Additives and Distribution
System Contaminants
The pollutants used as additives or that arise from the
distribution system and contribute to the degradation of
water are primarily metals: aluminum used as a clarifying
agent; copper, lead, and antimony from water pipes, joints,
and solder. The primary target organs for these substances
are the nervous and reproductive systems and the kidney.
Dose-Response and Exposure Assessment. Re-
search efforts in this area will focus on these target organs,
concentrating on the uptake and distribution of me-
tals"primarily nickel, aluminum, and copper from the
gastrointestinal tract"and their transport to the reproduc-
tive and nervous systems. Studies will be conducted
under both high-dose and more realistic environmental
conditions as a basis for extrapolating from high to low
3.3 OFFICE OF WATER (WATER
QUALITY)
The Federal Water Pollution Control Act of 1972, as
amended by the Clean Water Acts (CWA) of 1977,1978,
1980, 1981, and 1987, require that the quality of the
nation's surface water supplies be maintained for their in-
tended purposes (i.e., that they be swimmable, fishable
and/or navigable). The states are responsible for defining
the uses for which surface waters are intended, issuing
permits, and monitoring discharges from industrial and
wastewater treatment sources to ensure that water quality
is maintained.
All sources and types of pollution of surface waters
(i.e., rivers, streams, lakes, bays, estuaries, most natural
wetlands and oceans) are covered by the Water Quality
Program. EPA regulations address "conventional pol-
lutants" (i.e., biological oxygen demand, suspended solids,
fecal coliform, pH, and oil and grease), and toxic pol-
lutants (i.e., 65 classes of chemical substances listed in the
CWA). From these 65 classes of toxic pollutants, EPA
has identified 126 compounds as priority pollutants. In
addition to the conventional and toxic pollutants, EPA
typically considers another 300 or so pollutants for regula-
tion that are known to have adverse effects and for which
accurate measurement techniques have been developed.
These are referred to as nonconventional pollutants.
EPA must publish water quality criteria for the
priority pollutants that set forth the maximum concentra-
tions consistent with the goals of the CWA. The criteria
are based solely on scientific data and judgment concern-
ing ecological and human health effects and must not take
into account economic or technological feasibility. The
criteria must specify the latest scientific knowledge on:
All identifiable effects in water on health and welfare,
including ecological effects and aesthetics
3-15
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Concentration and dispersal of pollutants or by-
products through biological, physical and chemical
processes
Effects of pollutants on biological community diver-
sity
The Agency has developed human health criteria for
108 of the 126 priority pollutants and aquatic life criteria
for 26 priority pollutants.
3.3.1 Office-Specific Research Needs
The full range of human health effects must be as-
sessed to develop water quality criteria documents and set
standards for effluent limitations. Because most of the
data used to derive these criteria are based on animal
lexicological studies, the Office of Water (OW) needs in-
formation that will help resolve the fundamental uncer-
tainties about extrapolating from the experimental animal
to the human situation and from the high-dose experimen-
tal exposures to the low concentrations generally en-
countered in the environment. Emphasis is placed on the
uptake and pharmacokinetics of the pollutants from the
gut after ingestion via water, the skin, or exposure to was-
tewater sludges.
From a practical perspective, human health data are a
low priority for the Office of Water for two reasons:
Human health criteria have been developed for most
of the priority pollutants, while ecological criteria
have been developed for only a few
Health effects criteria for water quality do not have to
be based on human data; adverse effects to wild
animal species provide a sufficient basis for standard
setting
Many animal species live in water, thus, they provide a
convenient means of setting the water quality standards
and monitoring compliance. The Office of Water does
need human data on health effects, however, to compare
with those gathered from animal species. Work is needed
on structure-activity relationship (SAR) methods and
databases for predicting effects in various species; in addi-
tion, extrapolation research is needed to link a variety of
effects in aquatic species to human disease outcomes.
Methods development for short, simple tests to
evaluate the effects of pollutant mixtures, especially in
wastewater and sludges, is a major health need for OW.
Tests are needed for monitoring the maintenance of water
quality and compliance with the standards. Tests and pro-
cedures are also needed to couple bioassays with chemical
fractionation to identify the toxic components of waste
waters and sludges. This information would be used to
develop targeted, cost-effective control technologies that
remove only those contaminants associated with biologic
activity.
3.3.2 Research Plan
The health research program in support of the water
quality program is quite small at this time, capable only of
providing technical support and guidance, and will be fur-
ther reduced over the next three to five years. In that time
period, work will be limited to identifying the needs for
health research in this area. This effort will include back-
ground analysis to identify needs for physiologically
based pharmacokinetics, SAR approaches, and databases;
it may also take the form of workshops to examine the
state-of-the-art of nonhuman bioassays for predicting
human effects, and vice versa, as a basis for defining fu-
ture needs and the most appropriate ORD health research
program to meet those needs.
3.4 OFFICE OF PESTICIDES AND TOXIC
SUBSTANCES
EPA's mandate to protect human health and the en-
vironment from pesticides and toxic substances is
provided under two acts:
The Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA), which gives EPA the authority to regu-
late the distribution and use of pesticides
The Toxic Substances Control Act (TSCA) of 1976,
which gives EPA the authority to prohibit or restrict
the manufacture, distribution, use, and disposal of
chemicals that present "unreasonable" environmental
or human health risks
Federal Insecticide, Fungicide, and Rodenticide
Act. Under FIFRA, EPA regulates the distribution and
use of pesticides, plant regulators, defoliants, and desic-
cants (hereafter referred to collectively as pesticides).
Pesticide companies are required to generate data on the
toxicity and environmental characteristics of the active in-
gredients in their products, i.e., the ingredients that bring
3-16
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about the product's intended function. The Agency
evaluates these data to determine whether a pesticide
should be registered (if it is a new pesticide) or
reregistered (if it is an existing pesticide, already
registered for use). If new data suggest that an existing
product may pose a hazard, EPA performs a special
review to determine whether the product should be taken
off the market
New Pesticides. All new pesticides must be
registered by EPA before they can be distributed and used.
To support a registration application, EPA may require the
manufacturer to perform as many as 150 different techni-
cal studies and data submissions. The health-related data
EPA can request include results of acute studies, sub-
chronic studies, chronic feeding studies, oncogenicity
studies, and metabolism studies. EPA can also request
data concerning the potential for human exposure. If a
product is identical or substantially similar to (e.g., has the
same active ingredient as) an existing pesticide, the re-
quirements for new testing data may be reduced.
EPA evaluates the data to determine "any un-
reasonable risk to man or the environment." At present,
there is no guidance or legal precedent to define the level
of risk considered acceptable under FTFRA. In practice,
for carcinogenic pesticides, a risk of 10~4 to 10"5 from
dietary exposure is a cause for concern, and the Agency
considers methods to reduce the risk, such as limiting the
product's use or denying registration. If EPA finds that
the product does not pose an unreasonable risk, the pes-
ticide is registered for use.
As part of the evaluation of applications, EPA sets a
negligible risk tolerance level for food-use pesticides on
raw agricultural commodities and a "no-risk" tolerance
level on processed food. A tolerance is an amount or con-
centration of the pesticide residue on food that will not
pose an excessive risk. EPA's authority to set tolerances
is provided by the Federal Food, Drug, and Cosmetic Act.
The act stipulates that tolerances be set at levels deemed
necessary to protect the public health, while taking into
account the need for "an adequate, wholesome and
economical food supply."
Existing Pesticides. The 1988 amendments to FIFRA
and the associated regulations require manufacturers to
submit more extensive testing data than previously re-
quired. To make sure that all pesticides currently in use
meet the new FIFRA requirements, previously registered
pesticides must be reregistered. Registrants seeking
reregistration must compare the data in their current
registration with the new requirements and supply any
missing data within prescribed deadlines. EPA must
review the data submitted to identify any further data gaps
and, after the final data submission by industry, must com-
prehensively examine all data submitted to support pes-
ticide reregistrations. Based on this review, the Agency
initiates appropriate regulatory action.
The 1988 amendments substantially increase the
number of chemicals to be evaluated by the Office of Pes-
ticide Programs. The reregistration program covers ap-
proximately 600 active ingredients in 35,000 pesticides.
The entire reregistration process must be completed in 3 to
9 years after enactment of the 1988 amendments (i.e., by
about 1991-1997).
Special Review. EPA's special review procedures are
initiated after evaluation of data supplied for reregistra-
tion, if the data indicate that a pesticide may present an un-
reasonable risk, including:
A risk of serious acute injury to humans
A risk of inducing in humans an oncogenic, heritable
genetic, developmental, or reproductive effect, or a
chronic or delayed toxic effect
A risk to humans sufficiently large to merit a deter-
mination of whether its benefits exceed its risks
A risk to nontarget organisms (e.g., acute or chronic
toxicity or adverse reproductive effects)
In a Special Review, EPA thoroughly evaluates the
risks and benefits of the pesticide, including acute and
chronic effects, and the exposure potential for farm
workers, bystanders, and consumers. The Agency then
decides whether to cancel, suspend, or modify the registra-
tion.
The Toxic Substances Control Act. TSCA gives
EPA the authority to regulate new and existing chemicals,
excluding pesticides, tobacco, tobacco products, nuclear
material, firearms, ammunition, food and food additives,
drugs, cosmetics, and devices. EPA can restrict manufac-
ture, processing, distribution, commercial use, or disposal
if the substance presents "an unreasonable risk of injury to
health or the environment." To evaluate chemicals, EPA
may require industry to submit extensive testing informa-
tion on the risks of particular substances. Under TSCA,
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EPA evaluates both new chemicals and existing chemi-
cals.
New Chemicals. Under Section 5 of TSCA, busi-
nesses must notify EPA at least 90 days before manufac-
turing or importing a chemical substance for commercial
purposes. This notification is called a premanufacturing
notification (PMN) and must include the following health
information:
Test data in the submitter's possession or control
(often, minimal data are submitted because many sub-
mitters have only minimal data in their possession,
and EPA cannot require them to generate data for the
initial PMN submission)
Descriptions of health and environmental effects data
that they know of or can reasonably ascertain
The reasonably anticipated manner and methods of
manufacturing, processing, distribution in commerce,
and disposal
Existing Chemicals. Section 4 of TSCA gives EPA
the authority to require testing of existing chemicals. To
implement this authority, EPA must find that
The chemical may pose an "unreasonable risk" to
human health or the environment; or that the chemi-
cal is produced in "substantial" quantities, which
could result in substantial or significant human ex-
posure or substantial environmental release, and
Insufficient data or knowledge exist about the health
or environmental effects of the chemical to reasonab-
ly determine or predict the impacts of its manufacture,
processing, distribution, use and/or disposal, and
Information on chemical identity
Testing is needed to develop such data
Information on the projected volume manufactured as
well as increase in magnitude and duration of ex-
posure and method of disposal
EPA must review the information within 90 days (un-
less for good cause EPA extends the review period for an
additional 90 days). If EPA identifies a potential un-
reasonable risk that can be evaluated through testing the
substance, the Agency can require the submitter to per-
form further testing of the substance before approving the
product for use in commerce. EPA can regulate the sub-
stance if it finds that it will present an unreasonable risk to
health or the environment. If EPA does not regulate the
substance or require testing, manufacture or import can
proceed as soon as the review period expires. Businesses
must also submit PMNs before manufacturing or process-
ing any chemical substance for a "significant new use"
(generally a change in use that may increase human ex-
posure). In making this determination, the Agency must
consider all relevant factors, including:
The projected volume of manufacturing and process-
ing
The extent to which a use changes the type or form of
exposure to human beings or the environment
The extent to which a use increases the magnitude
and duration of exposure to human beings or the en-
vironment
If EPA makes all these findings for a specific chemi-
cal or category of chemicals, the Agency may issue a rule
requiring industry to test the substance(s). The rule may
prescribe the effects to be investigated, the tests to be con-
ducted, and the experimental test guidelines to be used.
The TSCA statute itself details many of the studies that
may be required. EPA periodically publishes test
guidelines and is required by TSCA to review each test
standard at least once a year and revise it where warranted.
3.4.1 Office-Specific Research Needs
3.4.1.1 Federal Insecticide, Fungicide, and
Rodenticide Act
Hazard Identification for New and Existing
Chemicals. Under FIFRA, EPA requires and evaluates
test data for new chemicals, but relies on industry to per-
form the tests. EPA's research needs therefore fall
primarily into two areas: 1) development and refinement
of methods to be incorporated into test guidelines and
protocols, and 2) evaluation of data. In setting test
guidelines, EPA must first determine the health endpoints
for which the product should be tested. Ideally, EPA
should be able to recommend, through guidelines, a bat-
tery of tests that will capture all the major health endpoints
of concern. EPA then develops reliable, valid tests for
these endpoints, and interprets the human health sig-
nificance of the data.
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Test method development and validation are ongoing
processes. As new methods are developed, they are
validated before they can be used to screen chemicals
and/or assess their toxicity. The validity of a test regards
what the test measures and how well it performs that task.
When validating testing guidelines, researchers assess the
test's validity in a number of areas:
Content: Does the test cover a representative sample
of the domain to be measured?
Construct: What is the extent to which the test
measures a theoretical construct or trait (e.g., intel-
ligence, anxiety)?
Criterion: How effective is the test in predicting an
outcome in specific situations (e.g., toxicity)? Two
types of criterion-related validity assessments are
measurable:
n Concurrent validity measures: To what extent do
other test methods yield similar results to the
method in question (e.g., diagnosis of existing dis-
ease status)?
n Predictive validity measures: To what extent do
effects noted in one experimental situation predict
the effects found in another (e.g., animal-to-human
extrapolation issues)?
The Agency has already developed several standard
tests that have been incorporated into testing guidelines.
Program officials are reasonably satisfied with some of the
current protocols; however, ongoing research is needed 1)
to refine existing test protocols in areas such as
neurotoxicity and reproductive toxicity, and 2) to develop
shorter-term, more cost-effective, and more accurate
methods capable of assessing a greater variety of noncan-
cer endpoints and multiple endpoints simultaneously so
that more information can be obtained from each protocol.
A tiered approach to testing has been considered, where
the first tier is used to rapidly screen substances for poten-
tial toxic effects, and subsequent tiers are used to assess
toxicity with greater accuracy and specificity. New
methods at both tier levels are needed to assess im-
munotoxicology, potential carcinogenicity for non-
genotoxic agents, reproductive toxicity, endocrine effects,
and neurotoxicity.
A testing strategy has been developed to assess the
possible toxicity of microbial pesticide control agents
(MCPAs) in human populations. Research is needed to
determine if MCPAs can survive and interact with rodent
and human tissue, to compare routes of exposure, and to
investigate observed mortality in mice.
Additional structure-activity research is needed to
help define patterns of activity among certain classes of
chemicals. This information will be used to help define
which tests are appropriate for particular classes of chemi-
cals.
In terms of microbial pesticide control agents
(MPCAs), the health research needs of the Office of Pes-
ticide Programs (OPP) are very specific. Issues include
the significance of persistance and/or lack of clearance of
MPCAs from animals exposed via inhalation; the sig-
nificance of the mortality observed with Bacillus thurin-
giensis in the current pumonary research; comparison of
intraperitoneal exposures to inhalation and intravenous ex-
posures; the significance of baculoviruses in vertebrate
cells; and determining whether a significant interaction ex-
ists between viral pesticides and vertebrate viral
pathogens. These issues are of high priority to the Office
of Pesticide Programs.
As part of the reregistration process, EPA must
specify tests and evaluate data for existing pesticides. The
research needs described here for new pesticides are there-
fore also valuable in supporting the reregistration activities
of the Office of Pesticide Programs. The large number of
chemicals that must be evaluated for reregistration will
place a greater burden on EPA's resources to evaluate
chemical data. Thus, the need for research to develop
rapid and reliable tests for chemical toxicity will likely in-
crease in the future.
Dose-Response Assessment for New and Existing
Chemicals. In the pesticides program, the potential
human health effects of pesticides generally must be
evaluated based on data derived from high-dose exposures
in whole animals or in vitro tests. Therefore, dose-
response issues are a high priority for the pesticides pro-
gram, particularly for the numerous evaluations that will
be done under the reregistration program set up under the
1988 amendments. Research on mechanisms/sites of ac-
tion and pharmacokinetics is needed in the many areas
relevant to interpreting and evaluating data, including:
Fate and disposition of pesticides, especially as re-
lated to dermal absorption and route-to-route ex-
trapolation
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Extrapolation of effects observed at high doses to ef-
fects observed at low doses
Extrapolation of animal data to humans
Extrapolation from acute to chronic exposure and
from one route of exposure to another
In some cases, observed health effects are temporary
or transient; the body can adapt to the insult or repair the
damage when exposure ceases. Thus, research is needed
to study injury and repair mechanisms following chronic
and acute exposure. These data would help the tolerance-
setting process and the ability to interpret the significance
of lexicological effects.
As noted above, EPA must specify tests and evaluate
data for existing pesticides as part of the reregistration
process. The research needs described for dose-response
for new pesticides are therefore also valuable in support-
ing these reregistration activities. The large number of
chemicals that must be evaluated for reregistration will
place a greater burden on EPA's resources to evaluate
chemical data. Thus, the need for research to perform re-
search to support accurate data interpretation, assessment,
and extrapolation will likely increase in the future.
Exposure Assessment for Existing Pesticides.
Biomarkers"measures of samples of human tissues or
fluids (e.g., blood lead levels)"provide information about
human exposure, response, and/or susceptibility to future
challenge. Biomarkers come directly from humans and
offer great promise for 1) evaluating exposure and
response in populations, 2) evaluating the effectiveness of
control technologies, and 3) enabling early intervention in
the cascade of events from exposure to effect to prevent
disease. When used in experimental animals and when
coupled with pharmacokinetic experimental and modeling
studies, they will provide a means to extrapolate between
doses and species, and to understand mechanisms of dis-
ease.
Biomarker research is important across the full array
of adverse outcomes. Research is particularly needed to
develop markers for exposure, reproductive function,
genotoxicity, cancer, and pulmonary effects, and for out-
comes associated with specific classes of pesticides. Such
biomarkers could be used to evaluate whether humans
respond to various levels of pesticide exposures. This in-
formation would provide a basis for refining tolerance
levels and evaluating worker safety.
Exposure research is needed to better assess the level
of exposure that is occurring from pesticides currently in
use, i.e., via consumption of contaminated drinking water,
inhalation of emissions from incineration of pesticides,
food consumption, and exposure through skin contact
during application and use. This research should focus on
improving comprehensiveness, developing better sur-
rogates (including biomarkers), and improving validation.
Research is also needed to develop methods for monitor-
ing exposure to MCPAs.
3.4.1.2 The Toxic Substances Control Act
Hazard Identification for New and Existing
Chemicals. Most PMN submissions contain little or no
substantial lexicological data, and EPA is not given much
time to review the data. For ihese reasons, the program
office relies heavily on an analysis of a chemical's struc-
ture to predict its activity. Substantial research, including
data base development is needed lo refine and quantify
struclure-aclivily relationships for use in predicting the
toxic effects of untested chemicals.
Currently, most testing guidelines are limited to con-
ventional toxicity endpoints. Test method development is
a major research need for the TSCA program. Validated
methods are needed for all important toxicologic
endpoints, including particularly neurotoxicology,
developmental/reproductive toxicology, genetic toxicity,
and immunotoxicology endpoints. Also, short-term tesls
(e.g., in vitro lesls) are needed for screening for effecls in
all largel systems. Short-term screening methods would
enable OPP to develop abbreviated protocols that would
help reduce the economic burden on industry (so as not to
provide a disincentive to manufacture or import). Several
in vitro tests look promising, based on early validation ef-
forts with a limited number of chemicals. Research is
needed to evaluate them using more stringeni criteria. In-
terlaboratory validation is also needed.
In support of PMN registration evaluation, tesls are
needed lo evaluate Ihe potential pathogenicity and loxin
production in a variety of GEMs. Eventually, rapid short-
term assays will be needed lo replace ihe more expensive
long-term assays now under developmenl.
A specific research area is the potential health impacts
of conventional microorganisms and GEMs released to the
environment for commercial purposes. Such research is
needed to better understand microbial characteristics
responsible for infection, pathogenicity, and genetic ex-
change. EPA will use Agency-sponsored biotechnology
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ecology studies, approved environmental releases, in-
dustrial and other federal agency data bases to generate,
evaluate, and validate appropriate test methods and risk
parameters.
Biomarkers of effect would also provide information valu-
able for extrapolating animal test data to human outcomes.
In particular, biomarkers that are predictive of neurotoxic
endpoints and carcinogenesis should be developed.
For existing chemicals, EPA must determine which
chemicals should be tested, develop test methods, and pro-
vide scientific support for any regulatory action. Screen-
ing, test method development, and data evaluation are
therefore important ongoing research needs for the pro-
gram office. Such research helps increase the efficiency
and acccuracy with which the program office can assess
chemicals and make regulatory decisions. In particular,
rapid, inexpensive in vitro assays are needed for screening
and for enhancing our understanding of mechanisms of ac-
tion.
Several compounds of interest to the program office,
such as dioxin and perchloroform, appear to be non-
genotoxic carcinogens (i.e., they cause cancer by some
method other than direct interaction with DNA). These
compounds cannot, therefore, be evaluated using the
standard risk assessment process for carcinogens. Re-
search is needed to elucidate the mechanism of action of
nongenotoxic carcinogens to facilitate risk assessment,
particularly animal-to-human extrapolation.
Dose-Response Assessment for New and Existing
Chemicals. Dose-response research is a high priority for
the TSCA program. An understanding of mechanisms of
action would aid in developing short-term tests for certain
endpoints, improve the evaluation of risk to humans, pro-
vide a cost-effective means of screening large numbers of
chemicals, and also prove useful in understanding why
different species react differently to the same chemical.
Such understanding would increase the accuracy of inter-
species extrapolation in chemical assessments. In par-
ticular, mechanistic studies are needed for noncancer
effects and nongenotoxic carcinogens. Research is also
needed to develop and improve quantitative biomathe-
matical modeling of dose-response data from noncancer
endpoints.
Exposure Assessment for Existing Chemicals. Re-
search is needed to develop biomarkers of exposure that
would provide important tools for more accurately assess-
ing the extent and degree of exposure in human popula-
tions. Such information would help OPP determine
whether to require testing under Section 4 of TSCA. Ex-
tensive pharmacokinetic research is needed to validate
biomarkers of exposure and, where feasible, to determine
whether they could also be used as biomarkers of effect.
3.4.2 Research Plan
The Health Effects Research Laboratory has
developed a research plan to address the critical needs of
the program office for pesticides and toxic substances.
The research focuses on two primary outputs: test
methods development/validation and data interpretation.
The method selected for development may be pesticide- or
toxic-substance-specific or may be more generic to cover
classes of chemicals. The research process involves
method selection, method development, refinement or
evaluation of the methodology, validation, guideline
development, and data interpretation. The research falls
under the broader categories of hazard identification and
dose-response assessment described below.
3.4.2.1 Pesticides
Hazard Identification. To address the ongoing need
for improved testing methods, HERL will conduct re-
search to investigate the full array of potential adverse ef-
fects from exposure to pesticides, including"but not
limited to"neurotoxic, reproductive, and immunotoxic ef-
fects. Basic research will involve refining existing techni-
ques and creating new ones in these scientific disciplines.
Applied research efforts will involve evaluating and inter-
preting industrial data (both review of raw data and
laboratory research to verify and clarify data reported from
industry).
Neurotoxicity. Many pesticides work by interfering
with nervous system function. Therefore, tests for
neurotoxic effects are particularly important in
evaluating the potential human health effects of pes-
ticides. Research will continue to develop and
validate tests capable of detecting effects at all levels
of the nervous system, including behavioral,
neurophysiological, cellular, and molecular levels.
Since neurotoxicology is a rapidly evolving field,
HERL will periodically review and, where ap-
propriate, refine existing tests for potential refine-
ments to make them simpler, more rapid, and more
accurate.
The current first tier test battery for neurotoxicity
focuses on detecting the ability of a chemical to cause
overt motor, sensory, or autonomic deficits. Research
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will be conducted to support new test guidelines for
evoked potentials, glial fibrillary acid protein (GFAP)
(a neurochemical marker of neural injury), and effects
on learning and memory. HERL will also investigate
a test for sensory evoked potential, currently being
used by some industrial laboratories, for potential use
under FIFRA.
Research will be initiated to evaluate the use of tissue
cultures for studying potential neurotoxicants in vitro.
Initially, the approach will be systematically validated
by evaluating a number of neurotoxicants known to
produce structural deficits in vivo and comparing
those effects to neuroactive or short-acting psychoac-
tive agents known not to be neurotoxic in vivo. Sub-
sequently, chemicals with unknown neurotoxic
potential will be compared using in vivo and in vitro
procedures. The overall goal of this research will be
to arrive at a validated in vitro tissue culture proce-
dure that can be used to assess chemicals for potential
neurotoxicity.
Neurotoxicologic research will continue to work
within a tiered approach to neurotoxic testing. A first
tier battery of tests is currently being validated. Fur-
ther studies will be conducted to refine the battery and
determine the conditions under which it should be
used. Eventually, based on the results of mechanistic
research described below, tests for effects at the cel-
lular and molecular level may be added to the battery.
Another future research area is the development and
validation of the second tier of tests, which would be
used to confirm the results of the first tier, determine
if the effect(s) is(are) primary or secondary, and
develop further information about the chemical's
toxicity, such as the lowest-observed-adverse-effect
level. This information will be important for reducing
uncertainty in the risk assessment process.
Reproductive toxicity. Currently, reproductive
toxicity relies on fertility as a measure of reproductive
damage; however, this endpoint is relatively insensi-
tive under certain circumstances. Also, present tests
generally do not provide information on the affected
sex, the likely target tissue, or the mechanism of
toxicity. Thus, a major need exists for improved test-
ing protocols, as several Agency-sponsored
workshops have concluded. HERL will continue re-
search to develop alternative reproductive test (ART)
strategies to replace or supplement the current multi-
generation tests. This research will develop improved
methods to assess testicular, ovarian, and uterine
function (e.g, semen evaluations, oocyte toxicity).
The research is designed to develop an ART protocol
that would detect developmental effects beginning
with the period of sexual maturation, and proceeding
through the entire reproductive life cycle. A long-
term goal of this research is to effectively characterize
the affected sex and target site, and information on the
homologous nature of the response.
I Immunotoxicity. Immunotoxicity testing under
FIFRA is currently limited to Subpart M pesticides
(i.e., biopesticides). However, immunotoxicity test-
ing guidelines for other pesticides, as well as toxics, is
being discussed. Improved testing methodologies are
being developed, particularly in species other than the
mouse. Also planned is research to improve our
capability to predict risk of increased disease based on
effects in immune function tests. Research will con-
tinue to develop a tiered approach to immunotoxicity
testing in several species with particular emphasis on
species comparisons and relationship between effects
in tier one and susceptibility to infectious, neoplastic
or allergic disease in tier 2. This research will include
development of a variety of host resistance models in
the rat and mouse for use in tier 2 and for species-to-
species extrapolation. Research will also be con-
ducted to assess the potential of various types of
compounds to produce allergenic and autoimmune ef-
fects. This work would be particularly applicable to
microbial pesticides, which contain proteins and are
generally more likely to be allergenic than chemical
compounds.
Microbial pesticide control agents. To better under-
stand the likelihood of opportunistic infection and
pathogenicity, researchers will explore the capabilities
of MPCAs to invade tissues and interact with ver-
tebrate cells and vertebrate viral pathogens. Research
will also be conducted to compare routes of exposure
and observed mortality in mice. Also of import is the
persistence or lack of clearance of the MPCAs from
the lung.
Dose-Response Assessment. Research will be con-
ducted to develop a better understanding of the
mechanisms underlying the effects associated with pes-
ticides for which tests are available. Such research will
help elucidate the cellular/molecular events that are as-
sociated with changes in physiology and function. This
research will also enhance understanding of the predictive
value of the tests, and the ability to extrapolate data from
one species to another. Also, such research may ultimate-
ly lead to the development of cellular/molecular tests that
could replace or supplement existing protocols. These ef-
3-22
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IlllliilB^
forts need to be ultimately linked to pharmacokinetic
models such that a complete description of exposure, dose,
and outcome can be used in the risk assessment process.
Neurotoxicity. Many pesticides work by inhibiting
cholinesterase, an enzyme essential for neuromuscular
activity. At low levels, cholinesterase inhibition ap-
pears to have little or no effect. At high levels, it can
be fatal. Research will include a major initiative to
study cholinesterase inhibition. Issues to be ad-
dressed include the relationship between
cholinesterase activity and other effects observed fol-
lowing pesticide exposure; assessing whether
cholinesterase inhibition is an adverse effect, a marker
of adverse effect, or a marker of exposure; determin-
ing whether the age of exposure affects the vul-
nerability of the organism to the consequences of
cholinesterase inhibition; pinpointing the consequen-
ces of long-term exposure to cholinesterase inhibi-
tion; and evaluating the developmental effects of
cholinesterase inhibition. Clinical and animal studies
will be conducted to identify what learning and
memory endpoints can be quantified in both rats and
humans. Ultimately, HERL hopes to be able to use
rats to model the potential learning deficits a chemical
may cause in humans. To facilitate animal-to-human
extrapolation, studies will be done to correlate
electrophysiological and cognitive measures in test
species and humans.
Reproductive toxicity. Tremendous gaps exist in our
knowledge of how human developmental toxicants
behave in animal models, yet many of these toxicants
have defined pharmacokinetic and phamacodynamic
profiles that could readily be compared across
species. One project being developed is a "retrospec-
tive risk assessment" to evaluate how a well-
developed animal database predicts known human
risk. Research will also be performed to develop
capabilities for quantitative dose-response modeling
of developmental effects, with the ultimate goal of
developing biologically based dose-response models.
These models will be largely statistically based in the
beginning, but will gradually incorporate more biol-
ogy, such as pharmacokinetic considerations,
mechanisms of toxicity, and the presence or absence
of thresholds. A critical component will be the estab-
lishment of confidence limits about predictive low-
dose effects. Such models will be developed first for
developmental toxicity and then for reproductive
toxicity.
Studies of developmental toxicity will examine the
significance of morphological and skeletal variants,
such as supernumerary ribs, that are commonly ob-
served in developmental studies. Issues to be ad-
dressed include determining whether the effects are
adverse and whether related effects can be noted,
studying the mechanisms for these effects at the
molecular and cellular level, and assessing whether
similar effects occur in other species. Approaches
pioneered in examining variations in kidney develop-
ment will be used as a model for these efforts. The
role of maternal stress in embryonic development will
also be examined. This research will help establish
alternative methods for setting the maximum tolerated
dose use in standard developmental assays.
The Office of Pesticide Programs has expressed con-
cern about the acute and chronic effects of pesticide
exposures on people who apply pesticides. In
response to this concern, researchers will examine the
reproductive outcomes associated with acute and
chronic exposures. HERL will investigate whether
these outcomes are adverse and/or reversible, and
what the tolerance is to exposure. Research will focus
on male reproductive effects (i.e., semen parameters)
as well as events occurring during normal estrous cy-
cling and early pregnancy. These mechanistic
studies, conducted at the cellular level, will help
determine appropriate parameters for evaluation as a
function of putative mechanisms that are triggered
under differing exposure conditions. In addition, be-
cause recent human data suggest that approximately
one-third to one-half of all pregnancies are lost in the
first two weeks after fertilization, HERL will examine
the correlation between ovarian and uterine function
in animal models and humans in an effort to develop
an animal model for pre-implantation effects. This
research will provide the basis for recommendations
on identifying hazards to early pregnancy. HERL
will also conduct research to study the sensitivity of
the nonpregnant female to disruptions in reproductive
capabilities.
Researchers in reproductive toxicology will explore
the development of different biomarkers. They will
examine endocrine parameters and semen quality as a
potential biomarkers of effect. Studies will inves-
tigate the relationship of sperm alterations to predic-
tion of altered fertility in human and test species.
Investigators will also develop biomarkers of genetic
change in human and animal cells and develop
methods for using DNA adducts as biomarkers of ex-
posure and/or effect. In particular, HERL will ex-
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iiiiiiB
amine the relevancy of DNA adducts for cancer initia-
tion. Research efforts will investigate the significance
of gap junctions (e.g., communicator regions between
cells) as potential markers for promotion and car-
cinogenesis. Other work will develop biomarkers of
allergenic potential. In particular, this research will
investigate the relationship between serum antibody
levels and pulmonary hypersensitivity. HERL scien-
tists will also develop biomarkers of increased sus-
ceptibility to infectious disease using several immune
function endpoints. Biomarkers research will also be
conducted to investigate sensitivities of susceptible
populations such as field workers, children, and preg-
nant women.
3.4.2.2 Toxic Substances
Hazard Identification. Research needs for toxic
substances are similar in nature to those for pesticides. To
address the ongoing need for improved testing methods,
HERL will conduct research to investigate the full array of
potential adverse effects from exposure to pesticides, in-
cluding"but not limited to"neurotoxic, reproductive, and
immunotoxic effects. Basic research will involve refining
existing techniques and creating new ones in these scien-
tific disciplines. Applied research efforts will involve
evaluating and interpreting industrial data (both review of
raw data and laboratory research to verify and clarify data
reported from industry).
Structure-activity relationships. Structure-activity
research will address the need for more effective
methods to screen PMN chemicals. HERL is
developing a molecular similarity index and special-
ized databases for use in SAR analysis that profile the
genetic and developmental activity of chemicals in
relation to their structures. HERL will also conduct
research to provide a quantitative basis for classifying
and prioritizing chemical structures according to their
activity. Causal molecular models will be developed
that predict the distribution, transformation, deposi-
tion, or biological activity of classes of chemicals
from computationally available molecular properties.
Additional SAR research will explore mechanisms of
action of nongenotoxic carcinogens, and developmen-
tal and reproductive toxicants, and examine correla-
tions between mechanism and activity. Mechanisms
for mutation in germ cells will also be examined in an
effort to correlate structure and activity.
Developmental toxicity. In anticipation of future
needs, HERL will initiate development and validation
of in vitro screening assays for detecting developmen-
tal hazard. A battery of assays is likely to be re-
quired, with guidance provided as to which assays are
appropriate for which chemical classes. Similar as-
says may be established for endpoints of reproductive
toxicity, particularly as they relate to the events en-
compassing spermatogenesis.
Genetic toxicology. HERL will continue the
development of test guidelines for evaluating chemi-
cally induced heritable damage in the germ line, as
well as examine the types of genetic damage that can
be caused in diverse organisms.
Immunotoxicity. Researchers will continue the
development of test guideline methods for im-
munotoxicity and develop immunotoxic endpoints as
biomarkers of susceptibility to infectious or allergic
diseases.
Genetically engineered microorganisms. To ad-
dress the research need for methods to evaluate
GEMs, research will be conducted to investigate the
health implications of exposure to GEMs. This will
include research on 1) the survival of GEMs in the in-
testines of rodents and humans; 2) the ability of
GEMs to invade other tissues and induce adverse
health effects; and 3) the genotoxic and metabolic
changes that occur following exposure to GEMs.
Dose-Response Assessment. Dose-response research
needs for toxics are similar to those for pesticides. Re-
search will be conducted to develop a better understanding
of the mechanisms underlying the effects associated with
toxics for which tests are available. Such research will
help elucidate the cellular/molecular events that are as-
sociated with changes in physiology and function. This
research will also enhance understanding of the predictive
value of the tests, and the ability to extrapolate data from
one species to another. Also, such research may ultimate-
ly lead to the development of cellular/molecular tests that
could replace or supplement existing protocols. These ef-
forts need to be ultimately linked to pharmacokinetic
models such that a complete description of exposure, dose,
and outcome can be used in the risk assessment process.
Genetic toxicology. The structure-function proper-
ties of biological receptors will be studied with the
goal of developing molecular-level models of the
mechanisms of toxicity. Research will include ex-
trapolation studies to establish the experimental basis
for relating molecular dose to DNA adducts to in-
3-24
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duced genetic effects, and to determine whether these
relationships established using laboratory animals can
be extrapolated to humans. Also, research will be
conducted to examine the validity of extrapolating
carcinogenic effects from rodents to humans, and to
establish the shape of dose-response curves. This re-
search will support the development of biologically
based dose-response models for carcinogens.
Mechanistic research will be conducted to determine
the role of environmental chemicals in the expression
of oncogenes and tumor suppressor genes. Such re-
search will establish a biological basis for developing
biomarkers of preneoplastic and neoplastic lesions in
exposed or susceptible human populations.
The use of DNA adducts or cytogenetic effects as
measures of internal or target dose will be explored.
This research is designed to improve the accuracy of
dose determinations for interspecies and route-to-
route extrapolation. Basic research will be conducted
to understand the fundamental interspecies differen-
ces between the response of rodent and human cells to
genotoxic agents. Research has already indicated that
human cells may be more capable than rodent cells of
repairing at least some DNA lesions, suggesting that
human cells may be less sensitive to genotoxic agents.
Molecular techniques will be used to define and quan-
tify the specific interspecies differences.
To address the need to understand nongenotoxic car-
cinogenesis, HERL will conduct research to identify
nongenotoxic carcinogens and their mechanisms of
action, and to develop extrapolation models for this
class of carcinogens. Areas to be highlighted are gap
junction intercellular communication, alterations in
gene expression, and indirect genotoxic car-
cinogenesis (e.g., peroxisome proliferators, free radi-
cal generators).
Developmental toxicity. Currently, developmental
research focuses on fetuses exposed in utero because
there are few acceptable procedures for evaluating ef-
fects of postnatal exposure. However, OPP is par-
ticularly interested in research addressing the
potential developmental effects in pups exposed via
lactation from mothers who were exposed either der-
mally or via inhalation. Exposing females by these
routes requires removing them from their pups at fre-
quent and prolonged intervals during the lactation
period. This maternal deprivation itself causes stress
to the pups that may confound study results. HERL
will examine the role of maternal stress on the
growth, physiology, biochemistry, and behavior of
pups. Results of this research will supplement
guidelines for developmental neurotoxicity testing.
Research will also be conducted with the goal of
developing procedures for pharmacokinetic and
biologically based dose-response assessments for
reproductive and developmental toxicity, as described
earlier.
Neurotoxicology. In support of toxic substances
dose-response research needs, HERL will conduct re-
search to better understand the human neurological
significance of exposure to toxic substances. The re-
search will focus on development of an animal model
of personality changes, learning and memory deficits
seen in humans exposed repeatedly to pesticides; the
mechanism of compensation or tolerance after
repeated exposure; and interactions of toxicants with
central nervous system depressants (tranquilizers).
Biomarkers. To meet the research need for
biomarkers of effect and exposure, research will be
conducted as described above (see Exposure Assess-
ment under Pesticides). Also gene mapping techni-
ques will be used to study genetic alterations in
human and rodent precancerous and cancerous lesions
as potential biomarkers of effect and/or exposure.
3.5 OFFICE OF EMERGENCY AND
REMEDIAL RESPONSE
Under the Comprehensive Environmental Response,
Compensation, and Liability Act (CERCLA) of 1980, and
subsequent reauthorization and amendments (Superfund
Amendments and Reauthorization Act [SARA] of 1986),
EPA is responsible for identifying sites from which
releases of hazardous substances might occur or have oc-
curred. With only a few exceptions, Superfund coverage
extends to all sources of releases and all means of entry of
a substance into the environment. Once a site is iden-
tified, the Agency must ensure that the site is cleaned up,
either by removal of the wastes or remediation.
The procedures through which Superfund is imple-
mented are outlined in the National Oil and Hazardous
Substances Pollution Contingency Plan (NCP). After the
Agency is made aware of the existence of a site, it per-
forms a site evaluation. Based upon that evaluation, the
site is slated either for a short-term removal action or
longer-term remedial action. Within predetermined inter-
vals after completion of a remedial action, EPA must
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evaluate the continued effectiveness of the action through
a post-closure review.
The OHR/HERL research program relating to Super-
fund is structured to address the special health research
needs of the site evaluation, removal, remedial, and post-
closure review programs. At an early stage in the Super-
fund process, the Agency for Toxic Substances and
Disease Registry (ATSDR) performs a health assessment
for each site. Based on this assessment, ATSDR may per-
form an epidemiological study. ATSDR's decision about
whether and when to perform an epidemiological study is
relatively independent of the EPA cleanup process. Be-
cause EPA and ATSDR interact, both formally and infor-
mally, during these processes, however, OHR/HERL
research program results may provide direct input to
ATSDR epidemiology studies. The OHR/HERL program
in this area is new; thus, details of program implementa-
tion have not yet been fully determined.
Site Evaluation Program. This program, sometimes
called the preremedial program, supports both the removal
and remedial programs by conducting preliminary assess-
ment (PA) and site inspection (SI) activities. Removal
PAs are based on readily available information, which
may include the preliminary ATSDR health assessment, if
available. Removal Sis are performed if more information
is needed, and if the National Contingency Plan does not
specify collection of any particular data for these ac-
tivities. No risk assessment is performed, although some
estimate of the magnitude of the threat is made.
Remedial PAs are similar to Removal PAs. If a
Remedial SI is performed, the lead agency collects addi-
tional data necessary to apply the Hazard Ranking System
(HRS); even more data are collected if necessary to better
characterize the release and facilitate the effective and
rapid initiation of the remedial investigation/feasibility
study (RI/FS; see Section 3.5.1.3). These activities fall
primarily under hi. In general, they are based on compar-
ing what is discovered about conditions at a specific site
against what is already known about contaminants and en-
vironmental levels of concern for those contaminants (in
the form of RfDs, cancer potency factors, and LDso).
When, as often happens, sufficient data are not available
for the specific contaminants, evaluations may rely upon
structure-activity relationships.
Removal Program. The removal program, which is
limited (with a few exceptions) by statute to one year and
$2 million, is geared toward mitigating, abating, minimiz-
ing, stabilizing, or eliminating a release or threat of a
release that may threaten public health, welfare, or the en-
vironment Among the criteria used to determine whether
a removal action is necessary are:
Actual or potential exposure of human populations,
animals, or the food chain to hazardous substances,
pollutants, or contaminants
High levels of hazardous substances, pollutants, or
contaminants in soils largely at or near the surface
that may migrate (proposed section 300.415 of NCP)
This information is obtained from site evaluations and
knowledge of the current site conditions.
An important concern in managing a removal opera-
tion is community relations. A designated spokesperson is
assigned to inform the community of actions taken,
respond to inquiries, and provide information concerning
the release. EPA must have accurate and appropriate in-
formation to provide to the community on the potential
risks associated with any site and on how the data should
be interpreted. At a minimum, EPA should be able to
communicate 1) information about exposure and the
relationship between ambient and internal exposure; and
2) information about the relationship between magnitude
of exposure and health effects (dose-response issues).
Based on the hazard assessment and other considera-
tions, ATSDR may consider conducting one of several
types of human health (e.g., epidemiology) studies at a site
at which a removal action is scheduled. A primary
criterion in ATSDR's decision to perform either an ex-
posure study or an epidemiological study is the
availability of an adequate test or biomarker of exposure:
the ability to incorporate biomarkers of exposure into a
study allows much more accurate assignment of the study
population to groups.
Remedial Program. This program implements long-
term solutions (i.e., reduces controls, or eliminates risks to
human health and the environment) to problems posed by
a site. A central part of this program is the RI/FS, which
assesses the potential risk to human health posed by the
site if no action is taken (baseline risk assessment), as well
as the relative risks likely to result following various
cleanup options.
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Factors considered in assessing risk at a site include:
The nature (e.g., toxicity, bioaccumulation potential),
medium, and concentration of substances
The location of nearby sensitive populations
When systemic toxicants are present, cleanup goals are set
so that even sensitive subgroups of the human population
could be exposed to the remaining toxicants for a lifetime
without appreciable risk of significant adverse effect.
When known or suspected carcinogens are present,
cleanup goals are set to ensure that concentration levels
represent an excess upper-bound lifetime cancer risk to an
individual of between 10"4 and 10~7, using information on
dose/response relationships.
Several features of the risk assessments performed
during the RI/FS make them atypical of other risk assess-
ments performed in the Agency. First, the hazard iden-
tification stage of the risk assessments may have to
address the potential toxicity of chemicals for which little
data are available. Thus, knowledge of SARs is par-
ticularly helpful. Second, if bioremediation is being con-
sidered as a cleanup option, the feasibility study may need
to evaluate the potential hazard posed by the microor-
ganisms (or substances they produce) employed in the
process. These two concerns are hazard identification re-
search issues.
In terms of dose-response relationships, risk assess-
ments performed as part of the RI/FS must consider the
pollutant mixtures found at most sites rather than in-
dividual pollutants. Assumptions must be made about how
to evaluate risk associated with exposure to mixtures.
Second, Superfund risk assessments must consider multi-
ple pathways/routes of exposure. For this reason,
knowledge about route-to-route extrapolation and
dosimetry are of particular importance.
As with the removal program, the remedial program
requires a community relations plan that handles public in-
formation needs appropriately. Again, EPA must have ac-
curate and appropriate information to provide to the
community on the potential risks associated with the site
and on the appropriate interpretation of the data available.
At a minimum, EPA should be able to communicate infor-
mation concerning 1) exposure and the relationship be-
tween ambient and internal exposure, and 2) the
relationship between magnitude of exposure and health ef-
fects.
In remedial as in removal situations, ATSDR may
consider conducting epidemiological studies. Again, the
availability of biomarkers of exposure is critical to that
decision: without them, the study population cannot be ac-
curately assigned into groups.
Post-Closure Program. The CERCLA amendments
require reviews at least every five years at sites where
remedial actions leave hazardous substances, pollutants, or
contaminants above levels that allow for unrestricted use
and unlimited exposure for humans. If a selected remedy
is no longer considered protective (e.g., standards have
changed), EPA will evaluate and take additional action at
affected sites to mitigate the threat. No procedures have
yet been promulgated to accomplish these post-closure
reviews, and the development of such procedures is an
emerging issue for the program.
In many respects, post-closure activities may be
viewed as pollutant-monitoring activities. Biomonitoring
tools are some of the most promising for ensuring safety at
remediated sites, at least until further advances in the fol-
lowing areas:
Predictions of pollutant transformation in the environ-
ment
Monitoring of all likely pollutants
Predictions of the health impact of exposure to
various mixtures of pollutants
These biomonitors would serve as sentinels for hazard
identification at the site.
3.5.1 Office-Specific Research Needs
3.5.1.1 Site Evaluation Program
Health research data are needed to increase the num-
ber of chemicals for which hazard potential may be ac-
curately identified.
3.5.1.2 Removal Program
Dose-Response and Exposure Assessment. Because
of the typically time-critical nature of its work, the
removal program relies on generally available informa-
tion. Health research is needed to provide background
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data that the Agency can use to interpret for the public the
relationship between:
Ambient and internal exposure
Internal exposure and risk of adverse health effects
In addition, health research is needed to provide
biomarkers of exposure and effect so that epidemiologic
studies can be performed when appropriate.
3.5.1.3 Remediation Program
Dose-Response and Exposure Assessment. Health
research is needed to improve:
The baseline risk assessments that are part of the
RI/FS
The risk assessments included in the evaluation of
cleanup options
Availability and interpretability of biomarkers of ex-
posure and effect
3.5.1.4 Post-Closure Program
Research needs in this area include the develop-
ment/validation of test systems for in situ biomonitoring.
3.5.2 Research Plan
3.5.2.1 Site Evaluation Program
Work in this area falls under the category of hazard
identification. HERL research efforts in site evaluation
are focused on improving the database used to identify the
hazard potential of constituents. In support of that goal,
research will concentrate on improving the use of SAR for
identifying hazard; this strategy was chosen because it is
the most efficient means to the desired end when data are
unavailable and testing cannot be mandated. In the short
term, efforts will be directed to genetic toxicity as an
endpoint, because of the substantial database available
from which to develop SAR theories and models. As
more data become available, the SAR approach will be ex-
tended to other forms of toxicity, including developmental
toxicity and neurotoxicity.
3.5.2.2 Removal Program
Dose-Response and Exposure Assessment. Re-
search in this area is focused on these goals: 1) providing
information to the Agency that can be used in the com-
munity relations program to explain and interpret the sig-
nificance of site-specific data, and 2) providing
information about biomarkers for incorporation into site-
specific human health studies. To achieve these goals,
work will concentrate on biomarkers, particularly in terms
of exposure and dose-response assessment. This strategy
was chosen because biomarker research will foster a better
understanding of the relationship between ambient and in-
ternal exposure (i.e., dose to the target tissue), and be-
tween internal exposure and effect. This information will
be useful in the community relations program.
Biomarkers of exposure could be used to evaluate ex-
posure to any of the hundreds of chemicals of concern at
Superfund sites. ATSDR health assessments (from the
288 NPL sites evaluated since enactment of SARA) indi-
cate that the most commonly found classes of substances
are:
Volatile organic compounds (VOCs) (64 percent of
sites)
Metals (47 percent of sites)
Polycyclic aromatic hydrocarbons (PAHs) (27 percent
of sites)
Polychlorinated biphenyls (PCBs) (25 percent of
sites)
HERL/OHR work will focus on substances 1) that are
most likely to be found at Superfund sites, and 2) for
which valid biomarkers of exposure are currently not
available. Identification of valid exposure biomarkers,
coupled with knowledge of their kinetics in tissues, cells,
and fluids, will not only facilitate the documentation of
exposure, but will allow researchers to reconstruct ex-
posure history and make quantitative estimates of ex-
posure.
Removal rather than remediation is likely to occur at
sites where exposures could be relatively high for a rela-
tively short period of time. At these sites, public concern
may be high. Human health studies, if initiated, would
benefit from objective measures of toxic effect; these
could be made in humans and could be related to indices
of exposure. While many endpoints are important (e.g.,
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neurotoxicity, reproductive toxicity), research in this area
will focus on those that are 1) of greatest concern from a
short-term exposure perspective (since in the removal pro-
gram exposures are likely to be relatively short in dura-
tion), and 2) most likely to reveal significant gains with
limited resources and limited time.
The field that appears to best fill these criteria is
reproductive toxicology. Concern about reproductive dys-
function is high, problems in this area are likely to be
manifest after reasonably short exposures, and test
methods are available for evaluating male reproductive
function in both humans and laboratory animals. Sig-
nificant research needs in this area include:
Validation of computer-assisted analysis of sperm as
a method for quantitative assessment of sperm
motility and morphology
Evaluation of the sensitivity of sperm analysis to
toxicant-induced disruption
Development of a better understanding of the
relationship between alterations in sperm and
reproductive dysfunction
In addition to serving as a biomarker of reproductive ef-
fect, sperm may provide a host for chemical adducts,
which could be used as biomarkers of male reproductive
system exposure to environmental chemicals. Future
work may focus on development of sperm adducts as
biomarkers of dose to a target tissue.
3.5.2.3 Remediation Program
Research in this area is focused on improving the risk
assessments performed during the RI/FS, and on improv-
ing the availability and interpretability of biomarkers of
exposure and effect. Work will concentrate on those
aspects of risk assessment that are relatively unique to the
Superfund process, and on biomarker research that has a
particularly high probability of being useful in human
health studies at Superfund sites. Through this strategy,
research efforts will be directed to three risk assessment
activities: hazard identification, exposure assessment, and
dose-response assessment.
Hazard Identification. Work in this area will focus
on developing SAR.
Dose-Response and Exposure Assessment. When
ambient concentrations are high enough that humans have
been or are being exposed, biomarkers of exposure can be
used to document that exposure. Most useful would be
biomarkers that allow exposure to pollutant mixtures to be
evaluated; some work will be done in this area, particular-
ly with protein adducts as the biomarkers under study.
The first protein adducts to be examined will be those
produced by exposure to PAHs; this class of compounds is
often found at Superfund sites, and the data available sug-
gest that this research may be fruitful. Subsequent efforts
will address exposure biomarkers for other classes of com-
pounds.
Three research issues will be explored in dose-
response assessment: route-to-route extrapolation,
biomarkers (for target dose), and pollutant mixtures.
Route-to-route extrapolation: Often data are only
available for one route of exposure, but plausible ex-
posure scenarios suggest multiple pathways and/or
routes. In the short term, research will focus on
dosimetry via the inhalation route of exposure. In the
longer term, research will focus on the relationship
between oral and inhalation exposure. Since
dosimetry is age-dependent, near-term research will
address the principles underlying this age-dependence
(e.g., geometry of the airways).
Biomarkers: Just because exposure has occurred
does not necessarily mean that the chemical(s) in
question reached its target site(s). Understanding the
relationship between exposure and dose to the target
site can reduce significantly the uncertainty in Super-
fund risk assessments. As part of the HERL
biomarkers strategy, research efforts in this area will
focus on already-developed biomarkers of target dose
(i.e., DNA adducts) to determine 1) their life in tis-
sues, 2) relationship to exposure, 3) levels in human
tissue, and 4) relationship to genetic effects. To the
extent possible, this work will proceed using in vitro,
laboratory animal, and human studies.
Pollutant mixtures: Most Superfund sites contain
mixtures of pollutants. As a result, risk assessments
performed during the RI/FS are chemical mixture risk
assessments, and research needs in this area em-
phasize the importance of mixtures. The Superfund
pollutant mixtures research strategy represents an in-
tegral part of the HERL mixtures research strategy.
To meet Superfund needs, HERL will perform re-
search directed at understanding the validity of the
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current assumption that the effects of components in a
mixture are additive.
Much of the pollutant mixtures work to date has
focused on short-term in vitro tests of specific genetic
endpoints, mostly following exposure to combustion
emissions. In the long term, this scope will be
broadened through an emphasis on in vivo exposures
and other (e.g., systemic) endpoints. In addition, re-
search will focus on mixtures likely to be found at Su-
perfund sites, and on the comparative potency of
residual toxicity following various cleanup options.
3.5.2.4 Post-Closure Program
Work in this area falls under hazard identification.
Present methods for in situ biomonitoring focus on 1)
genetic endpoints, and 2) plant-based systems. In the near
term this focus will continue. Plant-based test systems
must be validated against more conventional test systems.
In addition, field studies must be validated against
laboratory studies that use 1) pure chemicals, and 2) field
samples. In the long term, efforts will turn toward ensur-
ing that biomonitoring tools developed for application
during post-closure review are protective against other
forms of toxicity as well as genotoxicity. This new goal
will almost certainly necessitate exploration of animal-
based test systems.
3.6 OFFICE OF SOLID WASTE
Under the Resource Conservation and Recovery Act
(RCRA) and its amendments, EPA manages a comprehen-
sive program to address the national problem of solid
waste. The program is designed to:
Protect human health and the environment from the
potential hazards of mismanaged waste
Conserve energy and natural resources
Reduce the amount of waste generated
Ensure that wastes are managed in an environmental-
ly sound manner
RCRA applies to all solid waste, which is broadly defined
as "garbage, refuse, or sludge or any other waste material,"
including liquids, solids, semisolids, or contained gases.
The criteria applied for managing a particular waste
depend on its characteristics. To accomplish the mandate
specified by RCRA and subsequent amendments, the Of-
fice of Solid Waste (OSW) must first characterize wastes.
This characterization then determines which program
covers the management of that waste"either Subtitle C
(hazardous waste) or Subtitle D (nonhazardous waste).
OSW programs are designed to track, permit, and enforce
requirements for waste management, OSW cannot request
health research from the regulated community, although it
may require industry to test the "characteristics" of the
waste. The OHR/HERL research program is structured to
fill that gap by addressing the special health research
needs associated with waste characterization, hazardous
waste, and nonhazardous waste. Because the research
program is new, details of program implementation have
not yet been fully determined.
Waste Characterization. Waste characterization is a
pivotal feature of the hazardous and solid waste program:
the results of this activity determine the regulatory path
taken by the waste. Wastes are defined as hazardous on
the basis of either
Their "characteristics"
Federal listing (in Appendix VIII of 40 CFR Part 261)
One of the characteristics used is toxicity, which was
previously defined according to the extraction procedure
(EP). This test evaluates the likelihood of any of 14 toxic
contaminants leaching from the waste placed in landfills.
A revision of the EP, the toxicity characteristic leaching
procedure (TCLP), allows 29 additional waste con-
stituents, mostly chlorinated compounds and solvents, to
be analyzed. The TCLP defines toxicity on the basis of
substance concentration (mg/L). The added compounds
were chosen primarily on the basis of available chronic
toxicity reference levels (RfDs for noncarcinogens and
RSDs for carcinogens), coupled with fate and transport
data.
Wastes are placed on the federal listing if they contain
substances that have been shown to have "toxic, car-
cinogenic, mutagenic or teratogenic effects on humans or
other life forms." Waste may also be listed if acute ex-
posure in low concentrations is likely to produce lethality
(acute LDso is < 50 mg/kg, acute LCso is < 2 mg/L, or
dermal LDso is < 200 mg/kg) or "is otherwise capable of
causing or significantly contributing to an increase in
serious irreversible, or incapacitating reversible, illness."
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Hazardous Waste (Subtitle C Program). This pro-
gram tracks hazardous waste from cradle to grave, and
sets standards for hazardous waste treatment, storage, and
disposal facilities (TSDFs). With few exceptions, TSDFs
must obtain permits; to do so, they must meet different
sets of standards for containers, tank systems, surface im-
poundments, waste piles, land treatment, landfills, and in-
cinerators. Ideal handling of hazardous wastes results in
complete disposal; standards become particularly impor-
tant, however, because treatment/disposal is typically in-
complete. For example, incineration results in products of
incomplete combustion (PICs), which are either released
into the environment or remain at the facility for sub-
sequent management (e.g., as ash).
Permitting hazardous waste treatment facilities re-
quires public hearings, which in turn catalyze an informal
risk assessment process to inform the public of the risk
posed by the site. Two features of these risk assessments
make them unique: the potential for multimedia ex-
posures and the potential for exposures to chemical mix-
tures.
Nonhazardous Waste (Subtitle D Program). The
Subtitle D rule, proposed in 1988, will include require-
ments for facility design and ground-water monitoring.
As with hazardous waste, incineration (municipal waste
combustion) is becoming an increasing popular treatment
method.
3.6.1 Office-Specific Research Needs
3.6.1.2 Hazardous Waste
Data are needed to improve the quality of the infor-
mal risk assessments performed. In particular, research is
needed to improve risk assessments that take into account:
Multimedia exposures
The potential for exposures to chemical mixtures
3.6.1.3 Nonhazardous Waste
Before siting a municipal waste combustor (MWC),
the associated risk must be assessed (though EPA uses an
informal process for these assessments). As with the Haz-
ardous Waste Program, health research can improve these
risk assessments. The risk from MWC emissions comes
from both primary exposure to emissions and from secon-
dary exposure"for example, through the food chain.
Specific health research needs include:
Determining the bioavailability of incinerator emis-
sions via the various exposure pathways
Evaluating the toxicity of the mixtures of PICs that
characterize incinerator emissions
Evaluating the comparative potency approach to per-
forming risk assessments on such complex mixtures
as incinerator emissions
3.6.1.1 Waste Characterization
Health data are needed to ensure that wastes are
properly characterized. In the context of the risk assess-
ment paradigm, this is a hazard identification problem. Of
particular value would be:
Methods for rapidly characterizing acute toxicity of
waste
RfDs for more noncarcinogens
RSDs for more carcinogens
A procedure for developing RSDs for noncarcinogens
3.6.2 Research Plan
3.6.2.1 Waste Characterization
Work in this area falls under the category of hazard
identification. Four research needs were identified in Sec-
tion 3.6.1; of these, the first (methods for rapidly charac-
terizing acute toxicity of waste) depends most directly on
laboratory research. The other three are best met by
reviewing and modeling existing data. Research efforts in
this area, therefore, are concentrated on improving
methods for rapidly categorizing the acute toxicity of haz-
ardous wastes. This effort, which will be relatively small,
will begin by pinpointing the research needs and then will
address those needs to the extent that they meet the criteria
listed above. Endpoints that may be investigated include
the use of in vitro tests for determining cytotoxicity as
well as cytotoxicity as a predictor of acute toxicity.
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3.6.2.2 Hazardous Waste
Work in this area falls under the category of dose-
response assessment. These research efforts are designed
to improve the health database from which risk assess-
ments are performed. Risk assessments, which are per-
formed through an informal process, must take into
account:
Multimedia exposures and the relationship between
environmental concentration and dose to the target for
each exposure route (to evaluate the dose-response
relationship through pharmacokinetics and dosimetry)
Pollution mixtures, particularly of metals
Thus, hazardous waste research is focused on these topics.
Because hazardous wastes are often deposited on soil,
research efforts must consider the possible contribution of
exposure through soil ingestion"a route of exposure that is
particularly important for children. In addition, many
compounds (e.g., lead) are known to be more toxic to
children than adults. Presently, 100 percent of toxicants
(e.g., metals) is assumed to be absorbed from soil. Con-
sensus of opinion suggests that this assumption is invalid;
but no agreement has been reached on how it should be
modified. Furthermore, no agreement exists on the extent
to which age affects bioavailability of substances from the
gut.
and open-fire smoky coal emissions) and lung cancer. Be-
cause incinerator emissions are likely to contain the kinds
of chemicals found in these other complex emissions, the
focus of research is on evaluating the relative potency of
MWCs compared to other combustion sources. The re-
search will place MWC emissions into a comparative
potency framework with other combustion emission sour-
ces (e.g., hazardous waste incineration, hospital waste in-
cineration). Based on these studies, OHR will be able to
provide the Agency with advice on how (or if or when) to
use the comparative potency approach to evaluate the
health risk of complex mixtures.
In addition to predicting effects, risk assessments
must also evaluate the dose of emissions that individuals
are likely to receive. While modeling and monitoring ef-
forts may allow prediction of ambient concentrations, ad-
ditional tools (e.g., biomarkers) are necessary to estimate
the dose. This research strategy, therefore, includes a
small-scale effort to develop exposure biomarkers that can
be used to estimate dose in a complex mixture context.
In the short term, research efforts will address these
issues, with particular emphasis on metals. In the longer
term, work will concentrate on two related pollutant mix-
ture issues: 1) how the presence of metals affects the ab-
sorption of other metals, and 2) the extent to which the
assumption of additivity of risk is valid for metals. This is
a new research program that will take shape following an
effort to identify and prioritize the knowledge gaps.
3.6.2.3 Nonhazardous Waste
Research in this area falls under dose-response assess-
ment. Research efforts will evaluate combustion emis-
sions using the comparative potency approach. This
approach may be viewed as a form of dose-response as-
sessment for pollutant mixtures, though efforts to develop
and validate the method fall under hazard identification
work. The link between in vitro/in vivo bioassay data and
human cancer following exposure to exogenous agents has
been demonstrated for certain complex emission sources
(e.g., roofing tar, cigarette smoke condensate, coke ovens,
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