EPA 600/R-03/069

September 2003

National Health and Environmental Effects Research Laboratory

Human Health Research Implementation Plan

US Environmental Protection Agency 

National Health and Environmental Effects Research Laboratory

Office of Research and Development

Research Triangle Park, NC 27711
 ii

Foreword

The National Health and Environmental Effects Research Laboratory (NHEERL), as part of the

Environmental Protection Agency’s Office of Research and Development (ORD), is responsible

for conducting research to improve the risk assessment of chemicals for potential effects on

human health. This research is intended to address key Agency problems in a timely and

responsive manner. To meet this responsibility, NHEERL is developing research

implementation plans to achieve the following objectives:

C Optimizing responsiveness of research activities to Agency needs,

C Sharpening the focus of research programs where needed,

C Providing a forum for engagement of scientific staff on issues and approaches,

C Focusing on multi-year planning explicitly linked to Agency performance goals,

and

C Providing a mechanism for prioritizing research.

This approach builds on the ORD planning process that identifies and prioritizes research needs.

Current areas for research include protection of susceptible subpopulations, harmonization of

risk assessment approaches, and improving aggregate and cumulative risk assessment in support

of the Agency’s Program and Regional Offices and legislative mandates, including the Safe

Water Drinking Water Act, Clear Air Act, Toxic Substances Control Act, and Federal Insecticide

Fungicide and Rodenticide Act. 

This document identifies the scientific problems and research that will be conducted concerning

human health. The ultimate goal of this research is to develop scientifically valid approaches for

improving human health risk assessment. This document was developed by representatives from

NHEERL research divisions and peer-reviewed by scientists from other ORD laboratories and

centers and EPA Regional and Program Offices. This document was also reviewed by scientists

external to the Agency. This implementation plan is intended to reflect research that will be

conducted over the next several years. As progress is made in achieving the goals outlined in

this document, it will be updated to address new and remaining human health challenges.

Lawrence W. Reiter

Director

National Health and Environmental Effects Research Laboratory
 iii

TABLE OF CONTENTS

Page

Foreword .........................................................................................................................................ii

List of Figures..................................................................................................................................v

Acknowledgments ..........................................................................................................................vi

NHEERL Human Health Research Executive Committee............................................................vii

Peer Review .................................................................................................................................viii

Acronyms .....................................................................................................................................ix

Executive Summary ................................................................................................... .................E-1

Section 1 Introduction .................................................................................................................1-1

Section 2 Research Approach......................................................................................................2-1

Section 3 Harmonization of Cancer and Non-Cancer Risk Assessment.....................................3-1

3.1 Problem.......................................................................................................................3-1

3.2 Goals...........................................................................................................................3-2

3.3 Critical Path................................................................................................................3-3

3.4 Program Projects ........................................................ ...............................................3-4

3.5 Gap Analysis and Links to Other Multi-Year Plans.................................................3-18

3.6 References..................................................................................................................3-21

Section 4 Susceptible Subpopulations.........................................................................................4-1

4.1 Problem.......................................................................................................................4-1

4.2 Goals...........................................................................................................................4-1

4.3 Critical Path................................................................................................................4-2

4.4 Program Projects .......................................................................................................4-3

4.5 Gap Analysis and Links to Other Multi-Year Plans.................................................4-36

4.6 References...................................... ..........................................................................4-38

Section 5 Cumulative Risk............................................................................................. .............5-1

5.1 Problem.......................................................................................................................5-1

5.2 Goal ............................................................................................................................5-2

5.3 Critical Path................................................................................................................5-3

5.4 Program Project ............................................. ............................................................5-3

5.5 Gap Analysis and Links to Other Multi-Year Plans...................................................5-7

5.6 References ..................................................................................................................5-8
 iv

Appendix A Research Allocation for Program Projects ..........................................................A-1

Appendix B Annual Performance Goals and Measures for Harmonization ..............................A-2

Appendix C Annual Performance Goals and Measures for Susceptible 

 Subpopulations ......................................................................................................................A-4

Appendix D Annual Performance Goals and Measures for Cumulative Risk ...........................A-8
 v

List of Figures

 Page

Figure 1 Mandates for Human Health Research..................................................................2-2

Figure 2 The Source-to-Outcome Continuum.....................................................................2-3

Figure 3 Critical Path for Harmonization Research ............................................................3-3

Figure 4 Critical Path for Research on Cell Signaling.........................................................3-6

Figure 5 Critical Path for Research on Luteinizing Hormone.............................................3-11

Figure 6 Critical Path for Research of P-450 and XMEs.....................................................3-16

Figure 7 Framework for Use of Mechanistic Data...............................................................3-19

Figure 8 Critical Path for Research on Susceptible Subpopulations....................................4-2

Figure 9 Critical Milestones for Research on Children’s Health.........................................4-5

Figure 10 Critical Path for Research on Windows of Vulnerability......................................4-11

Figure 11 Critical Milestones for Research on Developmental Environment.......................4-14

Figure 12 Critical Path for Research on Susceptibility Associated with Older Adults.........4-20

Figure 13 Critical Path for Research on Asthma...................................................................4-25

Figure 14 Critical Path for Research on Oxidative Stress......................................................4-29

Figure 15 Critical Path for Research on Genetic Polymorphisms.........................................4-34

Figure 16 Critical Path for Research on Cumulative Risk ....................................................5-2
 vi

Acknowledgments

Preparation of the NHEERL Human Health Implementation Plan was coordinated by the

NHEERL Human Health Research Executive Committee whose members are listed on the next

page. Research themes for the proposals described in this document were developed at a

scientist-to-scientist meeting held at Research Triangle Park in November 2001. Participants at

that meeting included scientists from NHEERL, as well as scientists from other ORD

laboratories and Centers which include:

•National Center for Environmental Assessment (NCEA),

•National Center for Environmental Research (NCER),

•National Exposure Research Laboratory (NERL), and the

•National Risk Management Research Laboratory (NRMRL).

Scientists from several Regional Offices, the Office of Air and Radiation, Office of Water, and

the Office of Prevention, Pesticides, and Toxic Substances were also in attendance. Based on

recommendations generated from the scientist-to-scientist meeting, research proposals were

generated and reviewed by the NHEERL Human Health Research Executive Committee, as well

as the following non-NHEERL scientists: 

•Rick Hertzberg (Region 4), 

•Kim Hoang (Region 9), 

•Marc Rigas (NERL), Chris Saint (NCER), 

•Jennifer Seed (Office of Toxic Substances), 

•Sophia Serda (Region 9), 

•Linda Sheldon (NERL), 

•Michel Stevens (NCEA), 

•Cynthia Sonich-Mullin (NCEA), and

•Edward Washburn (Office of Science Policy).
 vii

NHEERL Human Health Research Executive Committee

Environmental Carcinogenesis Division

R. Julian Preston

Andrew Kligerman

Experimental Toxicology Division

Linda S. Birnbaum

Hugh A. Barton (Chair)

Jane Ellen Simmons

Human Studies Division

John Vandenberg

Tony Huang

Neurotoxicology Division

Stephanie Padilla

Stan Barone

Reproductive Toxicology Division

Robert Kavlock

David Dix

Research Planning and Coordination Staff 

Hugh A. Tilson
 viii

PEER REVIEW

Dr. Vicki Dellarco, U. S. EPA Office of

Prevention, Pesticides, and Toxic

Substances, Washington, DC

Dr. George Daston, Proctor & Gamble,

Miami Valley Laboratories, Cincinnati, OH

Dr. Alan Ducatman, Chair, Department of

Community Medicine, West Virginia

University School of Medicine,

Morgantown, WV

Mr. Ravi Rao, U. S. EPA Region 4, Atlanta,

GA

Dr. Winona Victery, U. S. EPA Region 9,

San Francisco, CA

Dr. James Gibson, East Carolina University,

Brody School of Medicine, Department of

Pharmacology and Toxicology, Greenville,

NC

Dr. Richard B. Schlesinger, Department of

Biological Sciences, Pace University,

Pleasantville, NY

Dr. Melvin Andersen, CIIT-Centers for

Health Research, Research Triangle Park,

NC

Dr. Ed Carney, Technical Leader,

Developmental & Reproductive Toxicology,

The Dow Chemical Company, Midland, MI

Dr. Jay Goodman, Michigan State

University, Department of Pharmacology &

Toxicology, East Lansing, MI

Dr. Moiz Mumtaz, ATSDR, Division of

Toxicology, Atlanta, GA

Dr. Penelope Fenner-Crisp, Risk Sciences

Institute, ILSI, Washington, DC

Dr. William Slikker, National Center for

Toxicological Research, Division of

Neurotoxicology, Jefferson, AR

Dr. John Moore, Hollyhouse, Inc.,

Arlington, VA

Dr. Diana M. Wong, U. S. EPA Office of

Water, Washington, DC

Dennis Pagano, U. S. EPA Office of Air and

Radiation, Research Triangle Park, NC

Dr. George Lambert, Director, Clinical

Research Center, UMDNJ-Robert Wood

Johnson Medical School, Rutgers

University, Piscataway, NJ

Dr. Marion Ehrich, Virginia-Maryland

Regional College of Veterinary Medicine,

Department of Biomedical Sciences and

Pathobiology, Virginia Polytechnic Institute

and State University, Blacksburg, VA

Dr. Michael I. Luster, NIOSH, Chief,

Toxicology & Molecular Biology Branch,

Morgantown, WV

Dr. Elizabeth G. Marshall, Department of

Pediatrics, UMDNJ-RWJMS, New

Brunswick, NJ
 ix

Acronyms

ADHD Attention Deficit Hyperactivity Disorder

AMD Age-Related Macular Degeneration

APG Annual Performance Goal

APM Annual Performance Measure

As Arsenic

BBDR Biologically Based Dose Response

CCL Contaminant Chemical List

CDC Centers for Disease Control and Prevention

ChE Cholinesterase or acetylcholinesterase 

CNS Central Nervous System

COPD Chronic Obstructive Pulmonary Disease 

CVD Cardiovascular Disease

CYP Cytochrome P-450 

2,4-D 2,4-Dichlorophenoxy Acid 

DEHP Diesel Exhaust Particles

DBP Disinfection By-Product

EBIFs Ergosterol Biosynthesis Inhibiting Fungicides

EDC Endocrine Disrupting Chemical

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FQPA Food Quality Protection Act

FSH Follicle Stimulating Hormone

GJC Gap Junction Communication

(-GCS (-Glutamyl Cysteine Synthetase/Ligase 

GnRH Gonadotrophin-Releasing Hormone 

GPRA Government Performance and Results Act

GST-O Glutathione-S-Transferase Omega 

HAAs Haloacetic Acids

HAPs Hazardous Air Pollutants

HO-1 Heme Oxygenase-1

iAs Inorganic Arsenic

IgE Immunoglobulin E

ICC Interagency Coordinating Committee

IUGR Intrauterine Growth Retardation

LH Luteinizing Hormone

LTGs Long-Term Goal

MAPK Mitogen-activated protein kinase

MOA Mode of Action 

MYPs Multi-Year Plans

NCEA National Center for Environmental Assessment

NCER National Center for Environmental Research

NCS National Children’s Study

NE Noradrenergic

NERL National Exposure Research Laboratory

NHEERL National Health and Environmental Effects Research Laboratory

NIH National Institutes of Health

NTFs Neurotrophic Factors
 x

NOAEL No-Observed-Adverse-Effect-Level

NOEL No-Observed-Effect-Level

NQO-1 NADPH Quinone Oxidoreductase-1

NRC National Research Council

NRF2 Nuclear Factor Erythroid-Derived 2, Like 2

NRMRL National Risk Management Research Laboratory

OCHP Office of Children’s Health Protection

OP Organophosphate

OPP Office of Pesticide Programs

OPPTS Office of Prevention, Pesticides, and Toxic Substances

ORD Office of Research and Development

OW Office of Water

PBPK models Physiologically Based, Pharmacokinetic Models

PCBs Polychlorinated Biphenyls

PCR Polymerase Chain Reaction

PD models Pharmacodynamic Models

PK models Pharmacokinetic models

PKC Protein Kinase C

PM Particulate Matter

POD Point of Departure

RfD Reference Dose

RPE Retinal Pigmented Epithelium

ROS Reactive Oxygen Species

SAR Structure Activity Relationship

SH Rats Spontaneous Hypertensive Rats

STAR Science to Achieve Results

TSH Thyroid Stimulating Hormone

TSCA Toxic Substances Control Act

US United States

WKY Rats Normotensive Wistar-Kyoto Rats

XMEs Xenobiotic Metabolizing Enzymes
 E-1

Executive Summary

This document describes the

implementation plan for human health

research within the National Health and

Environmental Effects Research Laboratory

(NHEERL) for the next 8-10 years

beginning in FY02. Human health research

in the Office of Research and Development

(ORD) of the Environmental Protection

Agency (the Agency) is based on needs and

goals established by the Agency in response

to the Government Performance and Results

Act (GPRA). The human health research

described in this document aims to provide

broad, fundamental scientific information

that will improve understanding of problemdriven issues arising from risk assessment in

the Agency’s Program and Regional

Offices. Human health research supports

the identification of fundamental

mechanisms of toxicity and development of

methods and models to elucidate key

scientific uncertainties important to

understanding and predicting the effects of

environmental agents on human health. 

This implementation plan on human

health research provides a detailed approach

that NHEERL will take to address scientific

uncertainties described in the ORD’s Human

Health Research Strategy document, which

includes harmonizing the use of mechanistic

data in risk assessment, protecting the health

of susceptible subpopulations, and

determining risk of exposure to multiple

chemicals. This document also outlines in

detail how NHEERL’s human health

research will meet the Annual Performance

Goals (APGs) and Annual Performance

Measures (APMs) described in ORD’s

Human Health Research Multi-Year Plan.

NHEERL’s human health program is

organized around program projects, which

are multi-investigator, multi-disciplinary

efforts designed to address major research

themes. Programs projects were developed

by teams and subject to internal and external

review.

Research on Harmonization of Cancer and

Non-Cancer Risk Assessments

The assessment of health risk from

exposures to environmental agents has

traditionally depended on whether the

response is a cancer or non-cancer health

effect. However, there is a growing

consensus that there is a need to develop a

consistent, flexible set of principles for

using and drawing inferences from scientific

information in risk assessment. NHEERL’s

research in this area will focus on

understanding the biological events that

precede toxic or adverse effects and

identifying common or similar modes of

action across cancer and non-cancer

endpoints that could provide the basis for a

harmonized approach for risk assessment.

Mechanistic information is also crucial for

reducing or replacing uncertainty factors in

risk assessment, especially for interspecies

extrapolation and for linking dosimetry

models such as pharmacokinetic (PK)

models with empirical or pharmacodynamic

(PD) models for effects of chemicals having

similar or differing modes or mechanisms of

action. Three program projects were

identified: (1) research on cell signaling

pathways, (2) neuroendocrine mechanisms,

and (3) P-450 and xenobiotic metabolizing

enzymes. 

Susceptible Subpopulations

The variability in responsiveness of

humans to environmental pollutants can be

associated with differences in biological

susceptibility arising from intrinsic factors

such as life stage and genetics or with

acquired factors such as disease. 

Approaches to better identify and
 E-2

characterize susceptible subpopulations by

describing the biological basis underlying

differential responsiveness is an overarching

goal of NHEERL. Research on susceptible

subpopulations will focus on developing a

scientific understanding of the biological

basis for differing responsiveness of

subpopulations within the general

population. NHEERL research on

susceptible subpopulations will also address

the role of life stage, genetic background,

and preexisting disease on responsiveness to

environmental agents. Seven program

projects were identified: four focusing on

life stage issues, one on asthma as a disease,

and two on genetic components of

susceptibility. 

Cumulative Risk

Cumulative risk assessment is a major

concern to many Agency Program and

Regional Offices. NHEERL’s research on

cumulative risk will focus on providing data

that may be used in the development and

refinement of approaches to predict

interactions of chemicals at low dose

concentrations. Research in this area will

utilize empirical and mechanistic data and

models to develop strategies to predict the

effects of chemical mixtures. The program

project identified in this area will explore

the limits of the additivity assumption that is

the standard assumption in non-cancer risk

assessment guidelines in the Agency. This

research will also address mixtures of

chemicals with common or dissimilar modes

of action and will evaluate the effects of

these mixtures in repeated exposure

situations. 

The research described in this NHEERL

Human Health Research Implementation

Plan not only involves interdisciplinary

teams within NHEERL, but in many cases

involves collaborations with scientists from

other ORD Laboratories and Centers or

Agency Program Offices. Each of the

following sections attempts to link research

in that program project to other Agency

problem-driven research areas (i.e., air

toxics, drinking water, pesticides, and toxic

substances), as well as providing a narrative

on the programmatic impact of the work and

describing collaborative efforts with other

Laboratories and Centers within ORD. 
 1-1

Section 1

Introduction

Purpose and Scope

This document, the NHEERL Human

Health Research Implementation Plan,

describes the framework and

implementation plan for human health

research within the National Health and

Environmental Effects Research Laboratory

(NHEERL) beginning in FY02. Human

health research in the Office of Research

and Development (ORD) of the

Environmental Protection Agency (the

Agency) is based on needs and goals

established by the Agency in response to the

Government Performance and Results Act

(GPRA). The human health research

described in this document is core research

that aims to provide broad, fundamental

scientific information that will improve

understanding of problem-driven human

health issues arising from risk assessment in

the Agency’s Program and Regional

Offices. Core research focuses on

developing and applying the best available

science for addressing current and future

environmental hazards, as well as on

developing new approaches for protecting

human health. NHEERL’s human health

research program supports the identification

of fundamental mechanisms and

development of methods and models to

elucidate key scientific uncertainties

important to understanding and predicting

the effects of environmental agents on

human health. 

This implementation plan on human

health describes the approach that NHEERL

will take over the next 8-10 years to address

the scientific uncertainties outlined in

ORD’s Human Health Research Strategy

document, which includes harmonized use

of mechanistic data in risk assessment,

protecting the health of susceptible

subpopulations, and determining risk of

exposure to multiple chemicals. This

document also outlines in detail how

NHEERL will meet the Annual Performance

Goals (APGs) and Annual Performance

Measures (APMs) described in ORD’s

Human Health Research Multi-Year Plan. 

Multi-Year Plans (MYPs) provide a

framework to integrate research across

ORD’s Laboratories and Centers, a basis for

creating annual plans, and a context within

which to understand how decision-making

in annual planning influences the ability of

ORD to meet future goals and outcomes. 

MYPs identify Long-Term Goals (LTGs)

that are to be addressed over an 8-10 year

period while APGs and APMs represent

milestones that must be accomplished in

order to meet the LTGs. 

Process for Developing this

Implementation Plan

This implementation plan was developed

through an iterative and interactive process

leading to the development of program

projects, which are multi-investigator, multidisciplinary efforts designed to address the

three major themes outlined in the ORD’s

Human Health Research Strategy. A

Steering Committee oversaw the

development of the program projects and

was composed of two representatives from

each health division (one manager, one

scientist) and representatives from other

ORD Laboratories/Centers and Regional

Offices. NHEERL representatives on the

Steering Committee served as the Executive

Committee. ORD’s Human Health

Research Strategy and the FY02 Human

Health Research Multi-Year Plan provided

background information and guided

strategic thinking as the planning effort 

progressed.
 1-2

A scientist-to-scientist meeting was held

in November, 2001, to fashion a limited

number of integrated program projects

responsive to the programmatic needs of the

Agency. This meeting was attended by

approximately eighty NHEERL scientists

and twenty scientists from other parts of the

Agency (i.e., ORD, Program and Regional

Offices). Following this meeting, sixteen

preproposals were submitted by research

teams in NHEERL and reviewed by the

Executive Committee. At least one review

per preproposal was provided by a nonNHEERL scientist. The Executive

Committee then recommended that eleven

of the research teams develop full proposals;

these evolved into the program projects

described in this document. 

The NHEERL Human Health Research

Implementation Plan represents a work-inprogress which puts forward current

thinking on major issues and ways to

address them. As the research progresses, it

will become increasingly apparent which

ideas and approaches are leading

successfully toward their goals and which

may not be. The Executive Committee will

convene a meeting of the researchers

involved in this effort approximately every

two years to present results and discuss

future directions. It is expected that

program projects will evolve over time in

response to the success of the research effort

and identification of new directions of

significance to the Agency. Changes in

work covered by the program projects at the

Laboratory level will be linked to strategic

revisions of the ORD Human Health

Research Multi-Year Plan.
 2-1

 Human Health Research Themes 

‘ Use of Mechanistic Data in Risk Assessment

‘ Protect Susceptible Subpopulations

‘ Assess Cumulative Risk 

Section 2

Research Approach

Context for Research

The Agency is charged with the

responsibility of protecting public health

and the environment. To fulfill this

mandate, the Agency uses the process of

human health risk assessment to identify and

characterize environmentally related human

health problems. Human health risk

assessment provides a qualitative and

quantitative characterization of the

relationship between environmental

exposures and effects observed in exposed

individuals. In 1983, the National Research

Council (NRC) described four primary steps

in the process of risk assessment: hazard

identification, dose-response assessment,

exposure assessment, and risk

characterization. Risk assessment is the

primary scientific input to the risk

management process, which involves the

recognition of a potential new risk and

development, selection and implementation

of Agency actions to address the risk. Risk

management often considers a wide variety

of other factors. The overall process of risk

assessment and risk management is often

called the risk assessment/risk management

paradigm. 

Risk assessment has become an integral

component of environmental decisionmaking under the statutes implemented by

the Agency. There are many uncertainties

associated with the risk assessment process

because of severe limitations in available

data and the complex interactions between

the sources and environmental

concentrations of contaminants, dose at the

target site, and response. These

uncertainties frequently result in the use of

default assumptions and uncertainty factors

in risk assessments. NHEERL’s human

health research program is based on the

assumption that major uncertainties in risk

assessment can be reduced by understanding

the fundamental determinants of dose and

the basic biological changes that follow

exposure to a chemical.

Based on input from Regional and

Program Office risk assessors, human health

research at NHEERL will focus on three

major themes as described in the ORD’s

Human Health Research Strategy and the

Human Health Research Multi-Year Plan: 

(1) the use of mechanistic data in risk

assessments, (2) protection of susceptible

subpopulations, and (3) assessment of

cumulative risk. Research on harmonizing

risk assessment approaches will lead to a

common set of principles and guidelines for

drawing inferences about risk from available

scientific information. The overall goal of

this research, which is described in Section

3 of this document, is that Program and

Regional Office risk assessors will use

mechanistic data in a harmonized manner

for risk assessments for all health endpoints. 

NHEERL will also focus on developing a

scientific understanding of the biological

basis for differing responsiveness of

subpopulations within the general

population. Research on biological

susceptibility, which is described in Section

4, will focus on the role of intrinsic factors

such as life stage and genetic background

and extrinsic factors such as preexisting

disease on responsiveness to environmental

pollutants. NHEERL’s human health
 2-2

Scientific 

Foundation for Risk 

Assessment 

Clean

Air TSCA FIFRA CleanWater

Safe 

Drinking

Water

Limits of 

Additivity

Biological 

Basis of 

Susceptibility

Use of Mechanistic 

Information in 

Risk Assessment 

Figure 1 Mandates for Human Health Research

research program will also address the fact

that humans are exposed to mixtures of

pollutants from multiple sources. This

research will provide scientific support for

decisions concerning exposure to a multiple

pollutants by assessing additivity as the

default assumption describing interactions

between chemicals in mixtures (see Section

5 of this document).

Authorization and Mandate for Human

Health Research 

One of the reasons for focusing on human

health research is that virtually all of the

Agency’s major legislative mandates (those

which require the Agency to promulgate

regulations to protect the public health and

welfare from environmental contaminants)

require that the Agency develop human

health risk assessments. These laws (and

amendments) include the Clean Air Act; the

Safe Drinking Water Act; the Clean Water

Act; the Toxics Substances Control Act

(TSCA); the Federal Insecticide, Fungicide,

and Rodenticide Act (FIFRA); the

Resources Conservation and Recovery Act;

the Comprehensive Environmental

Response, Compensation, and Liability Act;

the Superfund Amendments Reauthorization

Act; and the Food Quality Protection Act

(FQPA). In addition, Congress enacted

legislation in 1988 which mandated that the

Agency undertake Research to Improve

Health Risk Assessment. NHEERL’s

research to harmonize the use of

mechanistic data in risk assessment, protect

susceptible subpopulations, and assess

cumulative risk will contribute to building

the scientific framework for human health
 2-3

Environmental

Release 

Environmental

Concentration

Exposure 

Concentrations 

Target Organ

Dose

Early Biological 

Effects

Figure 2 The Source-to-Outcome Continuum Adverse Outcome

Fate/Transport 

Models/Data

Exposure 

Models/Data

PBPK 

Models/Data

BBDR 

Models/Data

Systems 

Models/Data

risk assessments required by various

legislative mandates (Figure 1). Of

particular relevance to the human health

research program is the that the FQPA

requires children’s risks to pesticide

exposures be considered during the

tolerance setting process. FQPA requires

the development of scientific information

necessary to “...ensure that there is a

reasonable certainty that no harm will result

to infants and children from aggregate

exposure to the pesticide chemical

residue....” Thus, information on children’s

exposure is required to (1) consider the

susceptibility of children to increased

exposure and (2) account for aggregate

exposures to pesticides from all sources,

including food, drinking water, and

applications of pesticides in homes, schools,

daycare centers, and other microenvironments. Because of these FQPA

mandates, the results of NHEERL’s research

on susceptible subpopulations will be

important for risk assessments by the Office

of Prevention, Pesticides, and Toxic

Substances (OPPTS). This Office is

required to establish allowable levels of

exposure to pesticides and toxic chemicals

and protect the most susceptible

subpopulations. The Safe Drinking Water

Act Amendments (1996) also requires

consideration of susceptible subpopulations,

including children, when setting standards

for chemicals and other contaminants in

drinking water. Section 408 of the Federal

Food, Drug, and Cosmetic Act, as amended

by FQPA, identified several research issues

related to the determination of risk of

exposures to pesticides, including aggregate

exposure and cumulative risk, susceptible

subpopulations, an additional factor of 10

margin of safety for children, and questions

related to extrapolation of data in risk

assessment.

Multidisciplinary Human Health

Research at ORD

 Research on human health at NHEERL

is viewed as part of a multidisciplinary

research program within ORD that addresses

linkages lying along a continuum from the
 2-4

source of an agent through exposure and

dose to adverse effects or outcomes (Figure

2). Research at NHEERL focuses on

developing physiologically based,

pharmacokinetic (PBPK) models to

determine target organ dose, biologically

based dose response (BBDR) models to

determine early biological effects in

pathways associated with toxic effects, and

models to predict adverse effects. NHEERL

human health research aims to reduce

uncertainties in the risk assessment process,

including extrapolation across species,

extrapolation from short- to long-term

lifetime exposures, and variability of

response within the human population. For

purposes of dose assessment, NHEERL

works with the National Exposure Research

Laboratory (NERL) to develop

pharmacokinetic (PK) models to estimate

internal dose metrics and establish the basis

for metabolic differences between species. 

The National Center for Environmental

Assessment (NCEA) performs complex risk

assessments of national interest and

develops risk assessment methods,

databases, and tools based on results from

NHEERL and other laboratories. The

National Risk Management Research

Laboratory (NRMRL) focuses on providing

the most effective and useful risk

management options and increasing linkage

between risk assessment and risk

management efforts. Intramural research

conducted by NHEERL is complemented by

extramural research sponsored by the

Agency’s National Center for

Environmental Research (NCER). Through

the Science to Achieve Results 

(STAR) Program, NCER supports grants

that focus on specific research needs

consistent with the mission of the Agency.

Specific linkages between NHEERL

research on human health and other

Laboratories/Centers within ORD, as well as

with other research groups, is described in

greater detail in the following sections. 

Resource allocation for each program

project based on FY03 FTE projections can

be found in Appendix A. 
 3-1

Section 3 

Harmonization of Cancer and Non-Cancer Risk Assessments 

3.1 Problem

The assessment of health risks from

exposures to environmental agents has

traditionally depended on whether the

response is a cancer or non-cancer health

effect. However, the 1997 report Risk

Management in Regulatory Decision

Making from the Commission on Risk

Assessment and Risk Management

concluded that the simple dichotomy

between cancer and non-cancer risk

assessment is not fully supportable by

current scientific evidence because it results

in expressions of risk that are not directly

comparable and differ significantly in

defining maximal exposures considered to

have negligible risk. In addition, the

National Research Council’s report on

Science and Judgment in Risk Assessment

(NRC, 1994) noted the importance of a risk

assessment approach that is less fragmented,

more consistent in application of similar

concepts, and more holistic than endpointspecific guidelines. The Agency’s Risk

Assessment Forum has also been actively

involved in reexamining approaches to

cancer and non-cancer risk assessment by

sponsoring internal colloquia to encourage

input and discussion between scientists and

risk assessors. One conclusion from these

discussions is that there is a need to develop

a consistent, flexible set of principles and

guidelines for using and drawing inferences

from scientific information in risk

assessment.

One issue related to the disparity in

approaches to cancer and non-cancer risk

assessments is the limited understanding of

the mode (MOA) or mechanism of action of

compounds. This lack of knowledge has

largely been due to limitations in the

experimental designs, methods, and animal

models that have been used to support

hazard identification and dose-response

phases of human health risk assessments. 

Therefore, understanding an agent’s MOA is

key to more accurate prediction and

characterization of hazard and risk and is the

basis for developing harmonized risk

assessment approaches for all health

endpoints. At present, risk assessment

guidelines for cancer and non-cancer

endpoints differ with respect to the use of

MOA information. Guidelines for noncancer effects such as reproductive toxicity

(US EPA, 1996) and neurotoxicity (US

EPA, 1998) use mechanistic information

largely in a weight-of-evidence framework

to strengthen the evidence that a human

hazard could exist. For example,

mechanistic data could be used to support

findings from an epidemiological study or

results from animal toxicity studies. 

Mechanistic data may also be used on a

case-by-case basis to reduce the magnitude

of uncertainty factors used to calculate the

Reference Dose (RfD), Reference

Concentration, or Benchmark Dose. For

example, if the same MOA could be

demonstrated to occur in humans and

animals, the uncertainty factor for animalto-human extrapolation could be reduced in

the risk assessment.

The use of MOA information is spelled

out in detail in the draft final Guidelines for

Carcinogen Risk Assessment (US EPA,

2003). As in the case of non-cancer

endpoints, MOA data can be used in a

weight-of-evidence framework to support

the evidence for carcinogenic hazard

potential based on epidemiological or

animal toxicity studies. On the other hand,

these guidelines note that MOA information

is key to addressing various default options

in the risk assessment. BBDRs using
 3-2

mechanistic information can be used to

relate dose and response on an agentspecific basis and to extrapolate to lower

dose levels, if needed. MOA data can be

crucial for establishing the point of

departure (POD) for risk assessment, the

linearity or non-linearity of the dose

response relationship, and the biological

plausibility of a response. MOA data are

also important for determining key events

that are of increased concern to susceptible

subpopulations, such as children, and

extrapolation from animals to humans. 

MOA information plays an important role in

developing PK models to estimate dose in

target tissues and helps define relative

sensitivity of various tissues to carcinogens. 

Unlike risk assessment guidelines for noncancer endpoints, the draft Cancer

Guidelines contain a framework for the

analysis of carcinogenic MOA information,

including steps necessary to define the

postulated MOA, identification of key

events, and assessment of experimental

evidence supporting the postulated MOA. 

There is a clear need to develop a

common approach for the use of MOA data

in risk assessment (Bogdnanffy et al., 2001). 

In the context of this implementation plan,

harmonization refers to developing a

consistent, flexible set of principles for

using and drawing inferences from available

information on MOA to support risk

assessment. “Mode of action” is defined as

a series of key events starting with

interaction of an agent with a cell and

proceeding through functional and

anatomical changes to result in an adverse

effect. “Mechanism of action” implies a

more detailed understanding and description

of the key events, including the molecular

level. Mechanism of action often describes

how a chemical works at all levels of

biological organization. For the purposes of

this document, mode and mechanism of

action are used interchangeably in this

document. “Key events” are defined as

empirically derived precursor steps linking

the proximal event to the adverse outcome. 

The sequence of key events leading to an

adverse effect at a given internal dose is also

known as the “toxicity pathway.” 

Toxicities arise from a range of factors that

may be specific to the chemical or chemical

class. The physiology of the organism and

other environmental factors may affect the

expression of toxicity. A framework for

harmonization of risk assessment

approaches must facilitate incorporation of

data that accounts for these factors. 

3.2 Goals

The overarching goal of mechanistic

research at NHEERL is to help derive a

commonly accepted set of principles

defining how MOA information can be used

in risk assessment, particularly as it relates

to extrapolation in risk assessment. The

NHEERL human health research

implementation plan addresses how MOA

information can be used in a more consistent

way to improve dose-response assessment

for the following:

• For various types of cancer and noncancer endpoints,

• For chemicals producing different

toxicities by a similar MOA, and

• For chemicals producing similar

toxicities by different MOAs. 

This research also addresses how

mechanistic data can be used in risk

assessments to

• Compare adverse outcome across

toxicities,

• Select the POD and approaches for

low-dose extrapolation, and

• Account for inter- and intra-species

differences in PK and PD capabilities

for all health endpoints.
 3-3

Develop 

Framework

to Define

Mechanism

Develop 

Methods to

Study 

Mechanism

Identify 

Common 

Modes

of Action

Framework for the Use of Mechanistic Information in 

Risk Assessment, Especially as it Relates to Animal to 

Human Extrapolation

Dose-Response

Studies

on Prototypic

Chemicals

Figure 3 Critical Path for Harmonization Research

3.3 Critical Path

Consistent utilization of MOA

information in risk assessment involves

several critical steps, as outlined in Figure 3. 

In order to achieve a more harmonized

approach for risk assessment based on the

use of MOA data, there must first be a

common framework for defining MOA. 

Questions to be addressed include the

following:

• How much evidence is needed to show

that a substance acts via a particular

MOA in animals and its applicability

to humans?

• How to show that two toxic

manifestations caused by the same

substance were produced by different

MOAs?

• How to show that a common effect

produced by two chemicals may have

been caused by two different MOAs?

• What should be the dose-response

assessment approach for a chemical

that produces multiple toxic

manifestations, but through a similar

MOA?

It is also highly probable that a key

element of research on the use of MOA data

in risk assessments will be the application of

emerging technologies, especially in

molecular biology. Methods and

approaches based on proteomics and

genomics will likely prove crucial in testing

hypotheses concerning common key events

that lead to both cancer and non-cancer

effects. Of particular concern will be

approaches to quantify changes in gene and

protein expression in toxicological studies

and to interpret such changes in a risk

assessment context. This will determine the

extent to which these emerging technologies

could be used to inform mechanistic

questions in risk assessment. A research

program applying molecular biological

techniques with mathematical and computer

models for prediction of effect and

understanding MOA is being developed by

ORD and is described in the draft document

A Framework for a Computational

ToxicologyResearch Program in ORD.

Once a framework has been developed

to define MOA for use in risk assessment,

work will be done to identify possible

common modes of action for chemicals

producing cancer and non-cancer effects. 
 3-4

Research will develop a clear understanding

of the biological changes that occur

following delivery of the active chemical

moiety to target sites and the relationship of

response to dose. Emphasis will be placed

on identifying possible common key events

(e.g., cell proliferation, receptor interaction,

response to injury or stress) for prototypic

chemicals that have both cancer and noncancer effects. The overall objective of this

research will be to demonstrate the

feasibility of determining potential common

precursor MOAs for cancer and non-cancer

effects. This information will provide the

basis for subsequent research to characterize

precursor steps for several prototypic

chemicals. Issues that will be addressed by

this research include the following:

• How MOA data at the low end of the

dose-response curve can inform

decisions about the most appropriate

risk assessment model,

• How MOA data can inform decisions

about the presence or absence of a

threshold, and

• How mechanistic information can be

used to select the POD. 

APGs and APMs related to research on

the use of mechanistic data in risk

assessment in the ORD Human Health

Research Multi-Year Plan cross referenced

to each program project can be found in

Appendix B.

3.4 Program Projects 

Research on harmonization of risk

assessment consists of three program

projects addressing MOAs and their use in

risk assessment. Program project 1

addresses the hypothesis that exposure to

different classes of chemicals will disrupt a

common key event, i.e., mitogen-activated

protein kinase (MAPK), in multiple tissues

leading to a range of toxicities. Program

project 2 addresses the hypothesis that

disruption of luteinizing hormone (LH)

secretion serves as a common MOA for

altered fertility, reproductive disease, and

cancer of the reproductive system in animals

and humans. Program project 3 addresses

the hypothesis that modulation of

cytochrome P-450s and other xenobiotic

metabolizing enzymes (XMEs) will lead to

common MOAs for multiple toxicities. A

fourth program project “Environmental and

Genetic Interactions in Hypertensive Rats: 

Oxidative Stress as a Common

Susceptibility Attribute for Non-Cancer

Risks” contains some mechanistic research

concerning oxidative stress as a common

MOA. However, the focus of this program

project is primarily on questions related to

differential responsiveness to chemical

exposure, i.e., susceptible subpopulations

(see Section 4 for details). These four

themes were selected because they involve

toxicity pathways associated with prominent

adverse health effects of high priority to risk

assessors (i.e., cancer, reproductive and

pulmonary toxicity, neurotoxicity). 
 3-5

Program Project 1: Harmonization of Cancer and Non-Cancer Risk

Assessment: Disruption of Mitogen-Activated Protein (MAPK) Signaling as

a Common MOA for Environmental Toxicants

Objectives:

• Use both in vitro and in vivo models to

demonstrate that diverse groups of

chemical compounds can disrupt MAPK

signaling in multiple tissues,

• Examine the disruption of MAPK

signaling as a common MOA leading to

the expression of multiple adverse effects

using pharmacologic and molecular

manipulations of the MAPK signaling

cascade as well as environmental

toxicants, and

• Demonstrate that changes in MAPK

signaling studied in vitro can be used to

extrapolate the MOA of chemicals in

vivo. Determine the degree of homology

of response between animals and humans.

Scientific Approach

The transmission of external signals to

the interior of the cell is a fundamental

process in the regulation of cellular

responses to physiological stimuli. Virtually

every reaction that a cell makes in response

to its environment is regulated by signaling

processes. These signaling pathways exist

in all cell types and determine the activity of

the cell ranging from quiescence to fully

stimulated. Cell signaling pathways are

known to be upstream mediators of a

number of basic biological processes

including cell cycling, proliferation,

migration, differentiation, plasticity,

inflammation, and cell death. Signal

transduction pathways are critical

intermediates, accepting and integrating

extracellular information with the nucleus to

affect gene transcription and invoke a

biological response. Thus, cell signaling

pathways have the potential to serve as early

and sensitive indicators of biological

perturbation (NRC, 2000). In addition,

understanding the signal transduction

pathways that underlie the biological

response to environmental and chemical

insult is critical to identifying the MOA

leading to adverse health effects. 

Protein kinases are signaling molecules

that interact with other messenger systems

and form a highly structured network to

integrate cell functions in an organism. 

These signaling pathways consist of proteins

grouped in “cascade” fashion which

typically originate in the cell surface and

reach into the cell cytosol and nucleus. 

Each of the proteins has a structural and/or

catalytic function that fits in the overall

pathway to attenuate the specificity,

magnitude, and duration of the signal being

transmitted. The pathway to be studied in

this proposal, the MAPK cascade, contains

receptor kinases as well as serine/threonine,

tyrosine, and dual specificity kinases with

multiple regulatory domains that determine

the specificity of the interactions (Chang

and Karin, 2001).

A number of studies conducted by

NHEERL scientists and others have

demonstrated that environmental

contaminants can disrupt signaling

processes in target cell types in a variety of

organ systems, including the lung, the

nervous system, the reproductive organs,

and the developing fetus. Although these

compounds differ markedly in their

physicochemical properties, they appear to

share a common MOA in their ability to

damage cell signaling by interfering with or

enhancing signal initiation, transduction, or

termination. Physiologically inappropriate
 3-6

Demonstrate that

Diverse Classes of

Chemicals affect 

MAPK

Determine if Disruption of MAPK

Leads to Multiple

Adverse Effects

Show that Effects

in In Vitro Models

Can Be Seen In 

Vivo

Demonstrate Applicability Across 

Species

Demonstrate Disruption of 

MAPK Underlies Toxicity

of Diverse Groups of 

Chemicals 

Project 1 Project 4 Project 2 Project 3

Figure 4 Critical Path for Research on Cell Signaling

signaling appears to result in a broad range

of toxic responses, including apoptosis,

inflammation, metaplasia, hyperplasia, 

hypertrophy, and neoplasia. Because

MAPKs are involved in these basic cellular

processes, it is proposed that disruption of

MAPK signaling may be a common MOA

that underlies the multiple adverse effects

observed in diverse tissues. The critical

path for research on cell signaling is

illustrated in Figure 4. 

Project 1 - Evaluation of MAPK

Signaling after Exposure to

Environmental Toxicants. External

stimuli play a major role in regulating

complex intracellular processes such as gene

expression, cell proliferation, growth,

differentiation, and death. These stimuli

include cell-substrate adhesion, growth

factors, hormones, neurotransmitters, and

cytokines that stimulate a plethora of

receptors. Environmental toxicants can act

at many of these sites to perturb intracellular

signaling, and, depending on exposure

conditions, tissue, and life stage, can result

in wide variety of adverse effects. In order

to harmonize risk assessment approaches it

will be necessary to determine whether the

array of possible upstream targets of

toxicants have a common downstream

response. In a multitude of cell types,

activation of a variety of receptors (e.g., Gprotein coupled receptors, ion channels,

receptor tyrosine kinases and cytokine

receptors) trigger the MAPK cascades which

serve as central downstream integrators to

modulate the cellular response to external

stimuli. Research in this project will test the

hypothesis that exposure to different classes

of chemicals will disrupt MAPK signaling

in multiple tissue types. This work is

intended to determine whether effects on

diverse upstream targets will lead to changes

in a common downstream endpoint, MAPK,

and provides the basis for continued testing

of MAPK signaling as a common MOA. 

The studies outlined in this project will

use several different in vitro and in vivo

models to understand the potential for

altered upstream signaling to lead to

changes in MAPK in embryonic tissue

(developmental toxicity), brain tissue

(neurotoxicity), pulmonary tissue (airway

disease), and skin epithelium (cancer). 

Because several different model systems

will be used, the chemicals to be examined
3-7

will vary. For each model system,

chemicals with known adverse effects will

be chosen based on existing data. Research

in project 1 will demonstrate that known

environmental toxicants which act on

diverse cellular targets in multiple tissues

ultimately lead to alterations in MAPK

signaling. Based on considerations of doseresponse and toxicity, chemicals and models

which show a change in MAPK signaling at

relevant doses will be chosen for further

study in project 2. Research in project 1

will include the following:

• Effects of arsenic (As) on cell

signaling in primary human

keratinocytes and bladder epithelial

and endothelial cells,

• Effects of particulate matter (PM) on

MAPK cascade components inhuman

airway epithelial cells,

• Effects of toxicants on ligand-receptor

interactions and subsequent MAPK

signaling events in the developing

embryonic palate,

• Effects of developmental toxicants that

alter protein kinase and MAPK

signaling in whole embryo cultures,

• Effects of toxicants that perturb

electrical excitability, integrin-receptor

interactions or calcium regulation of

MAPK signaling events in neuronal

tissue, and

• Use of flow cytometry and confocal

microscopy to detect MAPK signaling.

Project 2 - Disruption of MAPK Signaling

as a Common MOA Leading to

Expression of Multiple Adverse Effects. 

The studies in project 1 are designed to

demonstrate that toxicant-induced effects on

a variety of upstream targets ultimately

converge to perturb MAPK signaling. The

critical question to be addressed next is

whether the observed changes in MAPK are

related to the expression of toxicity in

multiple tissues. It is clear that MAPK

signaling is an important mediator of critical

cellular functions including embryogenesis,

cell proliferation, cell differentiation, and

cell death. Thus, alterations in MAPK

signaling is expected to be part of the MOA

that leads to changes in critical cellular

functions which are ultimately expressed as

the adverse effect in the different tissues and

model systems described above. This work

will attempt to tie the changes in MAPK

signaling identified in project 1 to

consequences downstream from this critical

signaling event. Research covered in project

2 will include the following:

• Studies on MAPK-induced disruption

of cell cycle progression leading to

augmented cell proliferation and

carcinogenesis by arsenicals,

• Effects of PM metals on MAPK

activation and subsequent gene

transcription,

• Studies on toxicants that interfere with

ligand-receptor interactions to alter

critical signaling events in the

developing embryo,

• Effects of disruption of MAPK

signaling and craniofacial

development by altering differentiation

of neural crest cells after exposure to

haloacetic acids (HAAs) in vivo and in

vitro,

• Research to determine if disruption of

cell signaling is a MOA by which

developmental neurotoxicants alter

neuronal differentiation and

synaptogenesis,

• Studies to determine if disruption of

cell signaling is a MOA by which

developmental neurotoxicants interfere

with activity-dependent plasticity in

intact neural circuitry, and

• Use flow cytometry and confocal

microscopy detection of MAPK

signaling to demonstrate that alteration

of MAPK affects basic cellular

processes, including proliferation and

apoptosis.
 3-8

Project 3 - Use of In Vitro Signaling Data

for Extrapolating the MOA of

Environmental Toxicants In Vivo. The

Agency depends on mechanistic

toxicological research to provide biological

plausibility to strengthen the knowledge

base used in risk assessments in a weight-ofevidence context. However, much of the

molecular and biochemical toxicological

research is presently conducted using in

vitro models which vary in their ability to

approximate toxic responses in the whole

animal or human. Therefore, evaluation of

the relevance of in vitro mechanistic

findings, such as MAPK activation, to in

vivo toxicity would lead to increased

confidence in the use of mechanistic data in

support of regulatory decision-making. This

project will determine if changes in MAPK

signaling studied in vitro can be used to

extrapolate the MOA of chemical

compounds in vivo. Research in project 3

will:

• Determine the effects of PM metals in

MAPK activation in vivo and in vitro,

• Compare the responses to

developmental toxicants in embryonic

organ culture to those observed

following in vivo exposure,

• Determine the role of integrinmediated initiation of MAPK signal

transduction in vivo in neurotoxic

effects in vitro and in vivo,

• Conduct in vitro and in vivo effects of

bioaccumulative chemicals such as the

polychlorinated biphenyls (PCBs) on

MAPK signaling in the developing

nervous system, and

• Compare cell signaling in the nervous

system in vitro and in vivo and

determine it relationship to cognitive

effects of environmental toxicants. 

Project 4 - Extrapolation of Perturbations

in MAPK Signaling from Animals to

Humans. MAPK signaling cascades are

highly conserved across species, indicative

of the critical role these signaling pathways

play in cell function. Due to this high

degree of conservation, it is proposed that

toxicants which perturb these pathways in

one species will have qualitatively similar

effects in the same tissue of another species. 

For the process of risk assessment, the

uncertainty associated with extrapolation of

data from animals to humans can be reduced

if it is demonstrated that the MOA is the

same in both species and if quantitative

differences can be accounted for through

data generated in tissues of humans and

animal models. Project 4 will determine if

effects of environmental toxicants on

MAPK in one species will be qualitatively

similar to those effects in other species. The

studies outlined in this project will use

several different in vitro models in which

comparable rodent and human tissue can be

obtained. These studies will also make use

of the information gained in projects 1 and 2

in order to focus on the upstream targets,

specific MAPK pathways, and critical

biological effects important to the

elaboration of pathogenesis for a particular

model. Research covered by this project

includes the following:

• Research to determine if MAPK

signaling is perturbed by methylated

trivalent arsenicals in mouse and

human primary keratinocytes,

• Research to determine the effects of

toxicants on MAPK signaling in

human and rodent embryonic palatal

cells, and

• Research to determine if alterations in

cell signaling produced by

neurotoxicants in rodent neurons are

similar in human neurons.
 3-9

Impact

A major goal of ORD’s research program

on human health is to develop a consistent,

flexible set of principles for determining risk

based on pertinent mechanistic data,

regardless of the nature of the toxicity (i.e.,

harmonize cancer and non-cancer risk

assessment approaches). There is an

emerging knowledge base which suggests

that the biochemical changes leading to

certain cancer and non-cancer health effects

are similar, and a more accurate prediction

and characterization of risk for cancer and

non-cancer endpoints can be based on

understanding a chemical’s MOA. In order

to harmonize the risk assessment process for

cancer and non-cancer health effects,

information is needed regarding the

application of mechanistic data for

chemicals that produce different toxic

outcomes by a similar MOA. The goal of

this program project is to identify

intracellular signaling pathways critical to

the expression of carcinogenic and

noncarcinogenic effects following chemical

insult. This research will show that

disruption of critical cell signaling pathways

is a common MOA that underlies the

toxicity of diverse groups of chemicals, that

can be used as an in vitro predictor of the

mechanism of toxic response in vivo and

extrapolate from experimental animals to

humans. This program project will provide

an example of how data on common MOA

can be used for comparative risk assessment

across biological endpoints and also reduce

the reliance on uncertainty factors by

providing a common measure for

extrapolation. In addition, the results of

these studies may identify scientifically

defensible biomarkers of exposure and

effect.

Cross-Agency Interactions

As this program project progresses,

consultations with scientists from NCEA

will be held on a periodical basis in order to

develop principles that can be used to

formulate guidance documents on the

harmonized use of MOA data in risk

assessment. 
 3-10

Program Project 2: Disruption of Luteinizing Hormone (LH) Secretion as

a Common MOA for Altered Fertility, Reproductive Disease, and Cancer of

the Reproductive System

Objectives

• Characterize the range of environmental

agents that alter LH secretion,

• Identify central nervous system (CNS)

mechanisms involved in this MOA,

• Compare effects in animal models to

those in humans, and

• Determine key events necessary for

altered reproductive function,

reproductive diseases and cancer of the

reproductive system in the rodent and

potentially the human.

Scientific Approach

One of the more difficult issues that

confront risk assessors is the interpretation

of toxicology data obtained in animal

species and applying them to human risk

assessment. A key to using such

information in risk assessment is the degree

of homology between humans and the test

species; that is, whether there exists a

common MOA. Within this context, the

research in this program project is intended

to derive a set of principles for application

to animal data pertaining to toxicants that

alter serum LH. This effort will focus on

how LH disruption, in both the test species

and humans, contributes to impaired fertility

and reproductive disease. These principles

can serve as a framework for evaluating the

effect of environmental chemicals that alter

the neuroendocrine control of LH. 

Moreover, as a valuable tool for examining

test data, it will serve as a prototypical

approach for examining dysregulation in

other neuroendocrine axes of importance to

human health risk assessment (i.e., prolactin

and reproductive tumors;

thyroid-stimulating hormone [TSH] and

thyroid function; and adrenocorticotropin

hormone and adrenal function).

Many xenobiotics have been shown to

influence the regulation of LH. However,

the actual mechanism (e.g., receptor,

enzyme) may be different depending on the

toxicant. Recently, alterations in LH

secretion have been identified as the MOA

for altered reproductive function (i.e.,

delayed puberty in both sexes, altered

ovarian function in the adult female and

pregnancy loss), premature reproductive

aging, and mammary gland tumor

development in the female following

chlorotriazine treatment (Cooper et al.,

1998, 1999, 2000; Laws et al., 2000;

Narotsky et al., 2001; Stoker et al., 2002). 

Similarly, exposure to selected xenobiotics

will produce changes in the secretion of LH

in the male resulting in decreased

testosterone; impaired fertility, or, as in the

case of linuron exposure, elevated basal LH

associated with an increased incidence of

Leydig cell tumors. These effects of altered

LH secretion generally require a protracted

treatment before the adverse effect is

observed. In contrast, it is important to note

that all environmental compounds that

modify LH secretion do not necessarily

result in the same sequence of adverse

reproductive or cancer effects. Thus, all

toxicants that alter the LH surge (the MOA)

may not lead to the same adverse outcomes. 

Based on these observations, the

fundamental hypothesis to be examined

herein is that, because the LH surge can be

modified by environmental agents that

interact with a variety of different CNS

mechanisms, the adverse outcomes observed

(in both rats and humans) will vary

depending on which CNS target site is

involved. Thus, a better understanding of

the dynamics involved in the disruption of

the LH surge will allow us to better predict

which adverse outcome(s) will emerge and
 3-11

Identify Range of

Chemicals that

Modify Regulation

of LH

Identify Mechanisms

Involved in 

Altering LH

Characterize Adverse

Cancer and NonCancer Effects

Mediated by LH

Study Effects of

Cumulative 

Exposures

Demonstrate How LH Disruption 

Impairs Fertility and Reproductive

Health in Humans and Animals

Project 1 Project 4 Project 2 Project 3

Figure 5 Critical Path for Research on Luteinizing Hormone

thus the relevance of each to potential

human reproductive disorders can be better

predicted. These studies will attempt to link

the key cellular events responsible for

changes in LH and adverse outcome in

animals and humans, as summarized in

Figure 5. 

Project 1 - Identification of the Range of

Chemicals that Modify the Regulation of

LH. The purpose of this project is to

determine the range of chemicals that may

influence the secretion of LH. Once a

chemical has tested positive in this

evaluation, the dose- and timeresponse associated with oral dosing will be

characterized using well-established

techniques. These studies will provide

important data describing the range of

environmental chemicals that alter LH

secretion, as well as the baseline

information necessary for at least two of the

projects described below. To date, several

groups of environmental compounds have

been shown to disrupt LH secretion in the

female rat; however, the universe of

chemicals that may possess such effects

remains to be determined. To identify

compounds for evaluation and testing, two

different approaches have typically been

used. First, those agents reported to affect

any of the hypothalamic targets known to

regulate gonadotrophin-releasing hormone

(GnRH) and LH secretion would be

predicted to affect LH in an ovariectomized,

estrogen-primed rat model. Second, those

compounds known to affect the estrogen or

androgen receptor would test positive in

assays using the intact female or male

respectively. Either one or both models can

be used to fully characterize the effect of the

compound on LH. A third approach has

been to identify compounds that, in vivo, are

reported to cause adverse reproductive

effects suggestive of LH disruption (i.e.,

altered cycling in the female rat, blockade of

the LH surge, Leydig cell hyperplasia in the

male).

Project 2 - Identify the Mechanisms

Involved in Altering LH. The regulation

of the LH surge is under the control of a

variety of neurotransmitters and

neuropeptides. Of particular interest is the

fact that several xenobiotics affect

noradrenergic and cholinergic
 3-12

neurotransmission because both these

neurotransmitters have been shown to

regulate the pulsatile release of GnRH and

subsequently LH secretion. Thus,

compounds that alter noradrenergic (NE)

synthesis (i.e., chlorotriazines [atrazine,

simazine, propazine] and dithiocarbamates

[thiram, metam sodium, disulfiram, carbon

disulfide]), block NE receptors (i.e.,

chlordimeform, amitraz, etc.) or

acetylcholine availability (i.e, carbamates)

have all been reported to alter both basal LH

secretion and the LH surge. Other

environmental agents have been reported to

alter the regulation of LH secretion, but the

mechanism(s) remains to be determined.

The purpose of project 2 is to develop a

database that documents the various

mechanisms through which the regulation of

LH secretion (both basal and pulsatile) may

be altered. Should a compound be shown to

alter the regulation of LH (project 1) by

modifying pituitary or circulating

concentrations, studies will be initiated to

determine the particular mechanism of

action underlying the observed effect. The

particular approach(es) employed will

depend upon published information about

the compound in question, including

available data about the compound’s effects

on other physiological processes using

similar mechanisms of regulation and

structure-activity (SAR) data available for

other members of its chemical.

Project 3 - Characterization of Adverse

Effects on Reproductive Function,

Reproductive Diseases, and Cancers of

the Reproductive System following

Modification of LH Secretion. This

research will use two approaches. The first

approach addresses the hypothesis that

environmental chemicals that change the

timing or magnitude of LH release will alter

reproductive function in both males and

females. The purpose of these studies is to

characterize the extent to which compounds

that interfere with the regulation of LH alter

a number of reproductive processes and to

provide an approach that will form the basis

for comparison between adverse

reproductive outcomes in the rodent versus

human. The second approach will address

the hypothesis that long-term alterations in

the endocrine milieu will lead to

reproductive diseases and/or cancer. These

studies will be dependent upon data

obtained from studies described in the

approach described above. For example, in

cases where the chemical causes long term

elevation of LH, additional endpoints will be

included in the male and female pubertal

studies to evaluate changes in ovarian and

testicular histology. These studies will

evaluate endpoints that are most relevant to

potential human health concerns associated

with this MOA.

Project 4 - Cumulative Exposures to

Environmental Compounds which

Suppress LH. There is concern that

exposure to multiple chemicals may alter the

effect in the target organ in ways not

predicted or expected based on the

dose-response curves of the single

chemicals and an assumption with regard to

additivity (i.e, toxicity may be greater or

less than expected based on the single

chemical information and an assumption of

either dose addition or response addition). 

In addition, there is concern that cumulative

exposure may cause toxicity in an

unexpected organ. The default assumption

for risk assessment of non-cancer endpoints

is that toxic chemicals with similar MOAs

will combine to cause toxicity in a doseadditive manner. However, there are

relatively few data supporting this

assumption, particularly for scenarios where

toxic chemicals with similar MOAs, but

different mechanisms will combine to cause

toxicity. This scenario warrants further

study. In this project, chemicals from

pesticide classes which are identified in

projects 1 and 2 as having the MOA in
 3-13

suppressing LH will be evaluated in a doserelated manner. Once the MOA and

portions of the specific mechanism within

each general class are determined, low-dose

cumulative exposure experiments will be

designed. This project is linked directly

with research on Cumulative Risk (see

Section 5). All APMs for this project are

reported in this Section on harmonization of

the use of mechanistic data in risk

assessment because the primary focus of the

research is on identification and

characterization of the LH MOA. 

Impact 

LH secretion is considered to be an

important MOA for risk assessment, and

chemical specific data will address key

issues such as whether or not LH secretion

is modified following exposure, the dose

response, the pattern of change that occurs

(chronic or acute disruption), the adverse

outcomes that develop, and the mechanism

involved. This information is critical for

determining the potential health risks of

exposure to the chemical in humans. By

determining the key events involved in

toxicant-induced chemical changes in LH

secretion, a better understanding of the

homology between the rat and human will

evolve. This research will also provide

significant information concerning the

conditions underlying altered reproductive

function and the role they may have in the

development of tumors of the reproductive

system (i.e., harmonization of cancer and

non-cancer endpoints).

The work evaluating cumulative effects

included in the current proposal will also

address important questions concerning the

evaluation of mixtures. There is a

substantial degree of confusion within the

Agency concerning how to select chemicals

to be used in a cumulative risk assessments. 

The approach to be taken in this program

project will be to link the cellular MOA

responsible for the change in LH and

adverse outcome. In these studies, the

working hypothesis will be that compounds

with similar mechanisms will show dose

additivity; whereas combined exposure to

compounds with different mechanisms

would be either additive and potentially

more than additive or less than additive.

Cross-Agency Interactions

The research efforts will involve ongoing

communications with OPPTS. The effect of

environmental agents on LH secretion and

the demonstrated role of altered LH

secretion in the development of adverse

reproductive and cancer outcomes was of

primary significance to the risk assessment

of the chlorotriazine pesticides by OPPTS. 

These studies have also played a major role

in the selection of compounds and the

development of the cumulative risk

assessment of the chlorotriazines again in

close collaboration with personnel in

OPPTS. 
 3-14

Program Project 3: Modulation of Cytochrome P-450s and Other

Xenobiotic Metabolizing Enzymes (XME) Leading to Common MOA for

Multiple Toxicities

Objectives

• Determine if P-450 and XME modulation

is a common key event for multiple

adverse effects in adult males,

• Determine if P-450 and XME is a

common key event for multiple adverse

effects in developmentally exposed males

and females, and 

• Develop computational toxicological

approaches to evaluate if alterations in P450 and/or XME are common key events

for cancer and non-cancer effects.

Scientific Approach

This program project directly addresses

the need to derive a commonly accepted set

of principles defining how MOA

information can be used in risk assessments,

particularly as it relates to extrapolation

issues. Evaluating whether chemicals have

a common MOA for their multiple toxicities

and the implications of that MOA for doseresponse assessment are two of the

fundamental problems in harmonization of

human health risk assessment approaches. 

This project addresses both qualitative and

quantitative methods that could be

eventually applied to risk assessment

activities.

Principles concerning the use of MOA

data in risk assessment will be derived

experimentally by examining a class of

prototypic chemicals, the conazole

fungicides (US EPA, 1999). Conazoles are

used both as pesticides and pharmaceuticals;

therefore, both experimental animal and

human toxicity data are available. These

fungicides are used in crop protection to

control fungal infections on fruits,

vegetables and cereal crops, and for seed

treatment of cereal crops. Medically, they

are used to treat local and systemic fungal

infections. Conazoles are a class of

ergosterol biosynthesis inhibiting fungicides

(EBIFs) which inhibit 14-"-sterol

demethylase (CYP51, lanosterol

14-"-demethylase). They contain either a

1,2,4-triazole or imidazole moeity which

prevents enzymatic activity through

interaction with the heme iron of P-450s. 

Because ergosterol is an essential

component of fungal membranes, the

inhibition of its biosynthesis leads to cell

death. Conazoles have also been developed

to selectively inhibit aromatase for the

treatment of breast and prostate cancer. In

vertebrate species, conazoles have complex

effects on the hepatic and non-hepatic

microsomal monooxygenase systems. They

can act as both inducers and inhibitors of

cytochrome P-450s depending on the tissue

and specific conazole considered (Morita et

al., 1990; Vinggaard et al., 2000). 

Many conazoles are hepatotoxic and

hepatocarcinogenic in mice. Some also

induce thyroid follicular cell tumors in rats,

and they are generally non-genotoxic using

standardized test systems. Several

conazoles are also hepatotoxic in humans. 

Both thyroid and liver cancer is thought to

be mediated by modulation of P-450 or

XMEs. Specifically, thyroid tumors are

thought to be a result of increased hepatic

metabolism and biliary excretion of

thyroxine as the glucuronide leading to

increases of TSH and overstimulation of the

thyroid (Hurley, 1998).

Hepatocarcinogenesis is thought to be a

result of increased P-450 levels leading to

oxidative stress, mitogenesis, and altered

foci development ultimately leading to

neoplasia.
 3-15

In the liver, conazoles affect the activity

and expression of a number of P-450s. For

example, the pesticide propiconazole

induces the activities of CYP1A1, CYP1A2,

CYP2B1/2, CYP2B6, and CYP3A4. 

Additionally, propiconazole inhibits

CYP2C11, and in reproductive and other

tissues also inhibits CYP19 (aromatase). 

The pharmaceutical ketoconazole also

induces several rat P-450s: CYP1A1;

CYP2B and CYP3A2. Furthermore,

ketoconazole inhibits the activities of

CYP1A1, CYP1A2, CYP2A6, CYP2C19,

CYP2D6, CYP2E1, and CYP3A4 in the

liver. In other tissues, ketoconazole inhibits

essentially all the steroidogenic P-450s. 

Ketoconazole most potently inhibits CYP17,

which is critical for two enzymatic steps in

the synthesis of androgens in the testis. 

Ketoconazole also significantly affects male

and female reproduction and is a potent

developmental toxicant due to disruption of

steroidogenesis.

Long-term regulation of steroidogenesis

is primarily at the level of transcriptional

regulation of the genes for the various

steroidogenic P-450 enzymes. The P-450

genes are regulated in tissue-specific,

developmentally programmed, and

hormonally regulated fashions. There are

substantial differences in the expression of

these genes and the activity of their gene

products among various mammals. 

Consequently, attention must be paid to

significant species differences between

humans and the rodent models. In the testis,

LH modulates the production of testosterone

in the Leydig cells, while follicle

stimulating hormone (FSH) modulates

estradiol synthesis (testosterone conversion

by CYP19 [aromatase]) in the Sertoli cells. 

Both LH and FSH are secreted by the

anterior pituitary, in response to the GnRH

from the hypothalamus. Thus, monitoring

LH and FSH will be critical in

understanding conazole effects on

steroidogenesis in vivo and subsequent

effects and responses to altered steroid

levels in the relevant reproductive,

endocrine, and neural tissues. In addition to

the sex steroids and the

gonadalhypothalamic-pituitary axis,

conazoles also affect glucocorticoid

metabolism and the adrenal gland, thyroid

metabolism and thyroid gland, and retinoid

metabolism significant to embryogenesis. 

The critical path for this program project is

summarized in the Figure 6. 

Project 1- Profiling of Toxic Effects, P450s, and Gene Expression in Multiple

Tissues Following Conazole Exposures in

Adult Male Rats and Mice. The purpose

of this project is to evaluate the hypothesis

that P-450/XME modulation is a common

critical event that contributes to the MOA

for hepatocarcinogenesis, thyroid

carcinogenesis, reproductive toxicity, and

neurotoxicity of selected conazoles in adult

male rodents. It is hypothesized that the

expression of numerous genes related to P450 expression and function will be affected

by conazole exposure in the various tissues

being studied and that these altered

expression profiles will provide mechanistic

insights useful to identifying common

MOAs. Research covered in this project

will include the following:

• Establish dose-responses for preclinical and toxic effects in the liver,

thyroid, testis and brain of young adult

male Sprague-Dawley rats and CD-1

mice following 14 daily doses by

gavage with various conazoles;

• Delineate conazole effects on P-450

protein expression and activity in liver,

thyroid, testis, and brain and evaluate

tissue dosimetry in these and selected

other tissues; and

• Profile gene expression of P-450s and

XMEs in testis, brain, thyroid, and

liver of mice exposed to conazoles.
 3-16

Project 1

Profile Non-Cancer Effects of Conazoles

in Multiple Tissues in Adults 

Project 2

Profile Cancer Effects of 

Conazoles in Thyroid and

Liver 

Project 3

Common Modes of Action

for Developmental Exposure

Project 4

Integrate Toxicity

Information for

Application in 

Risk Assessment

Demonstrate that Modulation of P-450s and 

XMEs are Common Mode of Action for 

Multiple Toxicities

Figure 6 Critical Path for Research on P-450 and XMEs

Project 2 - Adult Exposures Resulting in

Thyroid and Liver Cancer. This research

will determine the effects of conazole in

thyroid and liver of rats exposed during

adulthood and will consist of the following

studies:

• Research on the MOAs of conazoles as

both thyroid hormone disruptors or as

progenitors of toxic intermediates with

both MOAs occurring through

conazole-induced enhanced

metabolism, and

• Studies to examine the early events in

conazole-induced hepatic neoplasia

and relate these findings to P-450

induction to exclude genotoxic

intermediate involvement as a result of

conazole-induced P-450 metabolism. 

Project 3- Determining Common MOAs

for Reproductive and Neural Toxicities

Following Developmental Exposure to

Conazoles. Developmental effects are

expected at various stages of growth as a

result of both gestational and postnatal

exposures to conazoles. It is possible that

differential responses could be observed

depending on age, gender, or physiological

status. The goal of this research is to

understand how common MOAs produce a

range of toxicities in developing tissues,

following exposure to conazole fungicides. 

These studies will focus upon modulation of

cytochrome P-450s and other xenobiotic

metabolizing enzymes to identify common

MOAs, but will also assess broader effects

on gene expression. The general hypothesis

is that disruption of steroidogenesis by

conazoles in developing neural, endocrine,

and reproductive tissues will result in

adverse effects at lower doses than in adult
 3-17

animals. Furthermore, because this

common MOA is shared across various

tissues, mechanistic data resulting from P450 and gene expression profiling will be

useful in developing new approaches for

harmonizing risk assessments across various

toxicities. These approaches should be

especially relevant and applicable in

assessing the risk of environmental

exposures for children. Research covered

by this project will evaluate the following:

• Effects of gestational exposure to

conazoles on gestational hormone

levels, gestation length, pregnancy

maintenance and pregnancy outcome;

• Effects of gestational exposure to

conazoles on female fetal

development;

• Effects of gestational and postnatal

exposure to conazoles on timing of

female puberty and adult reproductive

function;

• Effects of gestational exposure to

conazoles on male fetal development;

• Effects of gestational and postnatal

exposure to conazoles on timing of

male puberty and adult reproductive

function;

• Effect of gestational and postnatal

exposure to conazoles on the

developing neurological system in

both male and female rats;

• Tissue dosimetry of parent compound

(and potentially active metabolites) to

assist in interpretation of results of

toxicity studies; and

• Selected cancer-related biomarkers in

rats developmentally exposed for 90

days for comparison with 90-day

exposures in young adults (project 2).

Project 4 - Computational Toxicology: 

Integrating Toxic Effects, P-450

Modulation, SAR Analysis, and Gene

Expression pRofiling for Application to

Risk Assessments. The term computational

toxicology will be broadly employed in the

context of this project to encompass a

variety of techniques and approaches for

accessing and modeling existing and newly

generated data on the conazoles, their varied

biological responses, and the activities of

structurally and biologically related

chemicals. The ultimate aim is to derive

useful generalizations with respect to

chemical structure, gene expression patterns,

and biological activation mechanisms that

can inform and harmonize future evaluation

of conazoles as well as, perhaps, other

chemicals with P-450-mediated toxicities

and common MOA characteristics.

The operating hypothesis is that shared

structural features and physicochemical

properties, as well as common patterns of

gene expression, can serve as useful

organizational principles and potential

predictors of metabolic and biological

response. More specifically, characteristic

profiles of structural/biological response

could suggest appropriate chemical

analogues, narrow the consideration of

potential adverse effects and animal models,

and aid in the design of a rational testing

strategy for new chemicals. Research

covered by project 4 will involve the

following:

• Provide insight into the structural basis

for the P-450-mediated toxicological

effects of conazoles and related

compounds from survey of

environmental and pharmaceutical

data sources and use of SAR models;

• Use of computational informatic

approaches for analyzing gene

expression profiles of P-450-mediated

toxicities across multiple test systems

(chemicals, species, tissues, doses)
 3-18

towards the goal of harmonizing risk

assessments; and

• Utilization of relational database

searching strategies to explore broader

associations between P-450 expression

and activity, gene expression profiles,

chemical structure, and biological

effects to provide guidance in future

assessments of conazole-like

chemicals and P-450-mediated

toxicities.

Impact 

This program project directly addresses

the need to derive a commonly accepted set

of principles defining how MOA

information can be used in risk assessments,

particularly as it relates to extrapolation

issues. Evaluating whether chemicals have

a common MOA for their multiple toxicities

and the implications of that MOA for doseresponse assessment are two of the

fundamental challenges in harmonizing

human health risk assessment approaches. 

This project addresses both qualitative and

quantitative methods that could eventually

be applied to risk assessment activities.

Cross-Agency Interactions

From its inception, this project has been

discussed and modified based on numerous

meetings with the Office of Pesticide

Programs (OPP). OPP has provided critical

insight into chemical selection, dosing and

bioassay endpoints, throughout the project’s

development. We have already planned

periodic meetings with OPP during the

conduct of this project to keep them

informed about progress as well as to garner

advice on future studies.

3.5 Gap Analysis and Links to Other

Multi-Year Plans

NHEERL research on the harmonization

of risk assessment will help identify

principles concerning the use of mechanistic

data in cancer and non-cancer risk

assessment through the study of common

MOAs and key biological events associated

with three specific toxicity pathways, i.e.,

cell signaling pathways, alterations of LH

secretion, and modulation of P-450 and

XMEs. Because these are only several of

the many toxicity pathways that would be of

relevance to risk assessors, this may seem to

be a limitation. However, the pathways

selected are relevant to a large number of

environmental pollutants. It should also be

noted that the mechanistic research

described in this implementation plan has

significant links to several ORD MYPs (e.g.,

Air Toxics, Drinking Water, EndocrineDisrupting Chemicals [EDCs]). Information

from all of these areas will contribute to the

overall goal of developing a framework for

the use of mechanistic data in risk

assessment (see Figure 7). 

For example, research on EDCs focuses

on mechanisms of estrogenic, androgenic,

and thyroid-mediated effects. EDC research

will characterize the effects of exposure to

multiple EDCs in various combinations such

as those with similar and different MOAs

and will determine the degree to which

effects of EDCs with defined MOAs can be

extrapolated across chemical class. EDC

research to develop standardized protocols

for screening chemicals for their potential

endocrine-mediated effects is also based on

an understanding of potential mechanisms

of MOAs underlying the tests. Thus,

research on EDCs will inform discussions

on the identification of potential common

modes of action and will provide supporting

data with which to develop a framework

concerning the use of mechanistic
 3-19

Framework for the 

Use of Mechanistic 

Data in Risk 

Assessment

Air Toxics/PM Drinking Water/ 

DPBs/CCLs

Harmonization

Research in 

Goal 8 

Endocrine 

Disruptors

Pesticides and 

Toxic 

Substances

Figure 7 Framework for Use of Mechanistic Data

information in risk assessment. 

Furthermore, emerging technologies

involving the use of proteomic and genomic

techniques are used to develop sensitive

screens and tests for EDCs and will

complement harmonization research to

determine the utility of emerging

technologies. 

In the area of drinking water, much of the

research on (As) involves elucidation of the

MOA of cancer and non-cancer effects and

the development of PK models based on

mechanistic studies. The results from these

studies will complement harmonization

research on other potential common MOAs

with other chemicals (i.e., cell signaling,

oxidative stress, hormonally-mediated

effects). Harmonization research will also

benefit from work on the mechanisms of

carcinogenicity of priority disinfection byproducts (DBPs), an evaluation of the

scientific basis for common MOAs of DBPs,

and their application to risk assessment. 

Research to provide a sound scientific basis

for the development of Contaminant

Chemical List (CCL) #3 will also provide

mechanistic data on cancer and non-cancer

endpoints for selected CCL contaminants. 

Finally, research to improve data and tools

for assessing and managing potential health

risks associated with drinking water

contaminants will involve a development of

a weight-of-evidence approach based on

common mechanisms and toxicity for

cumulative risk assessment for drinking

water contaminants. Thus, mechanistic data

from research on several drinking water

contaminants will provide significant input

into the development of a framework for the

use of mechanistic data in risk assessment. 

Research on Air Toxics involves the

development of mechanistic data to support

risk assessment, including research to

estimate human health effects and aggregate

exposures to hazardous air pollutants

(HAPs), to extrapolate animal-to-human

data for selected HAPs, to determine the

shape at low doses of the dose response

curve, and to develop models for

characterizing and predicting toxicity of

selected air pollutants and mixtures based on

MOA information. Harmonization research

to develop a framework for the use of

mechanistic data in risk assessment will also
 3-20

benefit from mechanistic work to identify

the mechanisms of toxicity for PM

constituents and/or its sources. MOA

research on potential common MOAs of

pesticides and toxic substances will further

the development of a framework for use of

mechanistic data in risk assessment.

Research on the use of mechanistic data

in risk assessment will also significantly

affect the other themes described in this

implementation plan, i.e., susceptible

subpopulatons and cumulative risk. For

example, risk assessment for cumulative risk

(see Section 5) is based on the risk of

combined toxic effects of chemicals with

similar or dissimilar MOAs. Mechanistic

research to identify common MOAs and to

develop emerging technologies will be

crucial to developing the tools needed to

assess cumulative risks associated with

exposure to multiple chemicals, mixtures,

and other factors across the Source-toOutcome Continuum. Research on common

MOAs using prototypic chemicals to

provide the scientific basis for the use of

mechanistic data in risk assessment will also

identify key events in specific toxicity

pathways that can be used to develop

framework and protocols for assessing

cumulative exposures and risks and to

develop methods and measurement data that

will support models of cumulative

exposures, dose, and effects.

Much of the research on susceptible

subpopulations (see Section 4) focuses on

the biological basis for differential

responsiveness to chemical exposures. A

crucial factor that must be determined is

whether the MOA of a chemical is the same

in the susceptible subpopulation as in the

general population. For example, to

evaluate the risks from childhood exposure,

it would be important to know if PK factors

resulted in an increased concentration of the

active chemical at the target site or if key

events in the toxicity pathway are of

increased frequency or expressed at a higher

rate in the susceptible subpopulation. It is

also possible that infants or children may

have differential sensitivity because a

chemical acts on a developmental process

that is not present in the adult. 

Harmonization research on potential

common MOAs and identification of key

events in representative toxicity pathways

from research on harmonization will be

important in determining the variation in

susceptibility to environmental agents as a

result of health status, aging, and genetic

factors. This research will also evaluate the

relationship between adverse health

outcomes and exposure to environmental

agents in utero and during infancy and

childhood and will elucidate the role of

environmental agents in the induction and

exacerbation of preexisting diseases such as

asthma. 
 3-21

3.6 References

Bogdanffy, M.S., G. Daston, E.M. Faustman, C.

A. Kimmel, G. A. Kimmel, J. Seed, and V. Vu. 

Harmonization of cancer and non-cancer risk

assessment: proceedings of consensus-building

workshop. Toxicological Sciences 61:18-31

(2001).

Chang, L., and M. Karin. Mammalian MAP

kinase signaling cascades. Nature 410:36-40,

(2001).

Cooper, R.L., J. M. Goldman, and L. Tyrey. 

The hypothalamus and pituitary as targets for

reproductive toxicants. In K. Korach (ed.),

Reproductive and Developmental Toxicology. 

Dekker, New York, pp 195-210 (1998).

Cooper, R. L., J. M. Goldman, and T. E. Stoker. 

Neuroendocrine and reproductive effects of

contemporary-use pesticides. Toxicology and

Industrial Health 15:26-36 (1999).

Cooper, R. L., T. E. Stoker, L. Tyrey, J. M.

Goldman, and W. K. McElroy. Atrazine

disrupts hypothalamic control of pituitaryovarian function. Toxicological Sciences

53:297-307 (2000).

Hurley, P. M. Mode of carcinogenic action of

pesticides inducing thyroid follicular cell tumors

in rodents. Environmental Health Perspectives

437-445 (1998).

Laws, S. C., J. M. Ferrell, T. E. Stoker, J.

Schmid, and R. L. Cooper. The effect of

atrazine on puberty in female Wistar rats: an

evaluation in the protocol for the assessment of

pubertal development and thyroid function. 

Toxicological Sciences 58(2):366-376 (2000).

Morita, K., T. Ono, and H. Shimakawa. 

Inhibition of testerone biosynthesis in testicular

microsomes by various imidazole drugs. J.

Pharmacobio-Dyn 13:336-343 (1990).

Narotsky, M. G., D. S. Best, D. Guidici, and R.

L. Cooper. Strain comparisons of atrazineinduced pregnancy loss in the rat. Reproductive

Toxicology 15:61-69 (2001).

National Research Council. Science and

Judgment in Risk Assessment. National

Academy Press, Washington, DC, 1994.

National Research Council. Scientific Frontiers

in Developmental Toxicology and Risk

Assessment, National Academy Press,

Washington, DC, 2000.

Stoker, T. E., D. L. Guidici, S. C. Laws, and R.

L. Cooper. The effects of atrazine metabolites

on puberty and thyroid function in the male

Wistar rat: an evaluation in the male pubertal

protocol. Toxicological Sciences 67:198-206

(2002).

U.S. Environmental Protection Agency. 

Guidelines for Reproductive Toxicity Risk

Assessment. Federal Register 61:56274-56322

(1996).

U.S. Environmental Protection Agency. 

Guidelines for Neurotoxicity Risk Assessment. 

Federal Register 63:26926-26954 (1998).

U.S. Environmental Protection Agency. 

Propioconzaole, Establishment of Time-Limited

Pesticide Tolerances. Federal Register

64(54):13080-13086 (1999).

U.S. Environmental Protection Agency. Draft

Final Guidelines for Carcinogen Risk

Assessment. Risk Assessment Forum,

Washington, DC. EPA/630/P-03/001A. 2003.

Vinggaard, A.M., C. Hnida, R. Breinholt, and J.

C. Larsen. Screening of selected pesticides for

inhibition of cyp19 aromatase activity in vitro. 

Toxicology In Vitro 14:227-234 (2000).
 4-1

Section 4

Susceptible Subpopulations

4.1 Problem

The variability in responsiveness of

humans to environmental pollutants can be

associated with differences in biological

susceptibility. As discussed in the ORD

Human Health Research Strategy, variation in

susceptibility depends on intrinsic factors such

as life stage and genetic factors, as well as

acquired factors such as disease. With regard

to life stage, there are specific periods or

windows of vulnerability during development,

particularly during early gestation, but also

throughout pregnancy and early childhood

through adolescence when toxicants might

permanently alter the morphology and/or

function of an organ system. Children may

also be more vulnerable to specific

environmental pollutants because of

differences in absorption, metabolism, and

excretion. In addition, children’s exposures to

environmental pollutants are often different

from those of adults because of different diets

and different activities (e.g., playing on floors

and in soil and mouthing of their hands, toys,

and other objects) that can bring them into

greater contact with environmental pollutants. 

The effect of aging on response to

environmental exposures is another area of

uncertainty. Older adults respond differently

than younger adults to environmental

exposures because of age-related changes that

limit the body’s ability to maintain

homeostasis and respond to injury. Research

at NHEERL will examine the effect of aging

on responses to environmental pollutants and

will develop predictive models that can be

incorporated into the risk assessment process.

The genetic factors that could predispose

human subpopulations to adverse effects from

exposure to pollutants include genetic

polymorphisms for metabolizing enzymes,

differing rates of DNA repair, and different

rates of compensation following toxic insult.

The main scientific question for this research

is whether such genetic differences

significantly influence risk at realistic, low

dose exposures. Information on genepollutant interactions as a result of long-term

exposure to environmentally relevant

concentrations of pollutants will be explored. 

Preexisting diseases may influence the

response to environmental toxicants by

altering xenobiotic metabolism or otherwise

altering the host’s response in a synergistic,

additive, or antagonistic manner. ORD

research has shown, for example, that mice

challenged with influenza have increased

mortality from exposure to several

environmental agents including dioxin, ozone,

and ultraviolet radiation. NHEERL research

will develop animal models of disease having

a high incidence in the human population

(e.g., asthma) and will determine the effects of

the disease on the dose-response curves for

high priority environmental agents (e.g., air

pollutants). 

4.2 Goals

The overarching goal of NHEERL research

on susceptible subpopulations is to identify

the biological basis underlying differential

responsiveness of susceptible subpopulations

of humans to pollutant exposure. This

implementation plan will address the

following issues:

• Identifying the adverse effects of

susceptible subpopulations that are

qualitatively or quantitatively different

from effects in the larger population,

• Determining the PK and PD basis for

differential responsiveness of

susceptible subpopulations to

environmental agents,
 4-2

Identify Potential

Subpopulations

Not Covered 

By 10X UF

Develop 

Analogous

Animal Models

Determine Biological Basis for

Sensitivity

Confirm 

Hypotheses in 

Human Models

Figure 8 Critical Path for Research on 

Susceptible Subpopulations

Improve Scientific Basis of Risk Assessment

for Susceptible Subpopulations

• Developing methods and models in

animals that can be used to predict

responses in human susceptible

subpopulations,

• Determining the relationship between

exposures earlier in life to adverse health

effects later in life,

• Determine the influence of critical

periods of development on expression of

toxicity, and 

• Determining the quantitative

contribution of genetic polymorphisms

to responsiveness to environmental

agents.

4.3 Critical Path

Research to improve the scientific basis for

risk assessment of susceptible subpopulations

will follow the steps outlined in Figure 8. 

Research must first identify which

subpopulations in the general population may

not be protected by current risk assessment

methodologies. For example, in the case of

non-cancer risk assessments, an uncertainty

factor of 10 is used by risk assessors to

account for variability within the general

population. Research has indicated that this

factor, under some circumstances, may not be

fully protective of children’s health. 

NHEERL research will continue to focus on

infants and children as a susceptible

subpopulation while exploring the relative

sensitivity of other subpopulations, especially

older adults, those with preexisting diseases

such as asthma, and those with specific

genetic polymorphisms. Once a potentially

susceptible subpopulation has been identified,

steps will be taken to develop animal models

that are as analogous as possible to the human

situation. This step is important to ensure that

the results from animal studies can be

extrapolated to humans and will reduce

uncertainties in the risk assessment process. 

Appropriate animal models will also be used

to investigate the underlying PK and PD

differences between the susceptible

subpopulation and the general population. 

Results from animal studies will generate

hypotheses that can be tested in human

epidemiological studies, and results from

human observational studies will be examined

in animal models to help determine

susceptibility factors and underlying MOAs. 

NHEERL research on susceptible

subpopulations will be consistent with the

APGs and APMs for Susceptible

Subpopulations identified in the ORD Human

Health Research Multi-Year Plan (Appendix

C).
 4-3

4.4 Program Projects

Four program projects address life stage

issues. Program project “Identifying and

Validating Biologic Indicators of

Susceptibility and Sensitivity among Children

to Assess Potential Risk of Adverse Health

Outcomes” is concerned with identifying why

children are differentially susceptible to

environmental pollutants. Program project

“Extrapolating Across Windows of

Vulnerability to Assess Children’s Health

Risks Using Rodent Toxicity Data” focuses on

how exposure, dose, and effect information

can be incorporated into risk assessment

methods to account for interindividual

variability. Program project “Long-Term

Effects of the Developmental Environment”

addresses the long-term consequences of

perturbations during the in utero period and

various diseases later in life. Program project

“The Aged as a Susceptible Subpopulation”

focuses on the differential sensitivity of the

older adults to environmental exposures.

The program project “Environmental Risk

Factors for Asthma” focuses on environmental

influences that act directly as allergens to

induce asthma or agents that enhance either

the induction or exacerbation of the disease

via non-specific stimulation of immune or

inflammatory responses. Two program

projects address genetic components that may

predispose subpopulations to exposure to

environmental agents. The program project

“Oxidative Stress as a Common Susceptibility

Attribute for Non-Cancer Risks” focuses on

the role of underlying oxidative stress as a

common susceptibility factor guiding

response to pulmonary, cardiovascular, neural

and reproductive toxicants. Program project

“Genotype and Phenotype in the Metabolism,

Toxicity and Carcinogenicity of Arsenic”

focuses on genetic capacity of individuals to

metabolize inorganic arsenic (iAs) as a

primary determinant in susceptibility. 
 4-4

Program Project 4: Identifying and Validating Biologic Indicators of

Susceptibility and Sensitivity among Children to Assess Potential Risk of

Adverse Health Outcomes Associated with Environmental Exposure

Objectives

• Identify and validate comparable biologic

indicators in both test animals and humans

that can be used to investigate the risk of

environmental exposures associated with

cancer and functional impairments of the

nervous, immune, and reproductive

systems; and

• Identify genetic and physiologic factors

that can modify the associations between

exposures and outcomes in a similar

fashion in both animal and humans.

Scientific Approach

There is substantial public health concern

that variation in the quality of the environment

causes adverse health outcomes in children. 

Many causes of childhood mortality, disease,

and disabilities affecting quality of life are

plausibly associated with environmental

factors. In order to better inform policy and

risk assessment, multifaceted scientific studies

are needed to provide bioindicators which

help identify environmental factors that are

harmful, harmless, or helpful in terms of their

effects on children’s health and development. 

The development and validation of

biomarkers that are applicable across species

are vital to improve the precision of risk

estimates. Thus, the major unifying theme for

this program project is identifying and

validating biological indicators of

susceptibility and sensitivity to assess the

potential risk of adverse child health outcomes

associated with environmental exposures. 

This program project will assess a broad

range of potential biomarkers. For example,

neurodevelopmental biomarkers will focus on

neurotrophic factors (NTFs),

neurotransmitters, proinflammatory cytokines,

and classical conditioned responses. Research

on immunological biomarkers will assess

antibodies, cytokine proteins, message

analyses, and phenotypic lymphocyte profiles. 

Reproductive function will be assessed using

mRNA and protein expression analyses,

immunoglobulins, proteins and other markers. 

Cancer risk will be examined by looking at

underlying mechanisms which might account

for inter-individual phenotypic variation in

response measured by a wide range of health

indicators. All projects in this program

project will focus on maximizing animal-tohuman extrapolation by identifying and

validating common biomarkers with a focus

on samples that could be obtained noninvasively from human subjects. While

human studies will be generally observational

with respect to exposure assessment, animal in

vivo studies and in vitro studies allow for

direct manipulation of exposure and

evaluation of biological indicators that are

predictive of adverse effects. This interactive

and iterative process between animal and

human studies will allow for the anticipation

of the types of health effects that might be

expected based on specific exposures and

gives important clues about the underlying

MOAs for complex disease-exposure

relationships.

The research in this program project is

linked to the National Children’s Study

(NCS), a multi-agency, multi-disciplinary

longitudinal study designed to evaluate

children’s health in the United States (US). 

Under the Children’s Health Act of 2000, the

Agency will work with other Federal

Agencies to plan and implement the NCS. 
 4-5

Develop Biomarkers

of Neurologic

Function 

Develop Biomarkers

of Immune Function

Develop Biomarkers

of Reproductive 

Health

Develop Biomarkers

of Cancer and

Cancer Precursors

Battery of Biological Indicators to

Assess Potential Risk of Adverse

Child Health Outcomes

Project 1 Project 4 Project 2 Project 3

Figure 9 Critical Milestones for Research 

on Children’s Health

Research at NHEERL is designed to

coordinate animal and human studies that

focus on endpoints where the burden of

illness, disability, or death is high for children

in the US and where an environmental factor

is implicated in the disease process. One of

the unique strengths of NHEERL is the ability

to coordinate human and epidemiological

studies with toxicological studies in test

animals. This research program will influence

the selection of biomarkers assessed in the

NCS. The critical steps to be followed in this

program project are summarized in Figure 9. 

Project 1 - Biomarkers of Neurologic

Function in Children. Neurological and

developmental disorders in children are a

major public health concern. The etiology of

most mental retardation and many other

neurodevelopmental disorders is unknown. 

Autism spectrum disorder may be increasing

in prevalence, and some neuropsychologic

conditions of childhood such as attention

deficit-hyperactivity disorder (ADHD) are

being diagnosed at an epidemic rate. 

Environmental exposures to known or

suspected neurotoxicants have been

implicated in these disorders of children. In

order to better understand the mechanisms

that link exposures to neurodevelopmental

outcomes in children, biomarkers in test

animals that can be applied to human

populations will be developed. Effects that

are observed in human populations will be

more fully evaluated by characterizing doseresponse relationships using animal models

(Barone et al., 1998; Lassiter et al., 2002;

White et al., 2002).

One series of studies will attempt to

demonstrate the association and predictive

validity of early postnatal serum measures of

NTFs and neurotransmitters with chemicalinduced brain damage in rats with the goal of

validating comparable early postnatal serum

measures with autism spectrum disorders,

ADHD, Downs Syndrome, and cerebral palsy. 

The hypothesis for this work is that alterations

in expression profiles of NTFs, neuropetides,

and cytokines are predictive for

developmental disorders and functional

impairments of the nervous systems. These

studies will:
 4-6

• Evaluate the association and predictive

validity of early postnatal serum

measures of NTFs, cytokines, and

neurotransmitters with chemicallyinduced brain damage in animal models;

• Assess the feasibility of measuring early

postnatal serum measures of NTFs,

neurotransmitters, and cytokines in

human infants; and

• Assess the feasibility of determining the

predictive validity of early postnatal

serum measures of NTFs,

neurotransmitters, and cytokines in

human infants with developmental

neurological disorders.

A second series of studies will focus on

developing analogous methods to evaluate

neurobehavioral function and animal models. 

The hypothesis for this research is that

developmental disorders in humans are linked

to fundamental processes that are common to

humans and experimental animals and can be

quantified. These studies will:

• Assess the feasibility of a field ready test

system based on classical conditioning

(e.g., eye blink conditioning) for use in

human infants;

• Investigate other potential measures of

neurobehavior (e.g., signal detection,

reaction time) applicable in both humans

and experimental animals;

• Establish the ontogeny of motor function

in an animal model ;

• Determine whether early (prenatal,

postnatal and/or perinatal) exposure to

toxicants alters the ontogeny of motor

function in animals and humans;

• Establish a conditioned-behavioral test

for quantifying susceptibility in animals

and humans; and

• Determine whether early exposure to

toxicants alters awareness and sensitivity

to the consequences of behavior in

animals. 

Project 2 - Biomarkers of Immune

Function, Developing Animal Models that

Predict Immune Function in Human

Children. Xenobiotic exposure during

critical windows of immune system

development can cause alterations in function

that persist into adulthood, and possibly for

life (Weisglas-Kuperus et al., 2000). 

Evidence obtained in experimental animals

strongly suggests that the developing immune

system is much more susceptible than that of

the adult. This project will test the hypothesis

that results of standard clinical immunological

assays (e.g., antibody responses, cytokine

protein and message analysis, and flow

cytometric phenotyping of lymphocyte

subsets) are useful predictors of changes in

host immunocompetence and provide detailed

information on the specific response to

antigens or vaccines. Included in the

definition of xenobiotics are environmental

microorganisms and infectious agents; thus,

the influence of early-life infection on

immunocompetence (both positive and

negative) will be evaluated. The results may

provide useful markers for increased

susceptibility to infectious disease and may

predict the loss of homeostatic control that

may lead to the development of immune

suppression, allergy, or autoimmune disease. 

Many disorders of childhood have an immune

function component and some, e.g., asthma,

are increasing and suspected to have

environmental antecedents. The hypothesis

for this work is that xenobiotic exposure

during critical windows of immune system

development will alter immunological

function that persist into adulthood and

possibly for life. Research in project 2 will

• Evaluate the effects of developmental

exposure to pesticides and assess the

predictive value of the animal model for

responses of children exposed to

environmental chemicals,
 4-7

• Assess the effects of developmental

exposure to endocrine-active xenobiotics

on immune function in animals, and

• Determine MOA of rodent

developmental immunotoxicants as a

means to identify candidate markers that

reflect possible effects in exposed

humans.

Project 3 - Biomarkers Related to

Reproductive Health Risks. Environmental

exposures during preconception and early

pregnancy can affect fertility, outcome of

pregnancy, and the health and development of

children from the pregnancy. Two approaches

will be used to evaluate reproductive health

risks. One uses samples obtained with

minimal invasiveness (e.g., hair, milk, and

blood) that may be accessible for collection

from human subjects and compares the results

between effects on surrogate and target-tissue

(e.g., gonads) markers of exposure and effect. 

The second approach looks at the placenta to

help identify useful biomarkers related to the

interface between the outside environment of

the mother and the developing fetus. Both

approaches are important for predicting the

eventual health of the offspring. In addition to

biomarkers that may be useful to predict the

offspring’s eventual reproductive capacity,

biomarkers that predict reproductive success

in the parents also have predictive value for

child health outcomes. Parents with

suboptimal fertility (such as those with a long

time-to-pregnancy) are at higher risk for

adverse pregnancy outcomes including low

birth weight and preterm delivery. The

biomarkers developed in this project may

have value in measuring children’s risk

associated with their parents reproductive

capacity as well as predicting the child’s

reproductive health. The hypothesis for this

project is that gene expression profiling of

reproductive tissues can reveal genetic

biomarkers of exposure and effect for

reproductive toxicants. Studies included in

project 3 are as follows:

• Evaluating the best methods to collect,

store and transport surrogate tissues

(blood, hair follicles, uroepithelial cells,

and semen) from humans and rodent

models such that sufficient quantities of

good quality RNA can be extracted for

gene expression analysis or

identification purposes, respectively;

• Confirming that historical, archived

placental specimens have sufficient

macromolecular integrity for utilization

in mechanistic/biomarker studies;

• Exposing fetal, juvenile, and adult rats

and mice to effective doses of known

and suspected reproductive toxicants

(i.e., disruptors of steroidogenesis such

as conazole fungicides) and using gene

expression analysis of target tissues

(testis, prostate, ovary, uterus) to

identify genetic biomarkers;

• Determining whether gene or protein

expression profiling in surrogate tissues

can provide reliable biomarkers for

exposure to reproductive toxicants (e.g.,

conazoles, PCBs, chloroatrazines) in

adult humans; and

• Determining whether gene expression

profiling in surrogate tissues from

children (blood, hair follicles,

uroepithelial cells) is possible using the

same methods and genes identified in

the fetal/juvenile rodents and adult

humans.

Project 4- Biomarkers of Cancer and

Cancer Precursors: Identification of

Genetic Susceptibility, Biomarkers of Gene

Expression, and Oxidant Status. Most

suspected associations between environmental

exposures and adverse health outcomes for

children are likely to have a component of

gene-environment interaction or variations in

susceptibility. These variations in

susceptibility make it difficult to assess risk in

the general population because we are unable

to effectively identify any at-risk subgroups. 

Consequently, the heterogeneous risk pool
 4-8

dilutes risk estimates and drives them towards

the null. The aims of this project are designed

to enhance our ability to determine the

important genetic factors involved in geneenvironment interactions and to assess

potential co-factors such as reactive oxygen

species (ROS) and antioxidant status and how

they relate to disease. The capacity ability to

measure similar processes in animal and

human studies will advance our ability to

determine which genetic and physiologic

factors are most important in assessing

children’s environmental health risk. 

Research in project 4 will:

• Evaluate whether dose, individual and

age-specific (adult vs children)

differences in sensitivity to chemicals

and mixtures can be assessed in human

blood, plasma, and/or individual blood

cells following in vitro treatment to

environmental agents;

• Examine various housekeeping genes

and their functional products, such as

those associated with DNA repair, cell

cycle control and apoptosis, to determine

if they respond differently to chemical

stressors in a manner that is dependent

on the life stage;

• Assess the utility of breast milk

epithelium and blood cells for

determining the contribution of genetic

variability (expression profiles and

polymorphisms) on an individual’s

sensitivity to environmental toxicants;

• Evaluate the extent to which plasma

concentrations of antioxidants

(ascorbate, urate, tocopherols and

glutathione) are cofactors that might

modulate risk factors in the initiation of

diseases in children;

• Validate use of human blood ex vivo in

the evaluation of dose, genetic, and agespecific factors that affect inter- and

intra-individual phenotypic disease

measurements; and

• Develop reliable assays to measure

endogenous and exogenous constituents

of breast milk that are implicated in

altering health status of women or their

breast-fed children.

Impact

Under the Children’s Health Act of 2000,

the Agency is working together with other

federal partners to plan and implement the

NCS. Within ORD, the importance of

research on children’s environmental health is

a long-standing priority with critical

implications for risk assessment. This

program project will demonstrate scientific

leadership in children’s environmental health

research by coordinating animal and human

studies that focus on endpoints where the

burden of illness, disability or death is high

for children in the US and where an

environmental factor is implicated in the

disease process. One of the unique strengths

of this implementation plan is the ability to

coordinate human and epidemiologic studies

with toxicology studies in test animals. This

research program can influence the selection

of biomarkers assessed in the NCS, which will

be the major study of children’s

environmental health of this generation.

Cross-Agency Interactions

This program project benefits from

substantial interaction and coordination with

Agency efforts in planning the NCS. In

cooperation with several other federal

agencies, under the leadership of the director

of the National Institute for Child Health and

Human Development, the Agency has a strong

role in developing this major child

environmental health study. The Agency

representatives to the Interagency

Coordinating Committee (ICC) of the NCS

are supportive of this program project and

maintain contact with the investigators to

insure that the biomarkers evaluated and
 4-9

validated will be of optimum utility. 

Members of the ICC come from across the

National Institutes of Health (NIH), Centers

for Disease Control and Prevention (CDC),

and the Agency. The Agency representatives

are from NHEERL, NERL, and NCEA. This

proposed research plan has been reviewed by

members of the ICC and the lines of open

communication are insured by the NCS webbased portal (a copy of this research proposal

and updates are maintained on the portal for

ready access by study planners). The study

planners also interact regularly with NCER

regarding extramural funding for NCS

projects. This program project is also

integrated with a series of Agency-funded

pilot studies for the NCS which brings

together investigators from across ORD to

support method development for the NCS. 
 4-10

Program Project 5: Extrapolating Across Windows of Vulnerability to Assess

Children’s Health Risks Using Rodent Toxicity Data

Objectives

• Focus modeling and laboratory research

around selected case studies including: 

volatile organics (including methanol),

organophosphate (OP) pesticides, and

conazoles;

• Obtain physiological, biochemical, and

anatomical parameters for rodents and

humans for selected windows of

vulnerability;

• Quantify metabolism, and perhaps other

PK processes such as absorption in humans

and rodents for the selected windows of

vulnerability with in vitro methods;

• Develop physiologically based PBPK

models and generate rodent PK data for the

case study chemicals during the windows

of vulnerability in rodents and humans;

• Develop human exposure models during

the windows of vulnerability to link to the

PBPK models; and

• Delineate the magnitude of age-related

differences in sensitivity to pesticides and

elucidate mechanisms (kinetic and/or

dynamic) underlying this difference in

sensitivity.

Scientific Approach

There are windows of vulnerability that

exist during various life stages during which

exposure to environmental chemicals may

result in permanent damage to biological

systems. These windows may exist at various

stages of gestation, as well as postnatally,

during infancy, childhood, or adolescence. 

One major source of uncertainty and potential

source of error in the assessment of the health

risk of children derives from developmental

events occurring in different time periods for

different species. This is particularly apparent

for events that occur in the early postnatal

(lactational) period for rodents and in utero

for humans. Thus, lipophilic chemicals such

as PCBs, which disrupt thyroid hormones,

cause developmental hearing toxicity in

rodents during the lactational period due to

massive mobilization from the mother’s fat

reserves to her milk during lactation. This

same effect would not likely occur following

postnatal exposure in humans because hearing

development occurs in utero. On the other

hand, the situation would be reversed if a

chemical were preferentially accumulated in

the fetus. Clearly, extrapolations between

animals and humans will be dependent upon

the developmental processes at risk. Because

animal toxicity data will continue to play an

important role in the assessment of a

chemical’s potential to affect children’s

health, there is a need to develop a systematic

approach to assess the relevance of animal

toxicity data occurring during different

developmental periods, both prenatal and

postnatal, to humans and to extrapolate the

response from animals to humans. It will be

necessary to first identify critical periods of

developmental processes in controls before

beginning studies using very specific

chemicals with known MOAs. The critical

steps for research in this program project are

summarized in Figure 10. 

Project 1 - Literature Review. The main

objective of this effort is develop a framework

that includes exposure scenarios and PK

processes associated with different windows

of vulnerability to assess children’s health risk

from animal toxicity data. The following

issues will be addressed by the literature

review:

• Physiological parameters for absorption,

distribution, metabolism and elimination

of selected environmental chemicals will
 4-11

PROJECT 1

Develop Framework

of Exposure and PK

Processes for

Different Windows

PROJECT 2

Delineate Magnitude

of Age-Related

Difference to

Insecticides

PROJECT 3

Determine 

Developmental PK 

for Fungicides

Establish Relevance of Animal

Toxicity Data from Developmental

Studies to Humans

Figure 10 Critical Path for Research on 

Windows of Vulnerability

be obtained for several animal species

in vitro PK parameters will be determined 

in order to:

- identify the enzymes involved 

in chemical metabolism and to quantify

their expression in adults and children

when that variance may contribute to

susceptibility to toxic injury and

- develop chemical-specific in vitroderived measures of tissue distribution 

of parent chemical and toxicologicallyactive metabolites.

• Develop PBPK models and rodent PK

information for various life stages. 

Methanol was selected due to ongoing

risk assessment activities in NCEA. 

Conazoles were selected in conjunction

with the developmental studies planned

for program project 3.

• In collaboration with other ORD

Laboratories/Centers, develop exposure

models.

Project 2 - Determine Age-Related PD

Changes. The objective of project 2 is to

delineate the magnitude of age-related

differences in sensitivity to pesticides and

elucidate mechanisms (kinetic and/or

dynamic) underlying this differential

sensitivity. The approach has involved

systematic comparisons of the dose-response

and time-course of pesticide effects in rats of

different ages, followed by development and

testing of hypotheses concerning the

mechanisms for these differences. Behavioral

and neurochemical (cholinesterase [ChE]

inhibition) evaluations have been used to

characterize the acute dose-related effects at

the time of peak effect of several

anticholinesterase pesticides. One outcome of

prior research in this area has been to

emphasize the effect of kinetic differences in

detoxification pathways and to suggest that

the age-related differences in kinetics can

predict overt toxicity. Research will continue

in this area to characterize the absorption,

distribution, metabolism, and excretion of
 4-12

ChE-inhibiting pesticides in young and adult

animal models.

Project 3 - Evaluate Developmental PK of a

Class of Prototypic Pesticides. This project

will evaluate developmental PK information

on selected conazoles associated with

developmental toxicities. It serves as a case

study (as described in project 1 above) and is

a component of the harmonization proposal

addressing modulation of P-450s and other

xenobiotic metabolizing enzymes using

conazoles as a case study (see Section 4). The

PK factors evaluated will be useful both for

better understanding extrapolations across age

groups and across species. The following

studies will be performed.

• Determination of the ability of selected

conazoles to transfer to mother’s milk in

rats, and

• Comparison of tissue dosimetry of

selected conazoles in pregnant and nonpregnant females, as well as assessing

the tissue dose to the fetus.

Impact 

This program project will identify

approaches to address PK and PD issues

related to extrapolation across windows of

vulnerability in multiple species. This will

provide options for risk assessment or for

additional research to improve those risk

assessments. This research will also define

for developmental endpoints and chemical

classes what risk assessment approaches are

feasible addressing windows of vulnerability

and what additional work is required to reach

a systematic set of guidelines for addressing

these issues of interspecies extrapolation for

exposure and dosimetry. Finally, the

development of computer simulation models

that can be parameterized for different species

and developmental time periods will be useful

for future development of guidelines dealing

with PK and PD issues across different life

stages. 

Cross-Agency Interactions

This program project involves major

collaborative efforts between NHEERL,

NCEA, and NERL arising, in part, from

internal funding. The selection of case studies

and the implementation of the research is

being done jointly.
4-13

Program Project 6: Long-Term Effects of the Developmental Environment

Objectives

• Develop animal models to evaluate effects

of developmental perturbation on adverse

health outcomes during adulthood,

• Assess key health outcomes during

adulthood following developmental

perturbation,

• Investigate mechanism(s) of adverse health

effects in adults following developmental

perturbation, and

• Provide input into interpretation of low

birth weight data from Agency testing

guideline studies.

Scientific Approach

Clinical and epidemiological studies have

shown significant correlations between

conditions during development and various

diseases later in life. In humans, low birth

weight has been used as a surrogate for

adverse developmental conditions, but the

specific conditions affecting birth weight are

usually unknown. Human studies demonstrate

inverse correlations between birth weight and

risk of later disease, and these correlations

hold even within the range of normal birth

weight. Adult-onset diseases with which birth

weight has been inversely correlated include

hypertension, coronary heart disease, diabetes,

obesity, schizophrenia, and early onset

chronic renal failure (Godfrey and Barker,

2001). There is also evidence that, for female

fetuses, the in utero environment can

significantly affect pregnancy outcome in

adulthood. Studies have shown that reduced

infant birth weight and other adverse effects

are more closely correlated with the mother’s

weight at her birth (i.e., maternal intrauterine

environment) than with her infant’s prenatal

environment (Rhind et al., 2001). While birth

weight has been used as a convenient and

available marker of developmental conditions

in humans, it is important to make clear that

adverse developmental conditions do not

always affect birth weight although such

adverse conditions may still have long term

effects. 

The most common adverse effect seen in

standard rodent developmental toxicology

studies is reduced fetal weight at term. 

Current Agency guidelines for interpretation

of developmental toxicity studies state that

fetal weight is an adverse outcome on par with

death and malformation, the other adverse

outcomes detectable in fetuses at term. 

However, the interpretation of reduced fetal

weight is a contentious subject, especially

when fetal weight is affected only at

maternally toxic exposures. Current study

designs do not examine the long term health

of offspring exposed during development (in

utero and/or postnatally). The utilization of

animal models will help to define the causal

factors and identify long-term effects under

controlled experimental conditions that are

impossible to duplicate in epidemiological

studies. Current developmental toxicity tests

do not address this important issue and may

be missing important adverse effects highly

relevant to the human condition. Maternal

undernutrition during pregnancy in rats has

been shown to cause hypertension, diabetes,

and other metabolic derangements, as well as

adverse neurological sequelae in offspring. 

This program project will examine the long

term health effects of toxic exposures during

development. The overall goal of this

program project is to determine the conditions

under which developmental perturbations will

lead to adverse health outcomes at adulthood. 

There is evidence that some environmental

contaminants may alter developmental
 4-14

Determine Conditions Under Which Perturbations 

During Development Produce Permanent Adverse Effects

Figure 11 Critical Milestones for Research 

on Developmental Environment

Project 1 Effects on PN 

Growth, Cardiovascular,

Renal, Glucose Metabolism

Project 2 Effects on PN 

Immune Function 

Project 3 Identify 

Placental Biomarkers

of Persistent Effects 

Project 6 Intergenerational

Effects on Female

Reproduction 

Project 5 Imprinting

of Key Metabolic

Pathways and Adverse

Outcomes 

Project 4 Effects on

Herbicides on Adult 

Adverse Outcomes 

Project 8 Effects on 

Development of 

Allergy 

Project 7 Molecular 

Pathways Involved in

Embryo/Fetal Programming 

programming in a manner that does not

necessarily result in malformations but affects

function later in life. The best-studied

examples of this are environmental agents

with endocrine activity, and these agents

typically affect the reproductive tract or

reproductive function. Effects of these agents

may not become apparent until puberty, or in

some cases, may only hasten the onset of

reproductive senescence much later in life. 

Latent health effects due to developmental

exposures to other classes of agents have

received scant attention, but there are

examples of neurological and

neurodegenerative effects of developmental

exposures. There is evidence that in utero

exposure to PCBs leads to altered thyroid

function and learning disabilities later in life. 

The mechanisms underlying these effects are

poorly understood, but well-designed studies

and new technologies such as gene expression

profiling should begin to reveal clues to the

developmental pathways involved. Studies

using this technology will be designed and

incorporated into this program project as

results emerge from the more descriptive

animal studies. Critical milestones associated

with this program project are summarized in

Figure 11.

Project 1 - Effects of Developmental

Toxicant Exposure or Undernutrition on

Adult Physiology. Significant decrements in

fetal weight are the most common adverse

outcome of rodent developmental toxicity

bioassays in which pregnant animals are

exposed to xenobiotics. Maternal

undernutrition has been studied extensively in

rodents and is an established method of

inducing lower birth weight in offspring

without exposure to toxic chemicals. It was

observed over 35 years ago that poor nutrition

during pregnancy led irreversibly to reduced

cell number in many tissues. It has since been

established that maternal protein restriction

affects islet cells and insulin-sensitive tissues

such as the liver, muscle, adipocytes, kidney

and brain in the offspring. Maternal

undernutrition also programs offspring for

hypertension, and this can occur even when
 4-15

the undernutrition is only transient during

early gestation. Hypertension has also been

noted as early as 4 weeks of age in rodent

models of fetal programming, and this

hypertension persists throughout life

(Langley-Evans et al., 1998). Among major

organ systems, the kidney appears to be

targeted specifically by maternal

undernutrition, leading to decreased kidneyto-body weight ratio and fewer nephrons in

the kidney at birth (Merlet-Benichou et al.,

1994). Prenatal malnutrition resulting in low

birth weight pups has also been associated

with behavioral abnormalities that persist into

adulthood. Cognitive deficits have been

reported in a variety of animal models using

both behavioral assessments of learning and

memory and neurophysiological indices of

synaptic plasticity (Kehoe et al., 2001). The

hippocampus subserves memory function in

humans, nonhuman primates, and rodents and

is also a target for glucocorticoids. Neonatal

stress induced by isolation induces changes in

transmitter function and neuroendocrine

activation and contribute to abnormal

behavioral and neurochemical responsiveness

later in life. Studies in project 1 will expose

pregnant rats to toxicants or undernutrition

during various developmental windows and

offspring will be observed during their entire

life span. This research will:

• Demonstrate that physiologic and

metabolic development of an organism

is shaped in part by its developmental

environment, and adverse developmental

conditions can produce permanent and

deleterious physiological changes which 

may become apparent later in life. 

Project 2- Long-Term Immune Function

Deficits Following Immune System

Perturbation During Development. 

Perturbation of immune system development

by various stressors has been documented to

result in functional decrements that may

possibly persist for life. For example,

perinatal and/or early postnatal exposure to

chemicals such as dioxin, organotins, and

organochlorine pesticides has been

demonstrated to suppress immune system

function later in life (Smialowicz et al., 2001). 

These results indicate that the developing

immune system is more susceptible to

perturbation by chemicals and pesticides than

is the adult immune system and that these

alterations may persist into adulthood, and

possibly for life. Research in project 2 will

further investigate the conditions under which

in utero and/or early postnatal stressors lead to

permanent and deleterious alterations of the

immune system at any stage of post-weaning

life. This research will:

C Demonstrate that results of standard

immunotoxicological assays (e.g.,

antibody responses, T cell-mediated

responses, cytokine protein and message

analysis, and flow cytometric phenotype

analysis of lymphocyte subsets) in the

rat are useful predictors of long-term

changes in immunocompetence that may

occur in humans.

Project 3 - Placental Tissue as a Potential

Source of Biomarkers Associated with

Intrauterine Growth Retardation (IUGR)

and Long-term Health Effects. There is

substantial evidence in the scientific literature

linking exposure to air pollution, cigarette

smoke and other agents to adverse pregnancy

outcomes including low birth weight,

intrauterine growth retardation (IUGR),

eclampsia, hypertension, premature delivery,

and effects on maternal health (Schardein,

2000). Many of these responses are

associated with physiological and

morphological changes in the placenta that

affect function, e.g., delivery of nutrients and

oxygen to the conceptus. Thus, the placenta

can be a source of biomarkers that relate to

exposure as well as to health effects. For

example, changes in gene expression, enzyme

induction/activity, and vascularization, among
 4-16

other endpoints, can be detected/monitored in

placental tissues from at-risk pregnancies and

compared with normal term placenta. Project

3 will develop a rodent model to evaluate the

effects of low birth weight on future health

and responses to toxicants. This research will

evaluate the role of altered placental

morphology, function, and gene expression in

the production of intrauterine growth deficits

and the long term health effects associated

with IUGR. This research will:

C Provide a basis for comparing animal

and human responses to toxicants for the

potential to impact intrauterine growth

and development. 

This project will also complement the

research proposed under program project 4

“Identifying and Validating Biologic

Indicators of Susceptibility and Sensitivity

among Children to Assess Potential Risk of

Adverse Health Outcomes Associated with

Environmental Exposures.” In that program

project, human placental specimens will be

obtained and examined with genomic,

enzymatic, protein expression assays, and

proteomic analyses. The identification of

biomarkers that occur in both species would

facilitate laboratory study of toxicants

implicated in IUGR and having long-term

health consequences. Research in project 3

will:

C Demonstrate that exposure to toxicants

affects growth and function of the

placenta, contributing to adverse effects

during gestation that persist throughout

life and can predispose to disease and

disability, and

C Compare human and rodent biomarkers

associated with IUGR and adverse

health outcomes.

Project 4 - Effects of High use Herbicides

on Breast and Prostate Development and

Life-Time Disease Risk in Humans and

Rodents Following Gestational or

Lactational Exposures. The overall

objective of this project is to determine the

effects of gestational exposure to

chlorophenoxy and chlorotriazine herbicides

on breast and prostate development and to

compare their adverse health risk outcomes in

the rodent and human. Historically, 2,4-

dichlorophenoxyacetic acid (2,4-D) and

atrazine have been the top two herbicides used

in US agriculture by acre. Although the two

families of compounds differ in potential

MOAs and metabolic clearance, they have

both been shown to have adverse health

outcomes following fetal or neonatal animal

exposures and little effect in adult animals. 

These studies will:

C Compare post-exposure health outcomes

in animal models with epidemiological

data in high chlorophenoxy and

chlorotriazine herbicide use counties

within the US.

Previous studies have shown that late

gestation exposure to atrazine caused impaired

mammary gland development in dams and

their female offspring, increased incidence of

prostatitis and lipomas in male offspring

(Fenton et al., 2002), and left the female

offspring with enhanced sensitivity to the

effects of a chemical carcinogen,

dimethylbenz[a]anthracene. Another study

found that atrazine, via modulation of

prolactin levels during early postnatal life,

increased the incidence and severity of

prostatitis in male offspring. Atrazine and its

individual metabolites induced adverse

reproductive tissue effects if delivered around

the time of puberty. However, there is little

information concerning the effects of 2,4-D. 

Studies in project 4 will:
 4-17

C Provide data on breast and prostate

developmental outcomes and cancer and

non-cancer endpoints following

exposure to chlorotriazine and

chlorophenoxy herbicides during periods

critical to target tissue or brain

development,

C Provide dose-response information for

atrazine and its metabolite mixtures and

chlorophenoxy herbicide exposures

during pregnancy and lactation in rats,

and

C Characterize adult risk to these

compounds following childhood

exposures in rodent models and in

humans.

Project 5 - Long-Term Metabolic System

Perturbations after Developmental

Exposures. Drug metabolizing systems are

known to be present to varying extent in

tissues of the embryo and fetus and can

influence the response of the conceptus to

toxic chemicals (Miller et al., 1996). Less

understood is how the ontogenetic profiles of

drug metabolizing enzymes are influenced by

exposure to toxicants and how toxicant

exposures during ontogeny of drug

metabolizing systems might affect their

expression and inducibility during adulthood. 

Permanent alterations in enzyme expression

and/or inducibility due to the developmental

environment could be a basis for changes in

susceptibility to toxic exposures in later life. 

Alterations in some of these metabolizing

systems could also adversely affect

metabolism of endogenous substrates

including steroid synthesis. Research in

project 5 will:

C Demonstrate that during pre- and

postnatal development there are critical

metabolic pathways which may be

imprinted by the developmental

environment, leading to permanent

changes in their expression and/or

inducibility. 

Project 6 - Intergenerational Effects on

Female Reproduction. There is evidence

that, for female fetuses, the in utero

environment can significantly affect

pregnancy outcome later in life. Studies have

shown that reduced infant birth weight and

other adverse effects are more closely

correlated with the mother’s weight at birth

(i.e., maternal intrauterine environment) than

with her infant’s prenatal environment. There

is a body of epidemiological data that

indicates a correlation between the perinatal

environment of a woman and her chances of

having an adverse pregnancy outcome. There

is concern about the relationship of the health

of mothers and the health of infants. Research

suggests that the incidence of abnormal

pregnancy outcomes including stillbirths in

women within lower social classes might be

associated with childhood stunting brought on

by a poor environment. It has also been

shown that the incidence of neural tube

defects, stillbirths, and low weight babies is

high in women born during periods of general

malnutrition. Research in this project will:

C Examine the effects of developmental

exposures on adult female fertility,

pregnancy maintenance and response to

developmental toxicants and

C Determine whether a female’s in utero

environment affects her ability to

maintain a normal pregnancy and

delivery in adulthood.

Project 7 - Gene-Expression Profiling for

the Elucidation of Developmental Pathways

Involved in Embryo/Fetal Programming. 

New technologies for rapidly screening for

changes in gene expression offer exciting new

avenues for the elucidation of pathways of

developmental toxicity. There are a limited

number of conserved signaling pathways

involved in the process of development, and

the functions of these pathways are becoming

better understood. Because the biology

underlying embryo/fetal programming is
 4-18

poorly understood, gene expression profiling

may be the best approach for beginning to

understand the process of embryo/fetal

programming. Gene expression microarrays

have already been used to examine

mechanisms of teratogenesis, but there are no

established approaches for carrying out such

work. These studies will be initiated only

after adverse effects have been established in

previous projects. Gene expression profiles

will be assessed using microarray technology

and real-time quantitative polymerase chain

reaction (PCR). The specific developmental

times and tissues/structures to be studied will

depend on the nature of the effects observed in

the descriptive studies. This research will:

C Demonstrate that embryo/fetal

programming involves changes in gene

expression leading to permanent

alterations in postnatal and adult

physiology, and these changes in gene

expression can be detected at early

stages using expression microarray

technology.

Project 8 - The Effects of Low Birth Weight

on the Development of Allergy. Research in

this project will determine if low birth weight

resulting from either maternal undernutrition

or in utero toxic exposure plays a role in

allergy development. Respiratory and oral

(food) allergies are thought to develop in early

life in a genetically predisposed population.

This project will:

C Determine if low birth weight affects

allergy development by increasing

susceptibility to sensitization, and/or by

enhancing the severity of allergic

responses. 

Impact

This project has the potential to change the

basic study designs for assessing

developmental toxicity and to reveal latent

toxic effects not seen before. If the results of

this study demonstrate long-term morbidity

associated with prenatal and/or postnatal

exposures, it may be necessary to extend

existing study designs or create new designs

to test for such effects. Perhaps some types of

morbidity associated with developmental

exposures are general in nature, while others

are chemical-specific. We may find that no

increase in disease is associated with

exposures that also affect fetal or early

postnatal growth, in which case we may not

need additional tests; however, such results

would clearly raise the significance of affects

on fetal weight or postnatal growth seen using

current study designs. These studies thus have

the potential to bring significant

improvements to the risk assessment process.

Cross-Agency Interactions

This project is being carried out in

collaboration with Dr. C. Kimmel of NCEA,

who will provide a critical risk assessment

perspective to the design, interpretation, and

application of these studies.
 4-19

Program Project 7: Susceptible Populations: Susceptibility Associated with

Older Adults 

Objectives

C Determine the characteristics that define

older adults as a susceptible subpopulation,

C Determine PK and PD basis for differential

responses of older adults, and

C Determine how susceptibility in older

adults varies across systems/tissues.

Scientific Approach 

By the year 2030, one in every five

Americans will be older than 65 years, nearly

double the current population. Given the

burgeoning of this subpopulation, it is critical

for planning for the care of the older adults to

determine whether environmental exposure to

toxic agents can result in an age-related

accelerated decline in function or increase in

diseases of aging such as cancer. Current

testing guidelines do not include toxicological

assessments focused on older adults. 

Research on aged human and animal models

will improve the scientific underpinning of

risk assessment decisions for the aged as a

susceptible population by moving risk

assessment toward more biologically-based

decisions about uncertainty factors. 

The goal of this project is to understand

whether responses to environmental insults

differ in the aged compared to the young adult

population. Research designed to reach this

goal will generate data that will identify and

prioritize those functions and mechanisms that

most lead to age-related decline. By doing so,

these studies will address the uncertainty of

whether toxicity data collected in young

adults provides a sufficient margin of safety to

protect against effects that may result from

toxic exposure to an aged individual. Older

adults comprise a subpopulation that may

have special susceptibilities to

toxicant-induced dysfunction or degeneration

due to the critical characteristics of their

life-stage. Understanding the biological basis

for a differential response in older adults can

provide the rationale for decisions on how to

appropriately incorporate the differential

sensitivity of older adults into an

environmental risk assessment framework.

The candidate mechanisms driving agerelated susceptibility that will be considered

include changes in PK, age-related alterations

in cell signaling, deposition and accumulation

of harmful metabolic by-products, natural

decline in reserve capacity, cumulative gene

replication error-induced alterations, changes

in processes specific to the cell cycle, DNA

repair, cellular homeostasis, and response to

oxidative stress.

This program project includes research in

two of the major areas of concern to older

adults, i.e. cancer and degeneration in the

central nervous system (CNS). The project on

carcinogenesis will bring to bear the many

aspects of initiation, promotion, repair

processes, and homeostasis on the issue of the

interaction between aging and toxicantexposure leading to tumor production. The

studies on neurodegeneration concentrate on

age- and toxicant-induced changes in response

and repair mechanisms and function in the

CNS and visual system. Work on response

and repair mechanisms in the CNS will be

compared to similar work in other tissue

systems in the cancer project to see if agerelated changes in repair and homeostatic

mechanisms are similar in these different

tissue types. This will allow us to prioritize

particular processes in specific systems. 

Figure 12 illustrates the critical path for this

program project.
 4-20

Figure 12 Critical Path for Research on 

Susceptibility Associated with Older Adults

Characterize Biological 

Differences 

Between Young Adult

and Older Animals

Determine Age-Related

Responses to 

Environmental Insult 

Framework to Inform Risk Assessment Decisions Concerning Aging

Evaluate Mechanisms of 

Susceptibility to Environmental

Insults

Project 1 - Mechanisms of Susceptibility to

Loss of Neural Function in Older Adults. 

This project is designed to investigate agerelated changes in the CNS and sensory

systems at multiple levels. Studies on

response and repair mechanisms in the CNS

will focus on differences in age- and toxicantinduced gene and protein expression in

different brain regions to begin to understand

the different processes that are associated with

functional losses in these brain areas. The

ultimate goal of these studies is to be able to

construct a model of the chain of events

leading from alterations at the molecular level

to alterations at the functional level. To

address the broad range of age-related

alterations that adversely influence the ability

to respond to toxic insult, we propose to

measure changes in gene and protein

expression for protective, repair, and plasticity

mechanisms in the brain as a function of the

interactions of age and toxic exposure. As a

focused example of the work linking changes

at the molecular and cellular levels to changes

in functional and morphological endpoints,

studies of age and toxicant-induced effects on

the retinal degeneration and visual function

will be examined. The studies on ocular

toxicity address the concern that pesticides

may be an environmental risk factor,

particularly to aging populations already

susceptible to retinal degeneration. 

Normal aging processes result in changes

in physiological systems that could contribute

to increased susceptibility to neurotoxic

exposures in older adults. The studies of gene

and protein expression will focus on the

critical pathways involved in toxic response

and detoxification/repair pathways. That is,

pathways involved in ROS generation and

elimination, proteins involved in maintenance

and repair, and genes that have wide ranging

function in regulation of cell processes such as

transcription factors or homeobox genes and

signal transduction pathways. Monitoring

global patterns of gene expression allows one

to examine multiple physiological
 4-21

mechanisms simultaneously, gain insight into

interactions between mechanisms, prioritize

them in order of importance, and to associate

them with functional alterations. Because the

CNS shares with the other organs an overall

decline in cellular and genetic repair and

homeostatic processes with aging, we hope to

determine if mechanisms that are affected by

aging in the brain and nervous system play a

similar role in age-induced susceptibility in

other systems and for other endpoints (e.g.,

cancer, non-cancer).

A second set of studies, incorporating some

facets of the gene and protein expression work

described above, will focus on effects in the

visual system in an aging model. The aging

visual system is susceptible to ocular toxicity

because of a number of factors. Anatomical

examination of retinas taken from individuals

free of ocular disease show an age-related loss

of rod photoreceptors and ganglion cells. 

Along with these losses is a lifelong

accumulation of non-degradable metabolic

by-products in and under the retinal

pigmented epithelium (RPE). In addition,

older individuals represent a growing

population uniquely susceptible to loss of

vision due to age-related macular

degeneration (AMD). AMD is the leading

cause of vision loss in individuals older than

65 years; the prevalence of AMD is estimated

to increase from approximately 1.6% before

age 64 to 28% eleven or more years later. The

pathogenesis of AMD is still largely

unknown, and it is not clear whether

environmental factors may push an individual

from the already compromised state due to

aging to a state of overt pathology. Broadlydefined chemical exposure, for example, has

been identified as a risk factor in AMD. 

Retinal degeneration has been reported in

humans exposed to OP chemicals and in

animal models of pesticide exposure and has

been linked to both aging and fungicide

exposure in epidemiological studies. In

addition, there is growing evidence that

inflammatory processes play a role in the

pathogenesis of age-related macular

degeneration and other important diseases of

older adults. The proposed studies examine

retinal physiology and a second messenger

(signal transduction) pathway in the retina

known to be affected by OPs in an older

animal model, the interactions of light and

environmental toxicants in models of retinal

degeneration, and a human population

exposed to pesticides and showing signs of

retinal degeneration. Increased age is a strong

co-factor. Research in project 1 will:

C Assess qualitative and quantitative

differences in response to toxicant

exposure between young and the aged in

tissue culture, animal, and human

models for neurodegenerative, protective

or repair and plasticity mechanisms at

functional, cellular, and molecular

levels. This work aims to identify

mechanisms of susceptibility, targeted

sites of neurotoxicant action, and the

resiliency of the CNS;

C Correlate age-related alterations with

functional endpoints, including brain

morphometrics / pathology, function,

and behavior;

C Evaluate whether effects observed

involve changes in the PK of the

chemicals studied; and

C Compare cell and molecular differences

found with those determined in other

tissues or resulting in other endpoints

(e.g., cancer versus non-cancer).

Project 2 - Mechanisms of Susceptibility to

Cancer in Older Adults. This project is

designed to investigate age-related changes in

the body that lead to increased occurrence of

cancer. Age is the strongest factor associated

with the incidence of cancer in humans. 

While the delay between an environmental

insult and the appearance of neoplastic growth

may explain some of this circumstance,

research is needed to determine what makes
 4-22

older individuals preferentially susceptible to

the occurrence of cancer. It is possible that

environmental agents have greater effect in

older individuals. There is general agreement

that cumulative damage caused by oxidative

stress is the fundamental cause of aging. 

Additional changes occur in the body when

hormone production and maintenance are

altered beginning at 40 to 50 years of age. 

Both of these processes, age-related changes

in response to oxidative stress and changes in

hormone levels, will be studied in this project

because they may be critical to the enhanced

cancer susceptibility of the older individual. 

Factors mediating increased susceptibility

may be present at all stages of carcinogenesis. 

The older population may be more susceptible

to cancer initiation because of age-related

alterations in DNA damage, detection and

repair–fundamental processes potentially

altered by some environmental agents. 

Damage in or inappropriate expression of

specific genes controlling cell proliferation or

other functions critical to maintenance of cell

homeostasis can lead to uncontrolled cell

growth resulting in cancer. Cancer promotion

processes may also be affected. These include

processes associated with chromosome

stability, DNA methylation, suppressor gene

activity, signal transduction processes and

spindle assembly, all components in the web

of promotion processes that can lead to

reduced growth control in cells of various

tissues. Disruption of a homeostatic process,

gap junction communication (GJC), which

exhibits tumor suppressor activity, can also

lead to increased cell proliferation and cancer

formation. 

Activities of some hormones are also

known to influence the occurrence of cancer. 

It is possible that changing hormonal levels in

older individuals affect the mechanisms of

cancer initiation and promotion by

environmental agents. For example, the

serum levels of melatonin, a known oncostatic

agent, decrease as a function of age in humans

beginning at 40 to 50 years of age. GJC will

be one process studied because it has been

shown to be augmented by physiological

concentrations of melatonin. In addition, the

occurrence of DNA damage will be studied

because melatonin has been shown to be one

of the most potent free radical scavengers in

the body.

Chemicals selected for study will provide

experimental validation of principles as well

as have direct value in risk assessment

analysis. The chemicals include the

following: (1) As, which damages DNA,

alters GJC, and changes DNA methylation;

(2) 2,4-D, which alters GJC and causes liver

tumors in rodents; (3) chloral hydrate, which

alters GJC but has not been found to cause

cancer; (4) potassium bromate and bleomycin,

which damage DNA; (5) selected chemicals in

the conazole class, which includes

pharmaceuticals and pesticides; and (6)

dimethyl benzathracene and N-nitroso-Nmethylurea, which cause DNA adducts either

directly or after metabolic activation, the latter

being influenced by melatonin. Ionizing

radiation may also be used because it causes

DNA damage that can lead to cancer in the

absence of metabolic conversion processes,

obviating PK concerns. As in project 1 of this

program project, much emphasis will be

placed on measuring age-related differences in

gene/protein expression and chemical toxicity

in order to evaluate susceptibilities of older

individuals. 

These studies will generate directly

comparable data across diverse experimental

platforms (i.e., whole animal studies, animal

cell culture studies, and human cell culture

studies). This enhances the value of these

studies in that these data may provide critical

information needed for extrapolation for the

purposes of human health risk assessment. 

Research in project 2 will:
 4-23

C Establish animal and cellular models to

assess whether older animals are more

susceptible than younger adult animals

to the carcinogenic effects associated

with exposure to specific environmental

agents,

C Evaluate the mechanisms of increased

susceptibility in older animals, and

C Establish biomarkers of response to

estimate risk in humans.

Impact

ORD seeks to provide adequate protection

to all segments of the human population,

including susceptible subpopulations. In

response to a legislative mandate, research has

concentrated on effects during development or

in the very young. Numerous gaps in the

toxicological database still exist, however, for

older populations. This program project seeks

to examine whether there are special

susceptibilities associated with older adults

compared to the healthy young adult

population. Older adults comprise a

subpopulation that may have special

susceptibilities to toxicant-induced

dysfunction or degeneration due to the critical

characteristics of their life-stage. The very

young have been recognized as a population

of interest in the FQPA and the Agency has

recently promulgated an Initiative on Aging

that will help guide research in this area in the

future. Research on older adults that

incorporates age-specific data on biologically

effective dose and health effects will improve

the scientific underpinning of risk assessment

decisions by moving risk assessment toward

more biologically-based decisions about

uncertainty factors. These studies will address

the uncertainty of whether toxicity data

provides a sufficient margin of safety to

protect against effects that may result from

toxic exposure to an aged individual. As such,

it should help our understanding of how to

appropriately incorporate the differential

susceptibility of the aged into the current risk

assessment framework of the Agency.

Cross-Agency Interactions

We anticipate interactions with the

recently-launched Agency-wide Initiative on

Aging, coordinated by Dr. K. Sykes of the

Office of Children’s Health Protection

(OCHP). Research on health effects of

environmental exposures to older adults has

already been identified in this initiative as one

of the critical elements. As this initiative

develops, we hope to see opportunities for

cross-agency interactions.
 4-24

Program Project 8: Environmental Risk Factors for Asthma

Objectives

C Identify and rank mold allergens which

may be important in the induction of

allergic asthma, and

C Assess the effect of air pollutant exposure

on the induction and exacerbation phases of

the disease.

Scientific Approach

More than 17 million people in the US had

asthma in 1998. The incidence of asthma has

doubled in the last 20 years; the largest

increase has been among children below the

age of 15 years of age (CDC, 1998). This

increase cannot be explained by changes in

diagnostic categorization or by alterations in

the gene pool and suggests a strong

association between environmental influences

and the incidence of disease. About 75% of

asthma is associated with allergy, and it

appears that the immune system can be

programmed to promote asthmatic responses

to certain antigens early in life. In nearly

100% of elementary school children with

asthma, allergens are the primary trigger for

asthma; and their disease is thought to result

from early exposure and sensitization to

common allergens in their indoor environment

(e.g., dust mite, cockroach, molds, animal

dander). While some of these allergens have

been studied extensively (e.g., dust mite and

cockroach), almost none of the mold allergens

have been characterized despite their

widespread distribution and potential

importance in the induction and exacerbation

of asthma. A recent National Academy of

Sciences Report concluded “There is

inadequate or insufficient evidence to

determine whether or not there is an

association between fungal exposure and the

development of asthma” (NAS, 2000). 

Epidemiological and clinical studies have

also demonstrated that asthmatic responses

can be exacerbated by ambient combustionrelated products and by domestic and

occupational exposures to airborne chemicals. 

While epidemiological studies have not

established a clear role in the induction of

asthma for air pollutant or pesticide

exposures, animal and clinical studies suggest

that ozone, nitrogen dioxide, residual oil fly

ash, and diesel exhaust particles can act as

adjuvants to enhance allergic sensitization. 

In addition, recent studies have found that

children in inner cities or living closer to

major highways have more asthma than

children in rural and suburban centers or

living further from heavily trafficked roads. 

Clinical and epidemiological studies have also

shown that children with asthma are

especially susceptible to the respiratory effects

of ambient combustion-related pollutants such

as sulfur dioxide and ozone. 

The recent Agency’s Asthma Research

Strategy (US EPA, 2002) describes research

needs related to susceptibility factors

contributing to asthma and risk assessment

issues such as dose and type of pollutant

which can increase the incidence or enhance

severity of allergic asthma. This NHEERL

Human Health Implementation Plan addresses

several classes of environmental pollutants

identified by the asthma research strategy

including bioaerosols (i.e., endotoxin, molds

and other allergens) and combustion-related

products formed from the burning of organic

fuels. The critical steps for this program

project are illustrated in Figure 13. 

Project 1 - Fungal Antigens as Initiators of

Allergic Asthma. The hypothesis for this

research is that the structure and function of

the protein constituents of mold isolates

determines their allergenicity and can be used
 4-25

Figure 13 Critical Path for Research on Asthma

Elucidate role of environmental 

agents on induction and 

exacerbation of allergic asthma

Project 1 Project 2

Identify and 

rank potency of 

fungal allergens

Confirm potency 

in naturally 

exposed humans

Remediation 

(NERL, NRML)

Determine adjuvancy of 

diesel & other pollutants

Quantitative model to 

extrapolate animal to human

Assess health effects of 

real-world exposures

to determine the relative potency of different

mold components. This project will use a

mouse model to develop a cause-effect

relationship between exposure to fungal

extracts (and specific protein components) and

asthmatic responses; characterize the specific

protein allergens from three fungi

(Metarhizium anisopliae, Stachybotrys

chartarum, and Penicillium chrysogenum),

look for common structural characteristics,

and assess the potency of these allergens

relative to known human allergens. Studies in

the project will:

C Identify those proteins in Stachybotrys

chartarum, and Penicillium

chrysogenum crude extracts that elicit an

allergic antibody response

(immunoglobulin E, IgE);

C Confirm that the proteins identified as

IgE inducers are the relevant allergens in

the mouse disease model and determine

if these proteins are allergenic in

humans;

C Characterize the physico-chemical

properties of the allergenic protein(s)

from the three fungi and compare to

other well-characterized protein

allergens such as dust mite allergens;

and

C Determine the relative potency of the

fungal allergens compared to other

allergens such as dust mite, cockroach,

and alcalase.

Project 2 - Effect of Xenobiotic Exposure

on the Incidence and Severity of Allergic

Asthma. The hypothesis for this research is

that endpoints reflecting pollutant-enhanced

severity of allergic asthma seen in

experimental animals can be successfully

modeled in vitro (cell culture systems) and

that quantitative relationships exist between

animal and human cell responsiveness.

Research in this project will develop

laboratory-based assays which can predict the

ability of various pollutants to enhance the

induction or exacerbate the severity of allergic
 4-26

asthma, in a dose dependent manner. This

approach will be tested using a prototype

pollutant (diesel exhaust particles [DEHP]) in

in vitro, in vivo (animal and controlled human

studies), and epidemiological studies. The

validated paradigm will be used to test the

relative potency of a variety of different air

pollutants and materials of interest. Research

in this project will:

C Compare the genetic expression and

release of pro-inflammatory

chemokines, receptors, and other

immunoregulatory molecules in healthy

and allergic airway epithelial cells and

macrophages from humans and rodents

after incubation with diesel exhaust;

C Correlate in vitro findings with in vivo

markers of disease (altered gene

expression and cytokine output, immune

function and patho-physiology) in rat

and mouse models of allergic lung

disease;

C Compare qualitative and quantitative

aspects of animal data with clinical

results from human studies investigating

the effect of DEHP exposure on nonasthmatic and asthmatic lung responses;

C Utilize in vivo animal techniques and in

vitro animal and human assays to screen

chemicals and obtain relative potencies

for air pollution particles and gases of

interest;

C Develop methodology for preparing,

storing, and transporting clinical

samples for subsequent immunological

analysis; and 

C Assess the prevalence of biological

markers of exposure to DEHP and

biological markers of immunological

function across intra-urban gradients of

combustion-related products and air

toxics among school-age children.

Impact

The results from this program project will

greatly contribute to the understanding of

environmental risk factors for asthma and will

address several key Agency needs including

species extrapolation and quantitative risk

assessment, identification of susceptible

subpopulations, aspects of children’s health,

as well as assessing the health effects of air

pollutants (both indoor and ambient) (ORD,

1999). This work will also contribute to the

identification of biomarkers of both exposure

and effect that will be subsequently used in

the NCS (see program project 4 in this

Section).

Cross-Agency Interactions

The first project has a strong interlaboratory collaboration between NHEERL

scientists and NERL engineers and biologists

in Cincinnati. In the second project, NRMRL

engineers are developing a diesel generation

and exposure system for use in the in vivo

studies performed by NHEERL scientists. 

The work from both these projects will be

transmitted to the Office of Air and Radiation,

as well as to risk assessors in NCEA.
 4-27

Program Project 9: Environmental and Genetic Interactions in Hypertensive

Rats: Oxidative Stress as a Common Susceptibility Attribute for Non-Cancer

Risks

Objectives

C Determine if oxidative stress is a common

susceptibility attribute for a variety of

environmental insults, including air

pollutants, pesticides, and herbicides;

C Evaluate potential mechanisms and

biological pathways responsible for

susceptibility to toxicants in the presence of

preexisting oxidative stress conditions; and

C Determine genetic contribution to oxidative

stress and susceptibility to toxicants.

Scientific Approach

Individuals with preexisting disease

conditions are likely more susceptible to

environmental exposures, and often the

uncertainty factor employed in risk

assessment may not be adequate to protect

these individuals. Although diseases such as

congestive heart failure, atherosclerosis,

Parkinson’s and Alzheimer’s diseases,

diabetes, infertility, and cancer have diverse

etiologies, they have one common element,

namely, the presence of oxidative stress

(Forsberg et al., 2001). Genetic susceptibility

to oxidative stress has been proposed in

individuals with diseases. Compensatory

antioxidant mechanisms are activated in

response to injury and the injury in healthy

individuals is followed by a normal tissue

repair. However, in genetically predisposed

individuals with chronic preexisting diseases,

these compensatory mechanisms are defective

either due to the presence of genetic

polymorphisms or a presence of disease;

therefore, these individuals are at increased

risk of morbidity and mortality from

environmental exposures.

One of the significant compensatory

antioxidant mechanisms involves regulation

of glutathione and a number of phase II

metabolism enzymes. Synthesis of

glutathione is regulated by γ−glutamyl

cysteine synthetase/ligase (γ-GCS). The gene

expression is under the control of nuclear

factor erythroid-derived 2, like 2, (NRF2)

nuclear translocation and binding to

antioxidant response element consensus

sequence present in phase II metabolism

enzymes. This protective transcriptional

pathway is induced by a variety of toxicants

which result in induction of γ-GCS,

glutathione S-transferase A1, heme

oxygenase-1 (HO-1), NADPH quinone

oxidoreductase-1 (NQO-1), superoxide

dismutase-1, thioredoxin, catalase, and uridine

diphosphoglycosyl transferase 1a6. Recently,

it has been shown that NRF2 knock-out mice

showed increased toxicity to a variety of

toxicants. It has also been shown that a

sensitive (C57BL/6J) mice strain is more

susceptible to oxygen-induced pulmonary

damage than the C3H/HeJ strain, and that the

difference in sensitivity appears to be

associated with mutation of NRF2 promoter

(T÷C, bp-336). This mutation is predicted to

add an Sp-1 transcription factor binding site in

the C57BL/6J mouse strain compared to

C3H/HeJ strain. A recent study also reported

a single nucleotide polymorphism at -588 in

human γ-GCS which decreases expression of

this enzyme and levels of plasma glutathione

in myocardial infarction patients. Thus, these

mutations are likely candidate single

nucleotide polymorphisms associated with

susceptibility to oxidative stress in animals

and humans.
 4-28

To understand the mechanism of

susceptibility, and the role of underlying

genetic oxidative stress as a common

susceptibility attribute, relevant animal

models exhibiting genetic predisposition to

oxidative stress can be employed. This

proposal involves the use of genetically

predisposed Spontaneously Hypertensive (SH)

rats as a model for increased oxidative stress

and compare to healthy normotensive Wistar

Kyoto (WKY) rats. SH rats are genetically

predisposed to increased oxidative stress and

have compromised compensatory ability to

increase tissue antioxidants in response to

mild to moderate tissue damage (expected

from environmental exposures). The

hypothesis underlying this program project is

that a rat model exhibiting phenotypic

predisposition to oxidative stress (associated

with hypertension/cardiovascular disease

[CVD]) will show measurable susceptibility to

a variety of insults affecting different organ

systems. The research done under this

program project will determine susceptibility

of SH versus WKY rats to known toxicants

that affect pulmonary, cardiovascular,

neuronal, ocular, and reproductive systems, as

well as investigate the roles for oxidative

stress in the exacerbation of the toxic

response. This will establish a base for

reducing uncertainty factors for individual

populations who are at increased risk of

environmentally induced disease because of

underlying genetic predisposition to oxidative

stress.

All individual projects listed below will

plan studies in three phases. In the first phase,

a dose-response characterization will be done

with acute exposure scenarios to determine

the level of susceptibility in SH versus WKY

rats in several organ systems using

appropriate organ specific toxicants. To

understand the role of oxidative stress and the

mechanism of increased susceptibility, the

second series of studies will involve

manipulating the oxidative stress of SH rats

using antioxidant treatment singly or as a

mixture (i.e., tempol, lazaroid, nacysteiline,

ascorbate, glutathione). Such agents will be

administered prior and/or during their

exposure to specific toxicants mentioned

under individual projects. The antioxidant

regimen that is most successful in reducing

systemic oxidative stress will be given

consideration for the purpose of this proposal. 

Antioxidants and biomarkers of oxidative

stress will be evaluated in all projects in

addition to determining organ specific toxicity

markers. Tissues harvested from these studies

will also be employed to investigate oxidative

stress-related signal transduction (MAP

kinases and protein kinase C [PKC]-mediated

signaling) and gene expression using

comprehensive commercial rat gene arrays. 

In the third phase of studies, tissues from SH

and WKY rats from selected studies will be

saved for isolation of DNA in order to screen

for possible polymorphism in select gene

regions (specifically genes for NRF2, NQO-1,

HO-1, and γ-GCS) based on historical

evidence of polymorphisms in susceptible

humans and animals. These studies will

establish the mechanistic basis for genetic

susceptibility to oxidative stress, and its

implication in risk assessment. The

polymorphisms will also be evaluated in

patients with CVD and Chronic Obstructive

Pulmonary Disease (COPD) which will

confirm the role of oxidative stress in human

susceptibility and establish a genetic link

between animal and human susceptibility

traits. The critical milestones for research in

this program project are summarized in Figure

14. 

Project 1 - Genetic Predisposition to

Hypertension-Associated Oxidative Stress

and Increased Cardiopulmonary

Responsiveness to Ozone and Zinc. This

research addresses the hypothesis that SH rats

will have exacerbated pulmonary injury and

cardiovascular effects from exposure to ozone

and zinc. Furthermore, this exacerbated
 4-29

Project 1

Relationship Between 

Oxidative Stress and 

Pulmonary?Cardiovascular Toxicity 

Project 2

Relationship Between 

Oxidative Stress and

Neurotoxicity

Project 3

Relationship Between

Oxidative Stress

and Ocular Toxicity

Project 4

Relationship Between

Oxidative Stress and 

Reproductive Toxicity 

Establish that Oxidative Stress is a Host Susceptibility

Factor Contributing to Exacerbated Toxicity for a

Variety of Toxicants in Diverse Organ Systems 

Figure 14 Critical Milestones for Research 

on Oxidative Stress 

Project 5

Determination of 

Promoter Mutation 

Frequency in Humans

response will be diminished in SH rats when

pretreated with antioxidants due to diminished

oxidant-mediated cell signaling and activation

of inflammatory mediators. Ozone has been

selected based on its specific oxidant action

and its importance as one of the six criteria air

pollutants, while zinc is selected based on its

likely cardiac effects and its potential role as

one of the causative constituent of ambient

PM. Research in Project 1 will:

C Determine relative susceptibilities of SH

and WKY rats to cardiovascular and

pulmonary effects of ozone and zinc

sulfate;

C Use antioxidant treatment to establish

putative mechanism of action for

oxidative stress in cardiovascular and

pulmonary effects of air pollutants;

C Assess cardiovascular function using

radiotelemetry prior to, during, and after

exposure to air pollutants; and

C Determine comprehensive gene

expression profile of the lung and the

heart and measure oxidative stress cell

signaling and specific nuclear factors

activation.

Project 2 - Susceptibility of Spontaneously

Hypertensive Rats to the Neurotoxic

Effects of Pesticides: the Role of Oxidative

Stress. Oxidative stress is a condition

affecting tissues and cellular functions in a

number of disease states. In healthy

organisms, ROS which mediate oxidative

stress, are generated during normal

mitochondrial bioenergetics and are

scavenged by various enzymatic and nonenzymatic antioxidants (e.g., glutathione,

catalase, xanthine oxidase, ascorbic acid, uric

acid). However, if the individual has genetic

errors which amplify the formation or reduce

the scavenging of ROS, this compensatory

response is reduced, resulting in a heightened
 4-30

state of oxidative stress to cells and tissues. 

Clinical and experimental evidence indicates

that oxidative stress underlies the

neurodegenerative condition of Parkinsons

Disease. This disease is thought to be a

multifactorial condition involving a genetic

predisposition to errors in energy metabolism

(i.e., mitochondrial complex I), environmental

exposure to chemicals that target energy

metabolism (e.g., certain pesticides) and a

high susceptibility of mesencephalic

dopaminergic neurons and glia to oxidative

stress and energy depletion. Project 2 will

examine the interplay of such factors (i.e.,

genetics, chemical exposure,

neurodegenerative diseases) in SH rats, a

strain which has a genetic predisposition to

oxidative stress. The first phase of this study

will compare behavioral and neurochemical

endpoints of oxidative stress in response to

ChE-inhibiting pesticides (carbamates, OPs)

and paraquat. Endpoints that are

representative of oxidative stress will be

measured. Brains taken from representative

animals showing both behavioral (i.e.,

reduction in locomotor activity) and

neurochemical changes will be examined for

neuropathological changes. In vitro

experiments will examine the effects of

complex I inhibitors (e.g., paraquat, rotenone)

on mesencephalic glia and neurons. The

effects of antioxidant pretreatment on

pesticide-induced behavioral and

neurochemical changes will then be examined

in whole animals, their brain tissues, and CNS

target cells in vitro. It is hypothesized that

because of their condition of chronic stress

and reduced ability to scavenge ROS, SH rats

will show an increased susceptibility to the

neurotoxicity associated with selected

pesticides. Research in this project will:

C Compare behavioral, neurochemical, and

neuropathological endpoints of oxidative

stress in response to various pesticides in

animals;

C Describe the morphological and

neurochemical response of PD target

neurons and microglia to complex I

inhibiting pesticides;

C Determine activation of PKC and MAP

kinase signaling and gene expression

patterns in response to pesticide

exposures; and

C Determine the protective effects of antioxidant treatment on pesticide-induced

neurotoxicity.

Project 3 - Ocular Toxicity and Oxidative

Stress. Visible light, the primary stimulus for

the visual system, is also its primary toxicant. 

Many xenobiotic compounds absorb light in

the ultraviolet or visible wavelengths and

produce ROS (singlet oxygen) in response. 

This project will investigate whether the

mixture of light-induced damage is a

mechanism for the generation of ocular

toxicity of the retina and the lens. The

hypothesis for this project is that xenobiotic

chromophores will absorb light and produce

damage through oxidative stress in ocular

structures and this damage will be exacerbated

in animal models deficient in protective

mechanisms due to either inherited

characteristics, e.g., as in SH rats, or due to

dietary deficiencies in antioxidants.

Antioxidant supplementation would likely

reduce damage. Research in this project will:

C Use physiological, morphological and

biomicroscopic endpoints to assay

damage to the retina and lens following

exposure to insecticides and fungicides;

and

C Determine levels of ROS in specific

ocular tissue to establish connection

between oxidative stress and retinal

damage.

Project 4 - Role of Oxidative Stress in the

Reproductive Function of Males and

Females. Epidemiological studies have

shown that certain subpopulations of people

have lower blood levels of glutathione and

may be more prone to oxidative injury in

somatic cells. Research in this project will

address the possibility that such individuals
 4-31

may be at increased risk of adverse

reproductive effects associated with the

induction of oxidative stress in gonadal

tissues. The hypothesis for this research is

that SH rats will have a diminished testicular

response to oxidative stress as compared to

the WKY strain and that lower amounts of

glutathione and decreased induction of

glutathione synthetic enzymes may be found

in the challenged SH animals. Differences in

levels of NRF2 would suggest a role for this

transcription factor in the altered response. 

Sperm motility may also be affected in these

animals due to increased membrane lipid

peroxidation in the presence of diminished

glutathione levels. These animals will also

serve as positive and negative controls for

subsequent experiments (DNA array analysis). 

The hypothesis for this research is that

diminished response to oxidative stress may

exacerbate the effects of known testicular

toxicants such as acrylamide and

thiocarbamate pesticides such as thiram or

molinate. Finally, the effects of

environmental toxicants on zygote formation

in SH and WKY rats will be tested. This

research will:

C Determine if the SH rat is significantly

more susceptible to toxicant-induced

oxidative stress/antioxidant levels than

normal rats,

C Determine oxidative stress-induced

responsive gene expression pattern in

testis following exposure to toxicants in

WKY and SH rats; and

C Determine the biomarkers and the role

of oxidative stress in susceptibility to

toxicants.

Project 5 - Oxidative Stress in CVD and

COPD Patients and Possible NRF2

Promoter Mutation. Lower levels of

antioxidants and systemic oxidative stress

have long been considered risk factors

associated with CVD, COPD, and other

chronic diseases. The presence of genetic

mutations in antioxidant compensatory genes

and their role in pathobiologic mechanisms of

disease development are not well understood. 

To determine if NRF2-mediated

compensatory antioxidant mechanisms are

involved in predisposition to oxidative stress,

selected gene mutations (specifically genes for

NRF2, NQO-1, HO-1, and (-GCS) that are

known to exist in humans and animals will be

evaluated in SH rats and humans with CVD

and COPD. This study will allow us to

establish the genetic basis to oxidative stress

and consideration of genetics in relevancy of

animal model to human diseases and will

C Correlate the detection of increased

mutation frequency in NRF2 promoter

and other related genes with that of the

severity of disease in humans and its

incidence in SH rats.

Impact

Currently an uncertainty factor of 10 is

employed in risk analyses in order to account

for differences in susceptibility due to host

preexisting conditions and genetics. 

However, recent epidemiological evidence

demonstrates that in many instances

susceptibility may lead to increased mortality

or disease such that an uncertainty factor of 10

may not be adequate. There is often

incomplete knowledge regarding the

existence, extent, and characteristics of

individual susceptibility about who is

susceptible and to what extent. Identifying

the mechanisms that underlie susceptibility to

many environmental toxicants that play a role

in the development or exacerbation of disease

leading to death is important in both basic risk

assessment and reducing the uncertainty. 

Research in this program project will also

utilize a relevant animal model to determine

increased susceptibility to a variety of toxic

insults and organ systems due to underlying

oxidative stress/disease. Knowledge of the

mode or mechanism of action for specific

agents will reduce uncertainties in their risk

assessment. Mechanistic work generating in

this program project may also lead to the
 4-32

identification of new biomarkers of toxicity,

as well as susceptibility to toxic insult.

This research will also lead to the

development and use of technologies to

evaluate gene expression profiles to

understand patterns of pathological processes

and identify potential gene polymorphisms to

better define susceptibility. These

technologies will lead to a greater

understanding of the key events associated

with adverse effects. Thus, the data generated

from the gene expression profiling studies will

be useful to the emerging Computational

Toxicology research program. 

Cross-Agency Interactions

As results are produced, collaborations with

NCEA and Program Offices will be

developed. 
 4-33

Program Project 10: Genotype and Phenotype in the Metabolism, Toxicity

and Carcinogenicity of Arsenic (As)

Objectives

C Identify genetic polymorphisms for

enzymes that metabolize As, 

C Examine the regulation of the gene and the

role of nutritional status in its regulation,

C Examine the function of arsenate reductase

as a critical factor in metabolism of As, and

C Examine As-protein interactions to gain

insight into the mode of toxicity for this

metalloid.

Scientific Approach

It is known that genetic polymorphisms and

mutations can greatly affect human

susceptibility to mutation and/or cancer (e.g.,

fast and slow acetylators of aromatic amines

and bladder cancer, xeroderma pigmentosum,

Cockayne’s syndrome and

trichothiodystrophy). Genetic polymorphisms

and mutations may significantly affect the

outcome of exposures to chemical stressors,

such as As. Given the relatively high

frequency of polymorphisms in the human

population, this proposal will address the

potential effect of specific polymorphisms on

responses to environmental exposures.

This research will study the role of genetic

factors in the expression of toxicity by

investigating a prototypic compound having

environmental relevance, i.e., As. There is a

consensus that understanding variability in the

metabolism of iAs in humans is critical to

understanding the response of humans to

chronic exposure to iAs (Lin et al., 2002;

National Research Council, 1999). Interindividual variation in absolute and relative

amounts of methylarsenic and dimethylarsenic

in urine has been attributed to differences in

ethnicity, age, sex, nutritional status, and

extant disease. In humans chronically

exposed to iAs in occupational and

environmental settings, considerable interindividual variation has been found in the

occurrence of specific signs and symptoms of

As intoxication. For example, only a small

fraction of individuals chronically exposed to

iAs in drinking water develop blackfoot

disease, a progressive occlusive disorder of

the peripheral vasculature. A role for

metabolic capacity in determination of

susceptibility is supported by studies in

chronically-exposed populations that find

absolute or relative increases in the amount of

metabolites of As in urine are associated with

increased occurrences of As -induced skin

lesions. 

This program project will determine

whether the inter-individual variation in

response is related to differences in the

capacity to metabolize iAs, if these

differences in metabolic capacity are primarily

determined by the kinetic properties of the

enzymes that catalyze the reduction of

pentavalent As and the methylation of

arsenicals, if the kinetic properties of these

enzymes are determined by their primary

sequence (hence, by the genotypes for these

proteins), and whether control of the

expression of these genes may be an important

in determination of the capacity for

metabolism of As. As a corollary to these

postulates, this research will determine if there

is a linkage between the genotypes for these

enzymes and the disease susceptibility

phenotype which is manifested by the capacity

of cells or organisms to metabolize iAs. It is

likely that polymorphisms in the genes which

encode these proteins determine their catalytic

activities and that the transcriptional

regulation of these genes affects their

expression. If susceptibility to the adverse

effects of chronic exposure to iAs is related to

capacity to produce methylated metabolites,

then genotypes for these critical enzymes are

highly relevant to predicting the hazard

associated with chronic exposure to this
 4-34

Identify Genetic

Polymorphisms 

Important for

Metabolism

Develop Necessary

Molecular Methods

for Screening 

Human Cells

Determine if Genes 

Influence Sensitivity

In Vitro

Demonstrate 

Susceptibility

and Sensitivity

In Vivo

Demonstrate Genetic Susceptibility

and Sensitivity in Humans and

Animals

Figure 15 Critical Path for Research on 

Genetic Polymorphisms

metalloid. Studies of genotypic-phenotypic

relations in human cells and in individuals are

important steps in estimating the role of

interindividual variation in determination of

susceptibility. The critical steps for research

on genetic polymorphisms are summarized in

Figure 15.

Project 1 - Genotype-Phenotype

Correlations for As Methyltransferase in

Humans. Research in this project will

examine the relation between the genotype of

Cyt19, a gene encoding the human As

methyltransferase and the phenotype for the

capacity to methylate iAs in human cells and

in individuals chronically exposed to iAs. The

hypothesis for the proposed research is that

the genotype of Cyt19 affects the phenotype

for As metabolism as exemplified by the

pattern and amounts of iAs and metabolites

produced by cultured human cells or present

in blood or excreted in urine by chronically

exposed individuals. The specific aims for

this study are (a) to examine in cultured

primary human hepatocytes, the relation

between the Cyt19 genotype and the

phenotype for the metabolism of As, as

reflected by the amounts of iA and

metabolites in cells and media; (b) to examine

genotypic variation in Cyt19 using DNA

isolated from peripheral blood of a number of

human volunteers; and (c) to examine the

relation between the Cyt19 genotype and the

phenotype for the metabolism of As as

reflected by the amounts of iAs and

metabolites in blood and urine from humans

chronically exposed to iAs in drinking water.

Research in project 1 will:

C Examine the relationship between Cyt19

genotype and phenotype in cultured

primary human hepatocytes from 20 to

30 donors,

C Examine the genotypes for Cyt19 using

DNA from a larger number of human

donors; and

C Study genotype and phenotype in

individuals who are chronically exposed

to iAs.

Project 2 - Genetic Susceptibility and

Sensitivity to As Exposures. This project

will address the hypothesis that cells, animals,

or individuals who might show

polymorphisms at the gene for glutathione

transferase omega (GST-O) will be

differentially susceptible to the genotoxic and

hence carcinogenic effects of As. Research in

this project will:
 4-35

C Determine if polymorphism in GST-O

exists and if it may be responsible for

differential sensitivity to arsenic

genotoxicity.

Project 3 - Altered Phenotype-Binding

Studies of Arsenicals and Purified Proteins

or Synthetic Peptide Amino Acid Sequences

of Importance in As Metabolism Reductase

and Cyt19) or Mechanism of Action. This

research address the hypothesis that there are

polymorphisms in As metabolism and

mechanism of action (Kitchin, 2001). Genetic

susceptibility to arsenicals will be revealed by

the binding of arsenicals to certain amino acid

sequences of proteins that are important in As

metabolism or mechanism(s) of action. If

there is a polymorphism in human Cyt 19,

there will be an altered amino acid sequence

that can be studied via binding experiments. 

If there is a genetic deletion or substitution of

key sulfhydryl(s) required for the binding of

the arsenical would be altered. These studies

will:

C Evaluate altered binding affinity and/or

amount of binding of arsenicals to

polymorphic animal or human proteins.

Project 4 - Interactions of Dietary Folate

Deficiency and As: Evaluations of Gene

Expression Changes in Association with

Enhanced DNA Fragility and Possible

Alterations in Methylation Patterns. As

toxicity may be enhanced in individuals with

folic acid (folate) deficiency. As this vitamin

is needed for normal DNA synthesis and

methylation, its deficiency can result in effects

which interact with those of As via different

pathways. Folate deficiency can result in

uracil misincorporation and increased DNA

fragility, an effect which could be increased

by As inhibition of repair enzymes. This

deficiency may also lead to a depletion of Sadenosylmethionine required for methylation

to result in DNA hypomethylation and

possibly decreased As detoxification. These

studies will:

C Use a mouse model to evaluate global

changes in gene expression associated

with folate deficiency, As exposure, and

most important, folate deficiency plus

As exposure.

Project 5 - Transcriptional Control. To

study the transcriptional control of Cyt19 (As

[III] methyltransferase) and the expression

pattern of Cyt 19 in various human tissues. 

Cyt 19 has been recently identified as the

methyltransferase that catalyzes the formation

of methylated and dimethylated As. 

Methylated arsenicals that contain As(III) are

more potent than other forms of arsenicals in

regards to genotoxicity (Mass et al., 2001) and

inhibition of various enzyme activities. Our

hypothesis is that the polymorphisms in the

transcriptional activity of Cyt 19 contributes

to human susceptibility to mutation and/or

cancer caused by As. By understanding the

transcriptional control of this gene, we are in

better position to understand the human

susceptibility to As. Methylation of As is a

critical feature of its metabolism, therefore,

characterization of Cyt 19 gene and its

expression pattern will improve our

understanding of the mechanism of As as a

toxin and a carcinogen. This research will:

C Delineate the regions and factors that

control the expression of the Cyt 19

gene,

C Study the tissue specificity of this gene

by looking at the presence or absence of

the expression factors in different

tissues, and

C Study the expression of this gene in

different populations.

Cross-Agency Interactions

We have ongoing collaborations with

NERL-Cincinnati to develop and validate new

analytical methods that are used in our work

on genetic polymorphisms of As metabolism. 

We also have a ongoing collaboration with

Region 10 to examine metabolism of As in

individuals ingesting a variety of marine
 4-36

organisms. This work may include studies of

genetic polymorphisms of As metabolism.

Impact

Although most attention has focused on the

potential health effects of chronic ingestion of

As-contaminated drinking water, substantial

exposure (and risk) occurs from other sources. 

As is a component of particulate material

generated by fuel combustion, is introduced

into the environment by the use of Ascontaining pesticides, and occurs in complex

organic species (e.g., arsenobetaine,

arsenosugars) in many foods. Hence, data

from the proposed research on the control of

the metabolism and toxicity of As will

contribute to risk assessments for chronic

exposure to As from water, air, and food. 

Because exposure to As occurs from each of

these media, the availability of new data on

the metabolism and effects of As will improve

the risk assessment for this element and assist

the Agency in its mission to protect public

health. 

In general terms, the proposed research will

emphasize three distinct, but related, aspects

of the health effects of As. Understanding the

role of genetic susceptibility in the control of

As metabolism will benefit the risk

assessment process by identifying

subpopulations of individuals who are

genetically predisposed to greater

susceptibility to the toxic or carcinogenic

effects of chronic As exposure. Second,

experimental evidence may be obtained that

show unusual sensitivity to As. Third, the

factors that are identified in the proposed

research as modifiers of the metabolism and

toxicity of As can be incorporated into

quantitative models which describe the

systemic and cellular metabolism of As and its

MOA as a toxin and carcinogen. 

This approach would extend current

modeling efforts that examine the fate of other

forms of As (i.e., total As or total methylated

As) and improve the utility of these models in

risk assessment. Taken together, the proposed

research effort examining the genotypic and

phenotypic control of metabolism, toxicity,

and carcinogenicity of As will contribute to

the Agency’s periodic reevaluation of risk

assessments for this element in water, air,

pesticides, and other media.

4.5 Gap Analysis and Links to Other MultiYear Plans

NHEERL’s implementation plan with

regard to susceptible subpopulations focuses

on life stage, genetic polymorphisms, and

asthma. NHEERL research on children is

consistent with research needs outlined the

Agency’s Strategy for Research on

Environmental Risks to Children (US EPA,

2000). NHEERL research on older adults

responds to the Administrator’s interest and

concern about the environmental health of

older Americans. Research described in the

program project on Aging has been included

in the recent inventory of Agency intramural

and extramural research projects organized by

the OCHP. Research related to older adults is

envisioned to increase over the next few years.

NHEERL research on genetic

polymorphisms deals primarily with a limited

number of possible genes associated with

differential sensitivity to the prototypic

chemical used in these studies, i.e., As. There

is considerable human and animal data

available on As, and it is clear that there is a

subpopulation of people that respond

differently to this metal. As indicated in the

program project on genotype and phenotype in

the metabolism, toxicity, and carcinogenicity

of As, there are several testable hypotheses

concerning potential gene-chemical

interaction that should provide basic

information needed to develop strategies for

addressing this issue with other environmental

agents from other chemical classes. Research

on a non-cancer class of chemicals, such as

the CHE-inhibiting pesticides, would provide

useful complementary information with

regard to gene/chemical interactions. Results
 4-37

from a cancer and non-cancer agent could be

used to help develop risk assessment

strategies that include potential gene/chemical

interactions in susceptible subpopulations.

NHEERL research with regard to

preexisting disease focuses on asthma and is

consistent with research priorities outlined in

the Asthma Research Strategy (US EPA,

2002). Other diseases such as autoimmune

deficiency diseases, cardiopulmonary

diseases, diabetes, and neurodegenerative

disorders such as Parkins disease could

potentially interact adversely with exposure to

environmental pollutants. NHEERL does not

plan to conduct extensive research on these

diseases at the present time, although

compelling epidemiological research

suggesting an interaction between a chemical

class and a disease could generate hypotheses

that could be tested in animal models.

Research on susceptible subpopulations is

also linked to several other MYPs. In the case

of Drinking Water, NHEERL research will

contribute to other studies to identify the risk

of birth defects due to drinking water

contaminants, provide fundamental

information on the basis of inter-individual

variation in As metabolism. For Air Toxics

and PM, NHEERL research will help identify

the role of specific polymorphisms in the

metabolism and repair genes and the relative

increase or decrease in tumorigenic risk

associated with exposure to air pollutants,

identify parameters for models describing the

deposition of fine and coarse mode particles in

older adults and moderate asthmatics, provide

basic research on the effects of concentrated

air particles in asthmatic humans and animals,

provide data for a report on the acute

respiratory effects of PM and co-pollutants in

asthmatic children, help describe the effects of

metals in animal model of allergic asthma,

and provide basic data to help describe the PK

of oxygenate mixes in young and older adult

populations. For EDCs, NHEERL research on

susceptible subpopulations will contribute to

the evaluation of toxicant-induced alterations

in mammalian reproductive development as a

function of critical window of exposure,

provide basis information for research on the

influence of life stage on the sensitivity and

reproducibility of estrogen cDNA

macroarrays, help evaluate critical windows

of exposure for alterations in mammary gland 

development and lactational function, and

characterize the effects of atrazine delivered

during critical windows of development.
 4-38

4.6 References

Barone, S., N. Haykal-Coates, D. K. Parran, and

H.A. Tilson. Gestational exposure to methyl

mercury alters the developmental pattern of trklike immunoreactivity in the rat brain and results

in cortical dysmorphology. Dev Brain Res

109(1), 13-31 (1998). 

Centers for Disease Control and Prevention. 

Forecasted state-specific estimates of selfreported asthma prevalence–United States,

1998. MMWR 47:1022-1025 (1998). 

www.cdc.gov/epo/mmwr/preview/mmwrhtml/0

0055803.ht .

Fenton, S. E., and C.C. Davis. Atrazine increase

dimethylbenz{a}anthracene-induced mammary

tumor incidence in Long Evans offspring

exposed in utero. Toxicol Sci 66, 185 (2002). 

Forsberg, L., U. de Faire, and R. Morgenstern. 

Oxidative stress, human genetic variation, and

disease. Arch Biochem Biophys 389:84-93

(2001). 

Godfrey, K.M. and D.J. Barker. Fetal

programming and adult health. Public Health

Nutr 4:611-624 (2001). 

Kehoe, P. K. Mallinson, J. Bronzino, and C.M.

McCormick. Effects of prenatal protein

malnutrition and neonatal stress on CNS

responsiveness. Brain Res Dev 132(1):23-31

(2001). 

Kitchin, K.T. Recent advances in arsenic

carcinogenesis: modes of action, animal model

systems and methylated arsenic metabolites. 

Toxicol Appl Pharmacol 172:249-261 (2001). 

Langley-Evans, S.C., D.S. Gardner, and S.J.M.

Welham. Intrauterine programming of

cardiovascular disease by maternal nutritional

status. Nutrition 14:39-47 (1998). 

Lassiter, T.L., L.D. White, S. Padilla, and S.

Barone. Gestational exposure to chlorypyrifos:

qualitative and quantitative neuropathological

changes in the fetal neocortex. Society of

Toxicology 2002 Abstract, Nashville, TN

(2002). 

Lin, S., Q. Shi, F.B. Nix, M. Styblo, M.A. Beck,

K.M. Herbin-Davis, L.L. Hall, J.B. Simeonsson,

and D.J. Thomas. J Biol Chem 277:10795-

10803 (2001). 

Mass, M.J., A. Tennant, B. Roop, W.R. Cullen,

M. Styblo, D.J. Thomas, and A. Kligerman. 

Methylated trivalent arsenic species are

genotoxic. Chem Res Toxicol 14:355-361

(2001). 

Merlet-Benichou, C., T. Gilbert, M. Muffat-Joly,

M. Lelievre-Pegorier, and B. Leroy. Intrauterine

growth retardation leads to a permanent nephron

deficit in the rat. Pediatr Nephrol 8:175-80

(1994). 

Miller, M.S., M.R. Juchau, F.P. Guengerich,

D.W. Nebert, and J.L. Raucy. Drug metabolic

enzymes in developmental toxicology. Fundam

Appl Toxicol 34:165-75 (1996). 

U.S. Environmental Protection Agency, Office

of Research and Development. Draft Strategy

for Research on Environmental Risks to

Children. 92 pp. (1999). 

http://www.epa.gov/ncea/pdfs/Draft21.PDF

National Academy of Sciences. Clearing the

Air: Asthma and Indoor Air Exposures. 

National Academy Press, Washington, DC. 

(2000).

National Research Council. Arsenic in Drinking

Water. National Academy Press, Washington,

DC. (1999).
 4-39

Rhind, S.M., M.T. Rae, and N. Brooks. Effects

of nutrition and environmental factors on the

fetal programming of the reproductive axis. 

Reproduction 122:205-214. (2001).

Schardein, J.S. Chemically Induced Birth

Defects, 3rd Ed., Marcel Dekker, New York. 

(2000).

Smialowicz, R.J., W.C. Williams, C.B.

Copeland, M.W. Harris, D. Overstreet, G.J.

Davis, and R.E. Chapin. Effect of

perinatal/juvenile heptachlor exposure on adult

immune and reproductive system function in

rats. Toxicol Sci 61:164-175 (2001).

U.S. Environmental Protection Agency, Office

of Research and Development. Strategy for

Research on Environmental Risks to Children. 

Washington, DC. EPA/600/R-00/068. 2000.

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?

deid=20068

U.S. Environmental Protection Agency, Office

of Research and Development. Asthma

Research Strategy. Washington, DC. 

EPA/600/R-01/061. 2002. 

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?

deid=54825

Weisglas-Kuperus, N., S. Patandin, G. Berbers,

T. Sas, P. Mulder, P.J.J. Sauer, and H.

Hooijkaas. Immunological effects of background exposure to polychlorinated biphenyls

and dioxins in Dutch preschool children: an

exploratory study of health effects from

“normal” environmental exposure. Environ

Health Perspec 108:1203-1207 (2000). 

White, L.D., T.L. Lassier, K.P. Das, and S.

Barone. Prenatal exposure to chlorpyrifos alters

neurotrophin immunoreactivity and apoptosis in

rat brain. Society of Toxicology 2002 Abstract,

Nashville, TN (2002). 
 5-1

Section 5

Cumulative Risk

5.1 Problem

Cumulative risk is of interest to a wide

variety of Agency’s Program Offices. For

example, the FQPA directs the OPP to include

in its assessments of pesticide safety the

risk(s) associated with the cumulative effects

of chemicals that have a common MOA. The

Office of Water (OW) is concerned with risks

from mixtures of DBPs and balancing any

risks associated with DBPs, as single

chemicals or mixtures, against the risks

associated with microbial agents in water. 

Recently, CCL chemicals have become a

concern of OW, leading to considerations of

cumulative risk for CCL chemicals as well as

mixtures containing DBPs and CCLs. The Air

Office is concerned with mixtures of criteria

air pollutants and volatile organic compounds. 

The Waste Program is concerned with

mixtures of chemicals from the same or

different chemical classes that are present in

the air, water and soil in and surrounding

waste sites. In addition to these

chemical-specific concerns, the Agency is

concerned with communities that may either

have elevated exposure to chemicals or may

be subject to stressors such as poverty, lack of

access to medical care, or inadequate

nutrition. 

NHEERL’s research will focus on

providing data that may be used in

development and refinement of risk

assessment approaches, particularly

addressing principles that may underlie

interactions of chemicals at low dose levels

(Teuschler et al., 2002). This research will

utilize empirical and mechanistic data and

models described below, as well as results

derived from work on harmonization (see

Section 4), to develop strategies to predict the

effects of chemical mixtures. Either or both

bottom-up (component-based) and top-down

(whole mixture) approaches may prove useful

for development of mechanistic data and

predictive models. 

The vast majority of risk assessments for

chemical mixtures rely on component-based

approaches. This raises a concern with

cumulative exposure because exposure to

multiple chemicals may interact in ways not

predicted or expected based on the

dose-response curves of the single chemicals

and on an assumption with regard to additivity

(i.e., toxicity may be greater or less than

expected based on the single chemical

information and an assumption of either doseaddition or response-addition). There is also a

minor concern with cumulative exposure that

toxicity may be seen in an unexpected organ. 

As dose-additivity for non-cancer health

effects is a default assumption on which many

Program Offices rely, including OPPTS for

OP pesticides, a major goal of this program

project is to examine those circumstances

under which dose-additivity is a reasonable

assumption. Included in this effort is the

development of efficient experimental designs

and statistical methods for mixtures more

complex than 2-3 chemicals.

Another very important research issue is

what happens when there are components of a

mixture which have different MOAs or

multiple mechanisms. As response-addition is

the risk assessment default assumption for

mixtures of chemicals with diverse MOAs,

another major research goal of the program is

the examination of the potential for additivity

of mixtures of chemicals with different

MOAs. Included in this research will be

comparisons of the appropriateness and

predictive value of response-addition versus

dose-addition for various mixtures.

Another important issue for either

component-based or whole mixture

approaches is whether the quality, e.g., nature

(greater than-, less then-, or additive) or
 5-2

Project 1

Test Dose-Additivity for

Agents with Common

Mode of Action

Project 2

Test Response-Additivity

for Agents with

Dissimilar Modes

of Action 

Project 3

Test Additivity

Assumptions following

Repeated Exposure

Framework to Predict Interactive Effects of Pollutants in Mixtures

Figure 16 Critical Path for Research on Cumulative Risk

Mechanism or

Mode of 

Action 

magnitude, of the interactions of chemicals

change as the dose level changes (Simmons,

1995) or as a function of repeated exposure. 

Data are needed to describe the dependence of

interaction magnitude on mixing ration, total

mixture dose and on component fractions. 

NHEERL research on cumulative risk will

explore the influence of dose and exposure

scenario on interactive toxicity. Historically,

most mixtures research has focused on shortterm exposure to higher portions of the doseresponse curve where such situations as

saturation kinetics might influence the

interactive outcome. In contrast to this

historical situation, NHEERL research will

include a targeted focus on the low end of the

dose-response range and on repeated

exposures.

NHEERL research will also address the

problem of determining a threshold response

for the endpoint(s) of interest at

environmentally relevant doses. These may

include doses at or below the No-ObservedEffect-Level (NOEL) or No-ObservedAdverse-Effect-Level (NOAEL) of the single

chemicals and doses representing either levels

found in the environment or at or near the

RfD. Doses will be selected to be as close to

environmentally relevant levels as permitted

by the limits on experimental sensitivity due

to the number of animals used. In vitro work

will make assumptions regarding the

extrapolation of experimental doses to the

target site concentration in vivo. The upper

end of the dose-response curves will include

effects which are determined to be adverse for

the endpoint in question. It should be noted

that experiments utilizing the mixture ray 

design explicitly measure responses over a

range of effect levels. Effects will not,

however, be characterized at dosages which

produce overt toxicity such as animal

lethality. The critical path for this research is

illustrated in Figure 16. 

5.2 Goal

The overarching goal of NHEERL research

on cumulative risk is to provide the scientific

basis for predicting interactive effects of

pollutants in mixtures and the most
 5-3

appropriate approaches for combining effects

and risks from pollutant mixtures.

5.3 Critical Path

Figure 16 outlines the critical steps that will

be taken for NHEERL research on cumulative

risk. First, either mechanistic data on selected

environmental pollutants or assumptions

about MOA will be used to design subsequent

research on mixtures (see Section 3). A

second step will be to study interactions

among chemicals with a known or assumed

common MOA to determine if the interactions

among them are dose-additive. A next step

will be to determine if interactions among

chemicals with dissimilar MOAs are

response-additive. The last step will be to

investigate whether the nature (i.e., additive,

greater than additive) of the interaction(s) and

their magnitude, if non-additive, that result

from acute exposure are different from those

that result from repeated exposure. Research

in this program project will be consistent with

APGs and APMs in the Cumulative Risk

Section of the ORD Human Health Research

Multi-Year Plan (Appendix D).

5.4 Program Project

Only one program project “Exploring the

Limits of Additivity” was developed. It is

concerned with the assumption of addivity use

in risk assessment of chemical mixtures.
 5-4

Program Project 11: Exploring the Limits of Additivity

Objectives

C Test the assumption of dose-additivity for

chemicals with a similar MOAs,

C Test the assumption of response-additivity

for chemicals with dissimilar MOAs, and

C Determine if the assumption of dose- and

response-additivity changes following

repeated exposure.

Scientific Approach

The Agency has published several

documents describing methods to perform

health risk assessments of chemical mixtures

(US EPA, 1986; 1990, 2001) in which three

approaches (i.e, actual mixture, similar

mixture or component-based) to quantifying

health risk for a chemical mixture are

recommended depending on the type of data

available to the risk assessor. The preferred

approach is to utilize data on the complex

mixture itself, but such data are often not

available. The second approach, which is

recommended when data limitations prohibit

the use of the first approach, uses data on a

“sufficiently similar mixture or a group of

similar mixtures.” As these data are also very

scarce, the overwhelming majority of

mixtures risk assessments are conducted with

component-based approaches. Thus, this

research will focus on component-based

approaches to provide information useful to

how mixtures risk assessment are actually

done in the present and in the future. In

addition to this component-based focus,

several research projects will take a “mixture

as the whole” approach.

The overall approach of project 1 of this

program project will be to test the assumption

of dose-additivity based on the evaluation of

several classes of chemicals, including OPs,

carbamates, pyrethrins, DBPs, persistent

organic pollutants, and azoarene-contaminated

water effluent. Project 2 will use the same

overall approach to determine if the default

assumption of response additivity holds for

chemicals having dissimilar MOAs. Project 3

will focus on predicting effects of exposure to

selected pesticides following repeated

exposure.

Project 1 - Develop and Apply Methods to

Examine Whether the Assumption of DoseAdditivity Holds for Mixtures Comprised

of Two or More Chemicals Acting via a

Common MOA. This project will use

relatively low exposure levels for the

component chemicals in the mixtures. Many

earlier interaction studies have explored the

consequences of high dose combinations, i.e.,

LD50s. Instead, these mixture studies will use

low doses, e.g., levels that do not produce

overt toxicity and approach more

environmentally relevant levels. In addition,

attention will be paid to the ratios of

chemicals present in the mixture because the

proportion of individual chemicals may

influence the nature of the interaction. 

Research in this project will focus on

determining whether an interaction between

pesticides in a mixture can be assessed by

their actions at specific biomolecular targets,

i.e., ion-gated or second messenger-linked

neurotransmitter receptors, various enzymes,

cellular macromolecules, DNA, proteins, or

even specific moieties of a chemical structure. 

This research will address the hypothesis that

compounds that interact with a common

biomolecular target (after adjustment for

potency and PK differences) will behave in a

dose-additive manner. Specific issues to be

addressed by these studies include the

following:

C Development of molecular and

mathematical models for the interactions

of pesticides with specific bio-molecular

targets,
 5-5

C Assessment of interactions of pyrethroid

pesticides on voltage-gated Na+

 channel

function and membrane excitability,

C Assessment of interactions between OP

and carbamate pesticides in rats 

C Assessment of interactions of mixtures

of multiple (3-5) OP pesticides in adult

and pre-weanling rats, and

C Assessment of the interaction of

multiple carbamate pesticides in adult

rats.

Research in this project will also use

diagnostic genotoxicity assays to examine low

dose additivity assumptions for mixtures of

compounds that act via a common MOA. 

This research will examine the following null

hypothesis: regardless of the genotoxic

MOAs and the specific type of xenobiotic

metabolism, mixtures of genotoxic

compounds that act by a common MOA, when

applied at low doses, do not depart from the

linear dose-additivity assumption. This

research will:

C Compare the low dose-response of

environmental mixtures and their

corresponding “totally artificial

mixtures” and the statistical (risk

assessment) prediction of the mixture

from the artificial mixtures for the

environmental mixture;

C Determine if the frequency of “rare

spontaneous mutations” for compounds

that elicit these rare events provides a

means for examining the dose-response

curve at extremely low doses;

C Examine the hypothesis that multiple

chemicals give an additive response

when each chemical is applied at below

detectable (response) doses but the

modeled additive response gives a

minimally detectable response; and

C Address the observation that genotoxic

mixtures also have other toxic effects

(e.g., immunotoxicity).

Research in this project will develop and

use experimental designs and statistical

analysis methods for mixtures of multiple

chemicals having a common MOA. Chemical

mixtures to be evaluated are a chemical

mixture comprised of 4 trihalomethanes

(chloroform, bromoform,

bromodichloromethane and

chlorodibromomethane) that are regulated by

the Agency; a chemical mixture comprised of

5 HAAs regulated by the Agency

(monochloro-, dichloro-, trichloro-,

monobromo-, and dibromoacetic acid); and a

9 chemical mixture that contains the submixtures. This research will:

C Extend existing methodology for simple

mixtures (Gennings et al., 1997) to

mixtures of up to 10-20 chemicals.

Additional research in project 1 will test the

interaction of persistent chemicals that are

similar in structure and assess their effects on

following developmental exposure. This

research will explore in in vitro and in vivo

models the effects of chemical mixtures such

as the PCBs and polybrominated

diphenylethers on key biological events

known to be critical for the development of

the nervous system, such as PKC. These

experiments will test the hypothesis that the

interactions of chemicals with a similar MOA

follow dose-additivity with regard to PKC and

will:

C Develop neuronal cultures (hippocampal

and cerebellar granule neurons) to

examine the effects of chemical

mixtures in vitro,

C Construct individual dose-response

curves in vitro for selected chemicals,

C Determine presence of dose-additivity of

binary mixtures, and

C Evaluate dose-additivity of complex

mixtures at the low end of the doseresponse curve.

Project 2 - Examine the Potential for

Response-Additivity of Mixtures of

Chemicals with Different MOAs. With few

exceptions, the research that has been

conducted examining the validity of the

assumption of additivity has used mixtures of
 5-6

chemicals that have either the same MOA or

the same target organ. Far less work has

examined whether additivity holds for

mixtures of chemicals that contain chemicals

with different MOAs (diverse mixtures), yet it

is to these mixtures that humans are exposed. 

Current risk assessments assume responseadditivity as the default for mixtures of

chemicals of dissimilar action. Thus,

predictive models that operate in the

observable effects region would need to

incorporate response-addition for dissimilar

chemicals. It is possible, however, that doseadditive and response-additive models may

yield the same answer in the very low dose

region. Research in project 2 will evaluate

models for assessment of mixtures of diverse

chemicals, again with emphasis on the lower

regions of the dose-response curves. Research

in project 2 will:

C Test the null hypothesis that additivity of

effects on membrane excitability will be

observed when mixtures of pyrethroids

and organochlorines are administered to

individual cells and cells in intact brain

slices;

C Test the null hypothesis that OPs and

carbamates will interact in a doseadditive manner;

C Use genotoxicity assays to examine low

dose additivity assumptions for mixtures

of compounds that act via a different

MOAs;

C Analyze and interpret the results of a

full-factorial study (5x5x5) of the organ

toxicity of three diverse chemicals,

including trichloroethylene, diethylhexyl

phthalate, and heptachlor, as well as all

possible toxicant combinations;

C Develop and use experimental designs

and statistical analysis methods for

mixtures of multiple chemicals having

different MOAs; and

C Test the interaction of persistent

chemicals (e.g., PCBs, pesticides,

polybrominated diphenyl ethers) and

metals (methylmercury, organotins) that

are not similar in structure and assess

their effects on neuronal PKC.

Project 3- Examine the Usefulness of the

Assumption of Additivity Under Repeated

Exposure Conditions. The existing literature

on the effects of chemical mixtures focuses

almost exclusively on acute exposure

scenarios. The results from these acute

experiments (additivity, synergy, antagonism)

must be used, for lack of better information, to

estimate the risk of chronic exposure to these

mixtures. However, it is well established in

biology that receptor systems will attempt to

maintain homeostasis; and, when subjected to

over-activation or chronic block, receptor

based systems will modulate accordingly to

return the system to its previous balance. This

has been observed as increases or decreases in

receptor or enzyme number; up- or downregulation of second messenger response

systems coupled to receptors; and alterations

in the expression of specific subunits

comprising the enzyme, receptor, or ion

channel. Beyond the level of the biomolecular

receptor, changes in metabolism may affect

the amount of active compounds in the

mixture. Thus, responses which were additive

following single, acute exposures, may

deviate from additivity following repeated

exposure to the same stimulus (mixture). 

Research in this project will:

C Develop models for mixtures of ChEinhibiting pesticides in adult animals

that determine the degree to which

repeated exposure to ChE-inhibiting

pesticides will differ from acute

exposure responses and

C Determine models for mixtures of

pesticides in developing animals.

Impact 

The results from this research effort are

expected to lead to improvements in the risk

assessment of chemical mixtures by (1)

testing assumptions of dose- versus responseadditivity, especially in the low dose region;

(2) examining changes in “additivity” from

acute to chronic exposure scenarios; and (3)

development of appropriate and efficient

statistical models for testing the additivity
5-7

hypothesis in mixtures comprised of larger

numbers of chemicals. Results from this

program project will include chemical specific

data, as well as new statistical methods and

experimental designs for the conduct,

analysis, and interpretation of mixtures

experiments. In addition, the information

from this research may be used to support

guidance for the risk assessment based on the

type of mixtures (e.g., common MOA

mixtures or dissimilar MOA chemicals,

simple mixtures or complex mixtures),

conditions (e.g., higher dose versus low dose,

acute versus chronic), and endpoints (e.g.,

genotoxicity, neurotoxicity, cytotoxicity) for

which the default assumptions of doseaddition or response-addition have been

validated.

Cross-Agency Interactions

This program project involves cross-ORD

collaborations between NHEERL and NCEA

explicitly in projects 1 and 2. The NCEA

collaborators are Ms. L. Teuschler and Dr. R.

Hertzberg. In addition to their specific work

on these projects, they will participate in the

program project research team meetings. The

planned workshops (use of dose addition to

predict interactive effects of mixtures with a

common MOA; use of response addition to

predict interactive effects of mixtures with

diverse MOAs; effect of dose on interactive

toxicity) will include experts and participants

from across ORD, the Program Offices, and

Regions.

5.5 Gap Analysis and Links to Other MultiYear Plans

In the past several years, cumulative risk

has taken on increased importance as

evidenced by several recent publications such

as Pesticides in the Diets of Infants and

Children (NRC, 1993) and Science and

Judgment in Risk Assessment (NRC, 1994). 

NHEERL research is consistent with research

needs identified in those documents and with

issues raised in the Supplementary Guidance

for Conducting Health Risk Assessment of

Chemical Mixtures (US EPA, 2001), as well

as the legislative requirement of the FQPA for

the Agency to focus on the cumulative effects

of pesticides that have a common mechanism

of toxicity. NHEERL research also focuses

on more recent concerns about predicting risk

from mixtures of chemicals with dissimilar

mechanisms, as well as issues related to

changes in mechanism as a function of

repeated exposure. NHEERL research will

examine mixtures from several chemical

classes, including the OPs, carbamates,

pyrethroids, halomethanes, persistent

bioaccumulative toxicants, conazoles, and

chloroatrazines. That NHEERL has little or

no research planned for aggregate risk is a

major gap although NHEERL will examine

the validity of route-to-route extrapolation for

selected air toxics within the Air Toxics MYP.

NHEERL research on cumulative risk is

linked to several other MYPs. In the case of

EDCs, NHEERL will examine the effects of

mixtures of dioxin-like chemicals on the

development in the rat and will examine the

mechanism(s) by which PCB mixtures affect

the development of the nervous system. In the

case of Air Toxics and PM, NHEERL

research on cumulative risk is linked to

research to characterize effects of volatile

organic compound mixtures using in vitro

assays and studies on interaction between PM

and gaseous co-pollutants in mediating

COPD. NHEERL research also is linked to

research to investigate the assumption of

additivity in assessing pesticides with

common MOAs (e.g., mixtures of OPs,

pyrethroids).
 5-8

5.6 References

Gennings, C., P. Schwartz, H. Carter, and J.E.

Simmons. An efficient approach for detecting

departure from additivity in mixtures of many

chemicals with a threshold additivity model. J

Agr Bio Env Stat 2:198-211 (1997). Erratum

5:275-259 (2000).

National Research Council. Pesticides in the Diets

of Infants and Children. Washington, DC,

National Academy of Sciences. (1993).

National Resarch Council. Science and Judgment

in Risk Assessment. Washington, DC, National

Academy of Sciences. (1994).

Simmons, J.E. Chemical mixtures: challenge for

toxicology and risk assessment. Toxicology

105:111-119 (1995).

Teuschler, L., J. Klaunig, E. Carney, J. Chambers,

R. Connolly, C. Gennings, J. Giesy, R. Hertzberg,

C. Klaassen, R. Kodell, D. Paustenbach, and R.

Yang. Support of science-based decisions

concerning the evaluation of the toxicology of

mixtures: a new beginning. Reg Toxicol

Pharmacol 36:34-39. (2002).

U.S. Environmental Protection Agency. 

Guidelines for the Health Risk Assessment of

Chemical Mixtures. Federal Register

51(185):34014-34025. (1986).

U.S. Environmental Protection Agency. Technical

Support Document on Health Risk Assessment of

Chemical Mixtures. Washington, DC. 

EPA/600/8-90/064. 1990.

U.S. Environmental Protection Agency. 

Supplementary Guidance for Conducting Health

Risk Assessment of Chemical Mixtures. 

Washington, DC. EPA/630/R-00/002. 2001.
 A-1

APPENDIX A 

Resource Allocation for Program Projects

HARMONIZATION

TITLE TEAM LEADERS FY03 FTE (ESTIMATES )

Harmonization of Cancer and Non-cancer

Risk Assessment: Disruption of MitogenActivated (MAPK) Signaling as a Common

Mode of Action for Environmental

Toxicants 

William Mundy (NTD)

Barbara Abbott (RTD)

 5.7

Disruption of Luteinizing Hormone (LH)

Secretion as a Common Mode of Action for

Altered Fertility, Reproductive Disease, and

Cancer of the Reproductive System 

Ralph Cooper (RTD) 3.1

Modulation of Cytochrome P-450s and

Other Xenobiotic Metabolizing Enzymes

(XMEs) Leading to Common Mode of

Action for Multiple Toxicities

Stephen Nesnow (ECD) 10.0

SUSCEPTIBLE SUBPOPULATIONS

Identifying and Validating Biologic

Indicators of Susceptibility and Sensitivity

among Children to Assess Potential Risk of

Adverse Outcomes Associated with

Environmental Exposure

Stanley Barone (NTD)

Pauline Mendola

(HSD)

 14.4

Extrapolating Across Windows of

Vulnerability to Assess Children’s Health

Risks Using Rodent Toxicity Data 

Hugh Barton (ETD) 2.7

Long-Term Effects of the Developmental

Environment 

John Rogers (RTD)

Chris Lau (RTD)

 8.5

Susceptible Subpopulations: Susceptibility

Associated with the Aged

Andrew Geller (NTD) 4.2

Environmental Risk Factors for Asthma Ian Gilmour (ETD) 4.8

Environmental and Genetic Interactions in

Hypertensive Rats: Oxidative Stress as a

Common Susceptibility Attribute for Noncancer Risks

Urmilla Kodavanti

(ETD)

 5.9

Genotype and Phenotype in the Metabolism,

Toxicity and Carcinogenicity of Arsenic 

Kirk Kitchin (ECD)

David Thomas (ETD)

 3.3

CUMULATIVE RISK

Exploring the Limits of Additivity Jane E Simmons (ETD)

Stephanie Padilla

(NTD)

 7.5 

 
A-2

APPENDIX B

Annual Performance Goals (APGs) and Measures (APMs)

 Harmonization Research

ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

APG - Identify potential common modes of actions that underlie

different toxic effects

2003

APM Report on immunohistochemical methods for the detection of

signal transduction activation in vivo in animals and humans

exposed to pollutants

2003 PP#1 MAP-Kinase

APM Report summarizing biological basis for common precursors for

cancer and non-cancer effects by prototypic agents

2003 All Program Projects

APG - Determine utility of emerging technologies in harmonizing risk

assessment

2008

APM Report on the development and application of emerging

technologies to detect changes in signal transduction in vitro and

in vivo

2005 PP#1 MAP-Kinase

APM Report on the use of toxicogenomic and related technologies to

define common modes of action of P-450 modulating chemicals

2006 PP#3 P-450 and XME

APM Summary report on the use of emerging technologies in risk

assessment

2008 All Program Projects

APG - Provide scientific basis for use of mechanistic data in

harmonized risk assessment

2012

APM Report on the use of mechanistic data to define common modes

of action for risk assessment of P450 modulating

chemicals

2004 PP#3 P-450 and XME

APM Report on the range of chemicals that modify regulation of

luteinizing hormone

2005 PP#2-LH

APM Report on the mechanisms involved in altering luteinizing

hormone

2006 PP#2-LH

APM Report on determining common modes of action for

developmental reproductive and neural toxicities induced by P450 modulating chemicals

2006 PP#3 P-450 and XME

APM Report summarizing use of cell signaling data as common mode

of action for harmonization

2007 PP#1 MAP-Kinase

APM Report on use of cell signaling data for extrapolation of mode of

action information from in vitro to the whole animal

2008 PP#1 MAP-Kinase

APM Report on the integration of toxic effects, P-450 modulation,

structure-activity analysis, and gene expression profiling for

application to risk assessment

2008 PP#3 P-450 and XME

APM Report on characterization of cancer and non-cancer

reproductive effects following modulation of luteinizing

hormone secretion

2008 PP#2-LH
ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

 A-3

APM Report summarizing data from prototypic compounds 

acting through p-450/XME modulation

2008 PP#3 P-450 and XME 

APM Report on evaluation of risk associated with multiple exposures

to chemicals having differential effects on secretion of luteinizing

hormone

2010 PP#2-LH

APM Report on the use of cell signaling data for extrapolation of mode

of action information for interspecies extrapolation

2010 PP#1 MAP-Kinase

APM Report summarizing harmonized risk assessment for chemicals

modifying luteinizing hormone secretion

2012 PP#2-LH

APM Report summarizing risk assessment for chemicals acting by

oxidative stress

2012 PP#9 Oxidative Stress

APM Summary report on use of cell signaling data for use in

harmonization of risk assessment

2012 PP#1 MAP-Kinase

APG - Provide a framework for harmonization of extrapolation factors

and mechanistic data in risk assessment

2012 All Program Projects
 A-4

APPENDIX C 

Annual Performance Goals (APGs) and Measures (APMs)

Susceptible Subpopulations 

ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

APG

2005

By 2005, provide risk assessors and managers with methods

and tools for measuring exposure and effects in children,

characterizing risk to children, and reducing risks to children

in schools from harmful environmental agents to support EPA

risk assessment and risk management

2005

APM Provide proof of concept for use of a biologically based, dose

response model of a specific cellular, developmental event to

predict the risk of adverse outcome (i.e., toxicant induced limb

defects)

2002 PP#4 Indicators

APM Report on the biological basis of childhood asthma 2004 PP#8 Asthma

APM Report on the health effects associated with exposure of

animals and humans with asthma to diesel exhaust particles

2004 PP#8 Asthma

APM External review draft report on conducting risk assessments

for children as a sensitive subpopulation and summary of

supporting ORD research

2005 PP#4 Indicators

APM Report on the health effects associated with exposure of

exhaust particles in asthma animal model

2005 PP#8 Asthma

APM Report on at least 1 method to test for effects in the National

Children’s Study, including the assessment of respiratory

health outcomes such as asthma

2004 PP#4 Indicators

APM Report on assessing health effects in children under five years

of age exposed to pesticides

2004 PP#4 Indicators

APM Report on methods to collect, store, and transport biologic

specimens from surrogate tissues in humans and rodent

models for protein and gene expression analysis

2004 PP#4 Indicators

APG

100

By 2009, complete two or more targeted epidemiological or

exposure studies on children to test hypotheses, collect data,

and validate methods and tools for measuring, characterizing,

and reducing real world risks from exposures to harmful

environmental agents to support EPA risk assessment and risk

management

2009

APM Report on determinants of susceptibility to the acute

respiratory health effects of combustion-related pollutants

among asthmatic children in 7 US communities

2003 PP#8 Asthma

APM Report on field ready test system based on classical

conditioning for use in testing developmental neurological

disorders in human infants

2004 PP#4 Indicators

APM Peer-reviewed scientific publication on intra-urban variations

in the prevalence of childhood asthma associated with intraurban gradients of combustion-related pollutants and air

toxics in El Paso, TX

2004 PP#8 Asthma
ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

 A-5

APM Report on the characterization of physicochemical properties

of the allergenic protein from selected indoor fungi and

compare to other well-characterized proteins for hazard

identification

2006 PP#8 Asthma

APM Report on prevalence of asthma and low pulmonary function

levels among 4th and 5th grade schoolchildren in a second

major urban area

2007 PP#8 Asthma

APM Report on analysis of National Children’s Study data on

relationship between exposure to environmental agents and

adverse health outcomes

2008 PP#4 Indicators

APM Report on a validation of field-collected diesel exposure

biomarkers

2008 PP#8 Asthma

APG

2012

By 2012, provide risk assessors and managers with methods

and tools for measuring exposure and effects in children,

including adolescents, characterizing cancer and non-cancer

hazards and risk to children, and reducing risks to children in

schools from harmful environmental agents to support EPA

risk assessment and risk management

2012

APM Report on the use of genomics for monitoring expression of

health status through analysis of accessible tissues and cells

2004 PP#4 Indicators

APM Report on the long-term persistent effects of developmental

exposure to environmental chemicals on cancer and noncancer endpoints

2004 PP#6 Developmental 

 Environment

APM Report on proteins from several indoor fungi that pose a

hazard with respect to allergenicity

2004 PP#8 Asthma 

APM Report on long-term effects of in utero insult on adult health

and reproduction to assess adequacy of current testing

guidelines for reproductive toxicity

2005 PP#6 Developmental 

 Environment

APM Report on the validation of a human blood ex vivo model to

evaluate dose, genetic and age specific factors impacting interand intra-individual phenotypic response measurements

2006 PP#$ Indicators 

APM Report on the assessment of the relative potency of several

indoor fungal allergens to obtain quantitative information for

risk assessments

2005 PP#8 Asthma 

APM Report on age-related pharmacokinetic and

pharmacodynamic changes in developing animals 

2006 PP#5 Windows 

APM Report on the reliability of assays to measure endogenous and

exogenous constituents of breast milk that are implicated in

altering health status of women or their breast-fed children

2006 PP#4 Indicators 

APM Report on feasibility of measuring early postnatal serum

measures of neurotrophic factors (NTF), neurotransmitters,

and cytokines in human infants

2006 PP#4 Indicators 
ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

A-6

APM Report on development of principles to be used to assess

cancer risks in children

2006 PP#4 Indicators 

APM Report on the application of mechanistic information in

assessing cancer risk in children

2007 PP#4 Indicators 

APM Report on the mechanism of chemical-induced childhood

asthma

2007 PP#8 Asthma 

APM Summary report on state of science and application to risk

assessment of pharmacokinetics and pharmacodynamics as it

relates to children’s health

 2008 PP#5 Windows 

APM Report on the predictive validity of early postnatal serum

measures of neurotrophic factors (NTF), neurotransmitters,

and cytokines in human infants for developmental

neurological disorders 

2010 PP#4 Indicators 

APG

135

By 2008, provide risk assessors and managers with methods

and tools for assessing differences in exposures and responses

to harmful environmental agents between the elderly and

younger adults. 

2008

APM

 

Report on modeling for age-dependent pharmacokinetics in

risk assessment

2004 PP#5 Windows 

PP#7 Aging 

APM Report on age-dependence of protective repair and plasticity

mechanisms in aged animal models

2005 PP#7 Aging 

APM Report on latent adverse health effects of developmental

exposures

2005 PP#6 Developmental 

 Environment

PP#7 Aging 

APM Report on differential response of older animals and cells to

environmental agents

2008 PP#7 Aging 

APM Report on gene expression changes correlated with latent

adverse health effects of developmental exposures 

2006 PP#6 Developmental 

 Environment

APM Report on mechanisms of susceptibility to environmental

insult in models of aging

2010 PP#7 Aging 

APG By 2007, analyze and demonstrate the role of genetic factors in

causing cancer and non-cancer endpoints

2007

APM Report on genetic polymorphisms that might alter human risk

of arsenic carcinogenesis

2002 PP#10 Genetics 

APM Report on the role of genetic factors in cancer endpoints 2007 PP#10 Genetics 

APM Report on gene array data from animal and human

polymorphisms and relation to disease susceptibility and

underlying oxidative stress

2008 PP#9 Oxidative Stress

APG

126

By 2006, evaluate the variation in vulnerability to

environmental agents as a result of health status as reflected

by nutritional status and pre-existing disease. 

2006
ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

 A-7

APM Report on the effects of pre-existing respiratory disease on

response to air pollutants

2005 PP#8 Asthma 

APM Report on comparative dose-response toxicity data for

susceptible hypertensive rats and role of oxidative stress

2006 PP#9 Oxidative Stress 
 A-8

APPENDIX D 

Annual Performance Goals (APGs) and Measures (APMs)

Additivity

ANNUAL PERFORMANCE GOALS AND MEASURES YEAR Program Project

APG 134 - Develop methods and measurement data to support models

of cumulative exposures, dose, and effects

2009

APM

 

Report on studies examining interactions of carbamate

pesticides in mixtures

2004 PP#11 Additivity

APM

 

Complete analysis and report on full factorial 5x5x5 study of

effects of three diverse chemicals on several non-cancer

endpoints

2004 PP#11 Additivity

APM Report on cumulative risk of exposure to two high use

pesticides on female reproductive system 

2004 PP#11 Additivity

APM Report of studies on modeling of dose-response curves of 

prototypic chemicals having similar or different modes or

mechanisms of action

2004 PP#11 Additivity

APM Develop in vitro preparations to study interaction of

pyrethrin pesticides in vitro

2005 PP#11 Additivity

APM Report on effects of disinfectant by-product mixtures in vivo

and in vitro

2005 PP#11 Additivity

APM Develop parameters to model dose-additivity for mixtures of

organophosphate and carbamate pesticides

2005 PP#11 Additivity

APM Develop in vitro methods to predict interactions of persistent

environmental toxicants

2005 PP#11 Additivity

APM Demonstrate effects of pesticide mixtures after repeated

dosing scenarios in vivo are different than those following

short-term exposure

2006 PP#11 Additivity

APM Report on studies to predict interactions between persistent

environmental toxicants

2006 PP#11 Additivity

APM Report on studies to predict interactions between

environmentally relevant mixtures of pyrethrins 

2007 PP#11 Additivity

APM Report on prediction of interactions of chemicals in vivo

based on principles developed in vitro

2008 PP#11 Additivity

APM Proceedings from workshop on principles of dose additivity

and response-additivity to predict interactions between

chemicals with similar and dissimlar modes of action,

respectively

2008 PP#11 Additivity

APM Summary report on methods and models to predict

interactions of environmental chemicals 

2009 PP#11 Additivity

APM Chemical mixtures report on the use of dose-additivity to

predict interactions of chemicals with a common mode of

action, and use of response additivity to predict interaction of

chemicals having different mechanisms of action

2011 PP#11 Additivity