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Environmental Neurotoxicology (1992)

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ISBN 978-0-309-04531-5 | DOI 10.17226/1801

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Committee on Neurotoxicology and Models for Assessing Risk, Board on
Environmental Studies and Toxicology, National Research Council
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Environmental Neurotoxicology

Environmental Neurotoxicology

Committee on Neurotoxicology and Models for Assessing Risk

Board on Environmental Studies and Toxicology
Commission on Life Sciences
National Research Council

NATIONAL ACADEMY PRESS

Washington, D.C. 1992

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

ii

NATIONAL ACADEMY PRESS 2101 Constitution Ave., N.W. Washington, D.C. 20418
NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose mem-
bers are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine.
The members of the committee responsible for the report were chosen for their special competencies and with regard for appropriate balance.
This report has been reviewed by a group other than the authors according to procedures approved by a Report Review Committee con-
sisting of members of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine.
The National Academy of Sciences is a private, nonprofit, self-perpetuating society of distinguished scholars engaged in scientific and
engineering research, dedicated to the furtherance of science and technology and to their use for the general welfare. Upon the authority of
the charter granted to it by the Congress in 1863, the Academy has a mandate that requires it to advise the federal government on scientific
and technical matters. Dr. Frank Press is president of the National Academy of Sciences.
The National Academy of Engineering was established in 1964, under the charter of the National Academy of Sciences, as a parallel
organization of outstanding engineers. It is autonomous in its administration and in the selection of its members, sharing with the National
Academy of Sciences the responsibility for advising the federal government. The National Academy of Engineering also sponsors engineer-
ing programs aimed at meeting national needs, encourages education and research, and recognizes the superior achievements of engineers.
Dr. Robert M. White is president of the National Academy of Engineering.
The Institute of Medicine was established in 1970 by the National Academy of Sciences to secure the services of eminent members of
appropriate professions in the examination of policy matters pertaining to the health of the public. The Institute acts under the responsibility
given to the National Academy of Sciences by its congressional charter to be an adviser to the federal government and, upon its own initia-
tive, to identify issues of medical care, research, and education. Dr. Kenneth Shine is president of the Institute of Medicine.
Environmental neurotoxicology / Committee on Neurotoxicology and Models for Assessing Risk, Commission on Life Sciences,
National Research Council
p. cm.
Includes bibliographical references and index.
ISBN 0-309-04531-2
1. Neurotoxicology. 2. Environmental monitoring. 3. Health risk assessment. I. National Research Council (U.S.). Committee on
Neurotoxicology and Models for Assessing Risk.
[DNLM: 1. Environmental Exposure. 2. Nervous System--drug effects. WL 100 E61]
RC347.5.E58 1991
615.9--dc20
DNLM/DLC
for Library of Congress 91-43537
CIP
Additional copies of this report are available from the National Academy Press, 2101 Constitution Avenue, N.W., Washington, D.C. 20418
S538
Printed in the United States of America First Printing, January 1992 Second Printing, August 1992

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

iii

Committee on Neurotoxicology and Models for Assessing Risk

PHILIP J. LANDRIGAN, Chairman, Mt. Sinai School of Medicine, New York

DOYLE G. GRAHAM, Vice Chairman, Duke University Medical Center, Durham, North Carolina
W. KENT ANGER, Oregon Health Sciences University, Portland
JEFFERY BARKER, National Institutes of Health, Bethesda, Maryland
TERRI DAMSTRA, National Institute of Environmental Health Sciences, Research Triangle Park, North
Carolina
DALE HATTIS, Massachusetts Institute of Technology, Cambridge
WILLIAM LANGSTON, California Parkinson Foundation, San Jose
HERBERT E. LOWNDES, Rutgers University, Piscataway, New Jersey
JOE MARWAH, National Institutes of Health, Bethesda, Maryland
PIERRE MORELL, University of North Carolina, Chapel Hill
TOSHIO NARAHASHI, Northwestern University Medical School, Chicago
PHILLIP P. NELSON, National Institutes of Health, Bethesda, Maryland
LAWRENCE W. REITER, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
PATRICIA RODIER, University of Rochester, Rochester, New York
JOSEPH RODRICKS, Environ Corporation, Arlington, Virginia
ELLEN K. SILBERGELD, University of Maryland at Baltimore, Baltimore
PETER S. SPENCER, Oregon Health Sciences University, Portland
BERNARD WEISS, University of Rochester, Rochester, New York
RONALD WYZGA, Electric Power Research Institute, Palo Alto, California
DONALD MATTISON, Liaison, Board on Environmental Studies and Toxicology
JOHN L. EMMERSON, Liaison, Board on Environmental Studies and Toxicology

Staff
RICHARD D. THOMAS, Program Director
KATHLEEN R. STRATTON, Project Director
MARY B. PAXTON, Senior Staff Officer
MARVIN A. SCHNEIDERMAN, Senior Staff Scientist
ANDREW M. POPE, Senior Staff Officer
NORMAN GROSSBLATT, Editor
ANNE M. SPRAGUE, Information Specialist
GARY J. BENNETT, Technical Adviser
IAN C.T. NISBET, Technical Adviser
HUGH TILSON, Technical Adviser
LINDA V. LEONARD, Senior Project Assistant

Sponsor
Agency for Toxic Substances and Disease Registry, U.S. Public Health Service

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

iv

Board on Environmental Studies and Toxicology

PAUL G. RISSER (Chairman), University of New Mexico, Albuquerque

GILBERT S. OMENN (Immediate Past Chairman), University of Washington, Seattle
FREDERICK R. ANDERSON, Washington School of Law, American University
JOHN C. BAILAR, III, McGill University School of Medicine, Montreal
LAWRENCE W. BARNTHOUSE, Oak Ridge National Laboratory, Oak Ridge
GARRY D. BREWER, Yale University, New Haven
EDWIN H. CLARK, Department of Natural Resources & Environmental Control, State of Delaware, Dover
YORAM COHEN, University of California, Los Angeles
JOHN L. EMMERSON, Lilly Research Laboratories, Greenfield, Indiana
ROBERT L HARNESS, Monsanto Agricultural Company, St. Louis
ALFRED G. KNUDSON, Fox Chase Cancer Center, Philadelphia
GENE E. LIKENS, The New York Botanical Garden, Millbrook
PAUL J. LIOY, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey
JANE LUBCHENCO, Oregon State University, Corvallis
DONALD MATTISON, University of Pittsburgh, Pittsburgh
GORDON ORIANS, University of Washington, Seattle
NATHANIEL REED, Hobe Sound, Florida
MARGARET M. SEMINARIO, AFL/CIO, Washington, DC
I. GLENN SIPES, University of Arizona, Tucson
WALTER J. WEBER, JR., University of Michigan, Ann Arbor

Staff
JAMES J. REISA, Director
DAVID J. POLICANSKY, Associate Director and Program Director for Applied Ecology and Natural Resources
RICHARD D. THOMAS, Associate Director and Program Director for Human Toxicology and Risk Assessment
LEE R. PAULSON, Program Director for Information Systems and Statistics
RAYMOND A. WASSEL, Program Director for Environmental Sciences and Engineering

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

Commission on Life Sciences

BRUCE M. ALBERTS (Chairman), University of California, San Francisco

BRUCE N. AMES, University of California, Berkeley
J. MICHAEL BISHOP, Hooper Research Foundation, University of California Medical Center, San Francisco
MICHAEL T. CLEGG, University of California, Riverside
GLENN A. CROSBY, Washington State University, Pullman
LEROY E. HOOD, California Institute of Technology, Pasadena
DONALD F. HORNIG, Harvard School of Public Health, Boston
MARIAN E. KOSHLAND, University of California, Berkeley
RICHARD E. LENSKI, University of California, Irvine
STEVEN P. PAKES, Southwestern Medical School, University of Texas, Dallas
EMIL A. PFITZER, Hoffman-LaRoche, Inc., Nutley, New Jersey
THOMAS D. POLLARD, Johns Hopkins Medical School, Baltimore
JOSEPH E. RALL, National Institutes of Health, Bethesda, Maryland
RICHARD D. REMINGTON, University of Iowa, Iowa City
PAUL G. RISSER, University of New Mexico, Albuquerque
HAROLD M. SCHMECK, JR., Armonk, New York
RICHARD B. SETLOW, Brookhaven National Laboratory, Upton, New York
CARLA J. SHATZ, Stanford University School of Medicine, Stanford
TORSTEN N. WIESEL, Rockefeller University, New York, NY
JOHN E. BURRIS, Executive Director

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Environmental Neurotoxicology

vi

The National Research Council was organized by the National Academy of Sciences in 1916 to associate
the broad community of science and technology with the Academy's purposes of furthering knowledge and
advising the federal government. Functioning in accordance with general policies determined by the Academy,
the Council has become the principal operating agency of both the National Academy of Sciences and the
National Academy of Engineering in providing services to the government, the public, and the scientific and
engineering communities. The Council is administered jointly by both Academies and the Institute of Medicine.
Dr. Frank Press and Dr. Robert M. White are chairman and vice chairman, respectively, of the National Research
Council.
The project was supported by the Environmental Protection Agency; the National Institute of
Environmental Health Sciences; and the Comprehensive Environmental Response, Compensation, and Liability
Act Trust Fund through cooperative agreement with the Agency for Toxic Substances and Disease Registry, U.S.
Public Health Service, Department of Health and Human Services.

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

PREFACE vii

Preface

There is convincing evidence that chemicals in the environment can alter the function of the nervous
system. The number of people afflicted with neurotoxic disease can only be estimated, because the number of
neurotoxic substances is unknown but probably plentiful, and the effects on the nervous system are many and
varied. Despite increasing attention to neurotoxicity in recent years, much work still needs to be done.
The Board on Environmental Studies and Toxicology of the National Research Council's Commission on
Life Sciences convened the Committee on Neurotoxicology and Models for Assessing Risk. Support was
provided by the Agency for Toxic Substances and Disease Registry of the U.S. Public Health Service. The
charge to the committee was to 1) assess the biologic bases of neurotoxicity with regard to establishing
underlying principles relevant to risk assessment and extrapolation across species; 2) review existing models and
indicators of neurotoxic action and disease, including structure-activity relationships, with respect to their
efficacy in identifying neurotoxicants from environmental, occupational, and other potential sources; and 3)
develop critical hypotheses for future research in neurotoxicology, particularly research that will lead to models
for assessing the risks of neurotoxic disease. Committee members represented the breadth of disciplines involved
in environmental neurotoxicology. Their expertise served the committee well as it endeavored to meet its charge.
The committee met seven times over 2 years. It focused on the magnitude of the problem, the use of
biologic markers, neurotoxicity testing, surveillance and epidemiology, and risk assessment. This report presents
the views of the committee members, and the conclusions and recommendations reflect the committee's
deliberations. The committee concluded that research is needed to improve understanding of the mechanisms of
neurotoxic action and the extent of neurotoxic disease. The committee encouraged the development of a
coherent, tiered testing strategy and the improvement of surveillance programs.
The committee acknowledges the tireless efforts of those without whom the report would never have been
completed. The committee thanks Hugh Tilson for his expertise and insight. The committee also acknowledges
NRC staff for their work in organizing and managing this undertaking. Devra Davis was instrumental in
conceptualizing the study initially. James Reisa, director of the Board on Environmental Studies and Toxicology,
provided much welcomed guidance and encouragement. In addition to his contributions as program director,
Richard

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Environmental Neurotoxicology

PREFACE viii

Thomas served as interim project director and gave invaluable support to the committee and staff. The
committee thanks senior staff officers Andrew Pope and Mary Paxton for their work in the formative stages of
the committee process and in helping with early drafts of the report, and Kathleen Stratton for seeing the report
through its final stages. The committee recognizes the tireless efforts of Anne Sprague of the Toxicology
Information Center and Linda Leonard, project assistant. Norman Grossblatt and Lee Paulson served as editors.
On behalf of the committee, I thank all who assisted in completing this report.
Philip J. Landrigan, Chairman
Committee on Neurotoxicology and Models for and Assessing Risk

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

CONTENTS ix

Contents

Executive Summary 1
Magnitude of the Problem 1
Neurotoxicity Testing 2
Surveillance and Epidemiologic Studies in Environmental Neurotoxicology 4
Biologic Markers in Environmental Neurotoxicology 5
Neurotoxicity Risk Assessment 5

1 Introduction: Defining the Problem of Neurotoxicity 9

Neurologic Responses to Environmental Toxicants 9
Magnitude of the Problem of Neurotoxicity 17
Detection and Control of Exposure to Neurotoxicants 18
Scope of this Report 19

2 Biologic Basis of Neurotoxicity 21

Cellular Anatomy and Physiology 21
General Aspects of Nervous System Structure and Function 27
Vulnerability of the Nervous System to Chemical Toxicants 30
Examples of Neurotoxic Mechanisms 33
MPTP 39
Summary 40

3 Biologic Markers in Neurotoxicology 43

Concepts and Definitions 43
Validation of Biologic Markers 48
Use of Biologic Markers in Risk Assessment 53
Summary 51

4 Testing for Neurotoxicity 53

Approach to Neurotoxicity Testing 54

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

CONTENTS x

Current Methods Based on Structure-Activity Relationships 58

Current In Vitro Procedures 59
Current In Vivo Procedures 65
Current Regulatory Approaches 86
Strategies for Improved Neurotoxicity Testing 88

5 Surveillance to Prevent Neurotoxicity in Humans 95

Neurobehavioral Test Batteries 97
Current Exposure-Surveillance Efforts 104
Current Disease-Surveillance Efforts 105

6 Risk Assessment 111

Approaches to Risk Assessment for Neurotoxicity 114
Curve-Fitting in Risk Assessment for Neurotoxicity 116
Mechanistic Models for Risk Assessment for Neurotoxicity 117
Summary 120

7 Conclusions and Recommendations 123

References 129

Index 149

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Environmental Neurotoxicology

CONTENTS xi

Tables and Figures

Tables
1-1 Partial List of Neurotoxicants 10
1-2 Human and Animal Neurobehavioral Effects Attributed to at Least 25 Chemicals 11
1-3 Selected Major Neurotoxicity Events 12
2-1 Nonneuronal (Glial) Cells of the Nervous System and Their Function 22
2-2 Neuron Type Classified by Neurochemical Released for Synaptic Transmission 27
3-1 Examples of Characteristics of Exogenous; Agents, Organisms, or Targets That Influence 44
Choice of Biologic Marker
3-2 Selected Markers of Neurotoxicity in Nervous System 45
4-1 In Vitro Neurobiologic Test Systems 62
4-2 Markers for Assessing Neurotoxicity in In Vitro Systems 63
4-3 Proposed Protocol for Developing and In Vitro Neurotoxicity Screening System 65
4-4 Neurotoxic Effects of Representative Agents in Humans and Animals 67
4-5 Examples of Behavioral Measures of Functional Neurotoxicity 69
4-6 End Points That Might be Included in a Functional Observational Battery 71
4-7 Areas of the Nervous System to be Used in Neuropathologic Evaluation 81
4-8 Tissues of the Nervous System to be Used in Neuropathologic Evaluation 82
4-9 Tests Used in NCTR Collaborative Study 83
4-10 Proposed Components for Evaluating In Vitro Neurotoxicity Screening Tests 92
5-1 Characteristics of Responses to Exposure to Some Neurotoxicants 98
5-2 Components of Clinical Neurologic Examination 100
5-3 Test Batteries 102
6-1 Some Neurotoxicants That Act on Receptors 118

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Environmental Neurotoxicology

CONTENTS xii

Figures
2-1 Diagrammatic representation of neuronal structure 23
2-2 Events in chemical synaptic transmission 26
2-3 Structures of type I and type II pyrethroids 34
2-4 Metabolism of hexane 37
2-5 Diagram of MPTP toxicity 39
3-1 Simplified classification of biologic markers 46
4-1 Biologic markers in the stages between formation and degneration of neural circuits 91
6-1 Effect of a shift in mean IQ score on the population distribution 113
6-2 Estimated developmental scores at various ages for three blood-level concentrations 115
6-3 Percentage of severe mental retardation among those exposed in utero by dose and gestational 119
age in Hiroshima and Nagasaki

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Environmental Neurotoxicology

EXECUTIVE SUMMARY 1

Executive Summary

The recognition that exposure to chemicals can cause neurologic injury evolved from studies of acute
illnesses in people exposed to high doses of environmental toxicants. These illnesses included encephalopathy in
children who ate chips of lead-based paint; blindness in persons who consumed wood alcohol (methanol); and
coma, convulsions, and respiratory paralysis after exposure to organophosphorus pesticides. Epidemics of
neurotoxic diseases related to environmental exposures have occurred: blindness and ataxia caused by organic
mercury in fish from Minamata Bay, Japan, and in fungicide-treated grain in Iraq; spinal-cord degeneration and
peripheral neuropathy caused by tri-o-cresylphosphate (TOCP) in cooking oil in Morocco; and tremors, motor
disturbance, and anxiety caused by the pesticide Kepone (chlordecone) in Hopewell, Virginia. In all, these
epidemics affected thousands of people and established clearly that toxic chemicals in the environment 1 can
cause neurologic and psychiatric illnesses.
In response to a request from the Agency for Toxic Substances and Disease Registry, the National Research
Council convened the Committee on Neurotoxicology and Models for Assessing Risk in 1988. The committee,
charged to review the biologic principles and mechanisms of neurotoxic action relevant to risk assessment,
produced this report, which discusses the magnitude of the problem of neurotoxic effects, testing strategies,
surveillance efforts, biologic markers, and risk assessment.

MAGNITUDE OF THE PROBLEM

Neurotoxicity caused by environmental toxicants results in a range of neurologic and

1 ''Environment'' is defined broadly in this document to encompass a wide range of external factors that can cause injury,

including diet, ethanol, tobacco, drugs, and occupational exposures, as well as toxic contaminants in what are ordinarily
considered components of the ambient environment—air, water, and soil. "Neurotoxicity" is defined as the capacity of
chemical, biologic, or physical agents to cause adverse functional or structural changes in the nervous system.

Copyright National Academy of Sciences. All rights reserved.

Environmental Neurotoxicology

EXECUTIVE SUMMARY 2

psychiatric disorders. Concern over the potential neurotoxic effects of chemical substances is greatest for agents
that cause irreversible or progressive changes. In addition to immediate and progressively developing effects,
there is increasing evidence that neurotoxic effects can occur after long latent periods. It is postulated that
intervals as long as many decades can elapse between exposure to a chemical and the appearance of neurologic
illness.
A major unanswered question—indeed, a central issue confronting neurotoxicology today—is whether the
causal associations observed in epidemics of neurotoxic diseases reflect isolated events or are merely the most
obvious examples of a widespread association between environmental chemicals and nervous system
impairment. Concern about subclinical neurotoxicity has brought this issue to its current prominence. Subclinical
toxicity refers to exposure-induced adverse effects that are too small to produce signs and symptoms evident in a
standard clinical examination. Subclinical neurotoxic effects can include alteration of a wide spectrum of
behaviors. Environmental chemicals associated with subclinical neurotoxicity include lead, organophosphorus
pesticides, some chlorinated hydrocarbons, some solvent mixtures, and mercury. Those are chemicals to which
many thousands of Americans are regularly exposed at work and to which even more are exposed in smaller
doses in the general environment. Although often subtle, subclinical neurotoxic effects are not necessarily
inconsequential; moreover, even subtle alterations can be irreversible. It has been hypothesized that an undefined
fraction of chronic neurologic and psychiatric illness in the human population can be exacerbated or even caused
by chronic, low-level exposure to environmental neurotoxicants.
The committee recommends that more accurate estimates of the extent of the problem of neurologic
and psychiatric dysfunction attributable to chemical agents in the environment be developed.
The estimates must be based on a combination of clinical, epidemiologic, and toxicologic studies coupled
with the techniques of quantitative risk assessment.

NEUROTOXICITY TESTING
The general reason to test substances for neurotoxicity is to identify neurotoxic potential before the
occurrence of human exposure. The ultimate goal is to prevent human disease. About 70,000 chemicals are used
in commerce, of which several hundred are known to be neurotoxicants. However, except for pharmaceuticals,
less than 10% of the chemicals in commerce have been tested at all for neurotoxicity, and only a handful have
been evaluated thoroughly. Furthermore, resources are not readily available to undertake across-the-board testing
of all chemical substances already in commerce.
New strategies for neurotoxicologic assessment of environmental chemicals will need to include
establishing testing priorities among chemicals for hazard identification (with emphases on new chemicals,
chemicals considered likely to be hazardous, and chemicals to which large numbers of people are exposed). The
new strategies will also require refining existing neurotoxicity test systems and developing sensitive, new testing
systems. To contain the labor and resource requirements of the testing strategy, developing quicker and more
economical approaches than are currently used must be encouraged, particularly for screening for potential
neurotoxicants.
A new strategy for neurotoxicologic assessment will build on and extend currently available test systems. It
will have a "tiered" structure—decisions to test chemicals at the higher tiers, as well as decisions concerning
types of testing, will be guided by data from the initial, or screening, tier. The first tier, or screen, will be used for
hazard identification. The results of the screen and a chemical's exposure pattern would guide further
characterization of dose-response relationships

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Environmental Neurotoxicology

EXECUTIVE SUMMARY 3

(second tier) and mechanisms (third tier).

No existing validated system satisfies all the necessary requirements for a screening program to detect the
neurotoxic potential of chemicals. The range of such a program should extend to the detection of
neurodevelopmental effects and effects on cognitive function and of neuroendocrine effects. No comprehensive
effort has been made to determine the predictive ability of individual screening tests by examining the
relationship between test results and data from long-term studies in animals or epidemiologic and clinical studies
in humans.
The screening tier will consist of a set of tests to measure multiple end points—including chemical,
structural, and functional changes—and a functional observational battery. Determination of the component tests
of the first tier is the most crucial aspect of the three-tiered approach, because truly positive substances will not
continue to later tiers unless detected at this point. The first tier is the heart of the screening aspect of
neurotoxicity testing; the later tiers might produce data of great value in advancing understanding of neurotoxic
processes and how the nervous system operates, but the first tier is the first line in preventing neurotoxic disease.
Much of the controversy over proper testing procedures arises from two intrinsically conflicting objectives—
minimizing the incidence of false positives (substances incorrectly identified as hazardous) and minimizing the
incidence of false negatives (substances incorrectly identified as nonhazardous). Too high an incidence of false
positives wastes resources. However, a high incidence of false negatives is potentially dangerous. For the
prevention of human disease, testing systems must be highly sensitive. Testing in the second and third tiers of
future test systems should expose false positives; the abandonment of an occasional new chemical on the basis of
what are actually false-positive screening results is the likely cost of this process.
The committee recommends that a rational, cost-effective neurotoxicity testing strategy be adopted.
It should allow an accurate and efficient progression from the results of hazard-identification studies
(screening) to the selection and application of appropriate test methods for defining mechanisms of toxicity and
for quantitative characterization of neurotoxic hazards.
A coherent testing strategy would incorporate a series of checks and balances. The results of the later phases
of testing would provide the data necessary to evaluate and validate initial screening batteries and thereby help to
identify tests that should be excluded from or incorporated into an efficient battery. Information generated by
such a strategy would reveal which types of data are most useful for accurate, quantitative prediction of the risks
to humans associated with exposure to similar chemical compounds.
Tests of neurotoxicity on chronically exposed animals might be carried out in conjunction with tests of
other chronic effects. Such testing might identify toxic effects of substances not previously known to be
neurotoxic. There is a particular lack of data on chronic and long-latency neurotoxic effects.
The committee recommends that for reasons of efficiency, integrative studies combining a variety of
end points be explored in the development of the neurotoxicity testing strategy.
To maximize detection of toxicity, some toxicity studies encompassing the full life span of experimental
animals should be encouraged.
Data are needed on the influence of dose, route of exposure, toxicokinetics, metabolism, and elimination on
the effects of a given neurotoxicant, and data are needed on the existence of interspecies differences. More
complete understanding of neurotoxic disease at a molecular level should also improve the ability to evaluate
new chemicals

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Environmental Neurotoxicology

EXECUTIVE SUMMARY 4

on the basis of structure-activity relationships. Existing in vitro test methods should be exploited more efficiently
than at present to identify and analyze the mechanisms of neurotoxic action at cellular levels. Development of a
mechanistic understanding of neurotoxicity might facilitate the discovery of biologic markers of exposure to
toxicants, as well as markers of early, subclinical neurotoxic effects.
The committee recommends that studies to define mechanisms of neurotoxicity in as much detail as
possible be encouraged, as well as studies to identify hazards.
A program needs to be undertaken to test the relationship between in vitro and in vivo findings and between
animal and human results for a set of well-defined substances.

SURVEILLANCE AND EPIDEMIOLOGIC STUDIES IN ENVIRONMENTAL

NEUROTOXICOLOGY
Epidemiologic and clinical studies of populations exposed to potentially neurotoxic chemicals are needed to
provide additional information on the human neurotoxic effects of environmental chemicals and to complement
in vitro and animal screening studies. High-risk populations must be identified and monitored. Public-health
surveillance systems to identify people who are potentially exposed to environmental neurotoxicants are not well
developed, and there is little information on the background incidence and prevalence of the major neurologic
diseases in the American population. People with diagnosed neurologic illnesses must be studied to identify
possible environmental etiologies and to complement and extend the knowledge gained through in vivo and in
vitro laboratory investigations.
Recognition of the neurotoxic effects of exposure to environmental chemicals through epidemiologic and
clinical studies is made difficult by the enormous variety of the possible reactions of the nervous system to toxic
insult. The changes are often subtle and subclinical, and months or years can elapse between exposure to a
neurotoxicant and the appearance of dysfunction or disease. Few attempts have been made to explore the
possible relationships between chemical exposures and chronic or progressive neurologic and behavioral
disorders. Populations known to be exposed to potential neurotoxicants should be followed for long periods in
prospective studies, and retrospective studies of people with neurologic illness must consider the possibility that
exposures occurred many years previously. Epidemiologic studies will increasingly need to use biologic markers
of exposure, of effects, and of susceptibility.
The committee recommends that exposure-surveillance systems cover a much broader range of
chemicals and use improved monitoring techniques for long-term assessment.
Existing disease-surveillance systems, such as those of the Social Security Administration, the Department
of Veterans Affairs, and the National Center for Health Statistics, should be modified to provide more useful
data on the incidence and prevalence of chronic neurologic and psychologic disorders, some of which are likely
to be of occupational and environmental origin. A broader range of neurologic disease end points should be
covered by surveillance programs. Anecdotal reports of neurotoxicity in humans need to be pursued vigorously
with clinical surveillance and followup. The incorporation in surveillance systems of the concept of sentinel
health events (SHEs) specifically for neurotoxic illnesses should be encouraged.
Recognition of the possible environmental origin of neurologic and psychiatric disease is hampered by the
inadequate training of most physicians and other health providers

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Environmental Neurotoxicology

EXECUTIVE SUMMARY 5

in occupational and environmental medicine. Greater uniformity in disease definition would improve
identification of diseases of neurologic interest.
The committee recommends that improved disease reporting be supported by the dissemination of
information on neurotoxic illnesses to physicians and other health professionals to increase their
awareness of environmental neurotoxicity as a possible explanation of specific illnesses or sets of symptoms.
All physicians should be trained to take a thorough occupational-exposure history and to be aware of other
possible sources of toxic exposure, such as hobbies and self-medication. Uniform clinical definitions of
neurotoxic disorders are needed to provide a common basis for reporting by physicians. Standardized national
reporting systems must be established for physicians to report outbreaks of suspected environmentally and
occupationally caused neurologic and psychiatric disorders.

BIOLOGIC MARKERS IN ENVIRONMENTAL NEUROTOXICOLOGY

As recently defined by a previous NRC committee, biologic markers are measures of changes or variations
in biologic systems or samples. It is useful to classify biologic markers into three types: markers of exposure, of
effect, and of susceptibility. A biologic marker of exposure in an organism is a measurable presence of an
exogenous substance, its metabolite, or the product of its interaction with some target molecule or cell. A
biologic marker of effect is a measurable biochemical, physiologic, or other alteration within an organism that,
depending on magnitude, can indicate potential or established disease. A biologic marker of susceptibility is an
indicator of an inherent or acquired variation in an organism's ability to respond to the challenge of exposure to a
specific substance.
Biologic markers provide a means to explore the relationship between exposures and effects. By virtue of
the incorporation of more relevant information about critical events and molecular mechanisms, biologic markers
contribute to a more complete scientific understanding of toxic injury. Biologic markers can be used to improve
sensitivity, specificity, and predictive value of detection and quantification of adverse effects at low dose and
early in exposure. Neuroscience and neurotoxicology can be enhanced by directing more research attention to
quantitative questions that information on biologic markers helps to answer.
The committee recommends that putative biologic markers in animal species be evaluated and
validated in in vivo and in vitro systems.
Biologic markers should be regularly incorporated into epidemiologic and clinical studies of neurologic
disease, particularly prospective studies. The primary goal of the incorporation of biologic markers into such
studies should be to validate their predictive accuracy and to test hypothesized quantitative relationships between
specific markers related to causal pathways involving neurotoxic outcomes.

NEUROTOXICITY RISK ASSESSMENT

Risk-assessment techniques provide a means for estimating the risks to humans associated with exposure to
toxic chemicals in the environment. The estimation of the risks most often involves extrapolation from high
experimental doses used in animal tests to lower environmental doses. Numerous assumptions are typically made
to bridge gaps in the available scientific data. Most

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Environmental Neurotoxicology

EXECUTIVE SUMMARY 6

risk-assessment procedures have focused on cancer as an end point, and techniques for assessing other types of
risk are relatively undeveloped. Commonly used paradigms for risk assessment do not accurately or adequately
model the risks associated with exposure to neurotoxicants. Virtually all neurotoxicologic risk assessment today
is limited to qualitative hazard identification and to the early stages of hazard characterization; neither sufficient
data nor adequate paradigms are available to permit quantitative evaluation of most neurotoxic risks.
Risk-assessment techniques that incorporate more quantitative information about dose-time-response
relationships and mechanisms of neurotoxicity are under development. Advances in this regard will improve not
only the assessment of risk, but also assessment of the benefits to be gained by reducing human exposure to
specific neurotoxic agents. The construction of new models for neurotoxicologic risk assessment depends on the
acquisition of new knowledge of the fundamental mechanisms of action of chemical toxicants on the human
nervous system. The molecular and subcellular mechanisms by which environmental neurotoxicants act need to
be delineated. Such information will improve prediction and quantification of risks, including risks that become
evident only long after exposure.
Neurotoxicologic risk assessment has been largely limited to the application of no-observed-effect levels
and uncertainty factors, so it has not generated information on specific risks for given magnitudes of exposure.
Experiments should include a range of doses that spans those relevant to expected human exposures. In addition
to providing a firmer basis for estimating human risk, such designs permit tests of the assumption that results
obtained at high doses predict the pattern of effects at low doses. Such variables as age, sex, duration of
exposure, and route of exposure need to be more systematically evaluated. Species-specific effects need to be
recognized and investigated. The usefulness of that kind of information for quantitative risk assessment would be
greatly amplified by serial measures of both neurotoxic end points and biologic markers. A single model will not
be adequate for all conditions of exposure, for all end points, or for all agents. It might be necessary to build risk-
assessment models to deal simultaneously with several end points produced by a toxicant. Such models should
incorporate biologic markers of neurologic dysfunction and be based on fundamental information on
mechanisms derived from experimental test systems and epidemiologic data.
The committee recommends the development of risk-assessment methods that capture the
complexities of the neurologic response, including dose-time-response relationships, multiple outcomes,
and integrated organ systems.
Experimental designs for studies of neurotoxic agents should provide information needed in the risk-
assessment process. Full exploration of relationships and development of risk assessment models would be
facilitated by original researchers' making complete data sets available to other investigators.
For the future, one of the major challenges for neurotoxicology will be to use insight from clinical
medicine, epidemiology, and toxicology to design effective systems for the prevention of neurotoxic disease in
the American public.

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Environmental Neurotoxicology

ENVIRONMENTAL TOXICOLOGY 7

Environmental Toxicology

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Environmental Neurotoxicology

ENVIRONMENTAL TOXICOLOGY 8

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 9

1
Introduction: Defining the Problem of Neurotoxicity

NEUROLOGIC RESPONSES TO ENVIRONMENTAL TOXICANTS

The human nervous system coordinates behavior; in perceiving and responding to external stimuli, it is
responsible for mediating communication with the external environment; and it coordinates the activities of all
other organ systems and thus plays an essential role in maintaining metabolic balance. The consequences of
damage to the nervous system can be profound. Massive injury can result in coma, convulsions, paralysis,
dementia, and incoordination. Even slight nervous system damage can impair reasoning ability, cause loss of
memory, disturb communication, interfere with motor function, and impair health indirectly by reducing
functions, such as attention and alertness, that ensure safety in the performance of daily activities.
Despite the nervous system's compensatory and adaptive mechanisms, many kinds of injury to the nervous
system are irreversible, because, after initial development, new nerve cells are not formed; resulting losses of
function can be permanent, as well as debilitating. Effects that are difficult to detect in an individual, such as a 5-
point decline in intelligence quotient (IQ), are of great concern if they occur throughout large portions of a
population. Prevention of damage to the nervous system is a major objective of social policy, medicine, and
public health.
"Neurotoxicity" is the capacity of chemical, biologic, or physical agents to cause adverse functional or
structural change in the nervous system. We use the term "environmental neurotoxicity" to refer broadly to
adverse neural responses to exposures to all external, extragenetic factors (e.g., occupational exposures, lifestyle
factors, and exposures to pharmaceuticals, foods, and radiation); it does not refer merely to the toxic effects of
chemicals that are present in the environment as contaminants of air, water, and soil. Table 1-1 lists compounds
for which there is evidence of neurotoxicity.
Possible effects of chemical toxicants on the nervous system are varied. For example, Table 1-2 lists
neurobehavioral symptoms that—according to clinical reports, epidemiologic investigations, and experimental
studies—are caused in humans or animals by at least 25 chemicals (Anger and Johnson, 1985; Anger, 1986).
Neurotoxicity can occur at any time in the life cycle, from gestation through senescence, and its manifestations
can change with age. The developing nervous system appears to be particularly vulnerable

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 10

TABLE 1-1 Partial List of Neurotoxicants

Acetone Excitatory amino acids
Acetonitrile Formaldehyde
Acrylamide Glycerol
Adriamycin Gold salts
Aliphatic hydrocarbons Hexane
Alkanes 2,5-Hexanedione
Alkyl styrene polymers Lead and lead-containing compounds
Aluminum Isophorone
Ammonia Lithium grease
N-Amyl acetate Manganese
Aniline Mercury and mercury-containing compounds Methanol
Antimony sulfide Methanol
Aromatic hydrocarbons Methyl acetate
Benzene Methyl nitrite
Butanol 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Butyl acetate 1-Nitrophenyl-3-(3-pyridylmethyl) urea
Cadmium Nitrous oxide
Carbon disulfide 6-OH-Dopamine
Carbon monoxide Organophosphates
Carbon tetrachloride Oubain
Chlordane Ozocerite
Chlordecone Petroleum distillates
Chlorinated hydrocarbons Pine oil
Chlorobenzene Polymethacrylate resin
β-Chloroprene Products of combustion
Chromium oxides 1-Propanol
Cresol Propylene gylcol
Cyclohexanol Pyrethroids
Cyclohexanone Ricin
Diacetone alcohol Selenium
o-Dichlorobenzene Shellac
Dichlorodifluoromethane Styrene
1,2-Dichloroethane Tetrachloroethylene
Dichloromethane Toluene
Dichlorotetrafluoroethane Trichlorobenzene
Dicylcopentadiene Trichloroethylene
Dimethylaminopropionitrile Trichlorofluoromethane
Dinitrobenzene Tricresyl phosphate
Diphenylamine Triethyltin; trimethyltin
Dyes Tungsten oxides
Ergot Turpentine
Ethanol Vincristine
Ethyl acetate Vinyl chloride
Ethylene glycol Xylene

Source: Adapted from Anger (1986).

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 11

TABLE 1-2 Human and Animal Neurobehavioral Effects Attributed to at Least 25 Chemicals
Effect No. Chemicals or Effect No. Chemicals or
Chemical Groups Chemical Groups
that Produce the that Produce the
Effect Effect
Motor Affective or personality
Activity changes 32 Apathy, languor, 30
lassitude, lethargy,
listlessness
Ataxia 89 Delirium 26
Convulsions 183 Depression 40
Incoordination, 62 Excitability 58
unsteadiness,
clumsiness
Paralysis 75 Hallucinations 25
Pupil changes 31 Irritability 39
Reflex abnormalities 54 Nervousness, tension 29
Tremor, twitching 177 Restlessness 31
Weakness 179 Sleep disturbances 119
Sensory General
Auditory disorders 37 Anorexia 158
Equilibrium changes 135 Autonomic 26
dysfunction
Olfaction disorders 37 Cholinesterase 64
inhibition
Pain 47 CNS depression 131
Pain disorders 64 Fatigue 87
Tactile disorders 77 Narcosis, stupor 125
Vision disorders 121 Peripheral neuropathy 67
Cognitive
Confusion 34
Memory problems 33
Speech impairment 28

Source: Anger (1986).

to some kinds of damage (Cushner, 1981; Blair et al., 1984; Pearson and Dietrich, 1985; Annau and Eccles,
1986; Hill and Tennyson, 1986; Silbergeld, 1986), but the results of some early injuries may become evident
only as the nervous system matures and ages (Rodier et al., 1975).
The observation that some neurologic and psychiatric disorders are of environmental origin is not new (see
Table 1-3). Initially, only striking manifestations of neurotoxicity were recognized; e.g., the association of coma,
convulsion, and colic with high-dose exposure to lead has been recognized at least since Roman times (Waldron,
1973; Nriagu, 1978). Other long-recognized associations include an increased frequency of depression and
suicide among workers in contact with carbon disulfide (Vigliani, 1954); spastic paraparesis (lathyrism) due to

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 TABLE 1-3 Selected Major Neurotoxicity Events
Year(s) Location Substance Comments
370 B.C. Greece Lead Hippocrates recognizes lead toxicity in mining industry (Klaassen et al., 1986)
Environmental Neurotoxicology

1st century A.D. Rome Lead Pliny warns against inhalation of vapors from lead furnaces (Waldron, 1973)
1837 Scotland Manganese First description of five cases of chronic manganese poisoning in factory workers handling powdered
manganese dioxide (Bellare, 1967)
1924 United States (New Jersey) Tetraethyllead In incidents at two plants processing the gasoline additive, over 300 (New Jersey) workers suffer
neurologic symptoms and five die; nonetheless, its use in gasoline continues for over 50 years (Rosner
and Markowitz, 1985)
1930 United States (Southeast) TOCP Compound often added to lubricating oils intentionally added to Ginger (Southeast) Jake, an alcoholic
beverage substitute; more than 5,000 paralyzed, 20,000–100,000 affected (Spencer and Schaumburg,
1980)
1930s Europe Apiol (with TOCP) Abortion-inducing drug containing TOCP causes 60 cases of neuropathy (with TOCP)(Spencer and
Schaumburg, 1980)
1932 United States (California) Thallium Barley laced with thallium sulfate, used as a rodenticide, is stolen and (California) used to make
tortillas; 13 family members hospitalized with neurologic symptoms; six die (Spencer and Schaumburg,
1980)
1937 South Africa TOCP 60 South Africans develop paralysis after using contaminated cooking oil (Spencer and Schaumburg,
1980)
1946 England Tetraethyllead People suffer neurologic effects of varied degrees of severity after cleaning gasoline tanks (Cassells
and Dodds, 1946)
1950s Japan (Minamata) Methymercury Hundreds ingest fish and shellfish contaminated with mercury from chemical plant; 121 poisoned; 46
die; many infants with serious nervous system damage (Spencer and Schaumburg, 1980)
INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY

1950s France Organotin Medication (Stalinon) containing diethyltin diiodide results in more than 100 deaths (Spencer and
Schaumburg, 1980)
1950s Morocco Manganese 150 ore miners suffer chronic manganese intoxication involving severe neurobehavioral problems
(Spencer and Schaumburg, 1980)

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12
 1956 Turkey Hexachorobenzene Hexachlorobenzene, a seed-grain fungicide, leads to poisoning of 3,000–4,000; 10% mortality
rate (Weiss and Clarkson, 1986)
1956–1977 Japan Clioquinol Drug used to treat travelers' diarrhea found to cause neuropathy; as many as 10,000 affected
over 2 decades (Spencer and Schaumburg, 1980)
Environmental Neurotoxicology

1959 Morocco TOCP Cooking oil deliberately and criminally contaminated with lubricating oil affects some 10,000
people (Spencer and Schaumburg, 1980)
1960 Iraq Methylmercury Mercury used as fungcide to treat seed grain is used in bread; more than 1,000 people affected
(WHO, 1986)
1964 Japan Methylmercury Methylmercury affects 646 people (Spencer and Schaumburg, 1980; WHO, 1986)
1968 Japan PCBs Polychlorinated biphenyls are leaked into rice oil; 1,665 people are affected (Goetz, 1985)
1969 Japan n-Hexane 93 cases of neuropathy follow exposure to n-hexane, used to make vinyl sandals (Spencer and
Schaumburg, 1980)
1969 United States (New Mexico) Methylmercury Ingestion of pork contaminated with fungicide-treated grain results in severe cases of human
alkyl mercury poisoning—first instance of such poisoning in United States (Pierce et al., 1972)
1971 United States Hexachlorophene After years of bathing of infants in 3% hexachlorophene, the disinfectant is found to be toxic to
nervous system and other systems (Klaassen, 1986)
1971 Iraq Methymercury Methylmercury used as fungicide to treat seed grain is used in bread; more than 5,000 severe
poisonings and 450 hospital deaths occur; effects on many infants exposed prenatally not
documented (Weiss and Clarkson, 1986; WHO, 1986)
1972 France Hexachlorophene 204 children become ill and 36 die in an epidemic of percutaneous poisoning clue to 6.3%
hexachlorophene in talc baby powder (Martin-Bouyer et al., 1982)
1973 United States (Ohio) Methyl n-butylketone (MnBK) Fabric-production plant employees exposed to solvent; more than 80 workers suffer
polyneuropathy, and 180 have less severe effects (Billmaier et al., 1974)
INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY

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13
 1974–1975 United States (Hopewell, VA) Chlorodecone (Kepone) Chemical-plant employees exposed to insecticide; more than 20
suffer severe neurologic problems, and more than 40 have less-
severe problems (Spencer and Schaumburg, 1980)
1976 United States (Texas) Leptophos (Phosvel) At least nine employees suffer serious neurologic problems after
Environmental Neurotoxicology

exposure to insecticide during manufacturing process (Spencer

and Schaumburg, 1980)
1977 United States (California) Dichloropropene (Telone II) 24 people are hospitalized after exposure to pesticide Telone II
due to traffic accident (CDC, 1978)
1979–1980 United States (Lancaster, TX) 2-t-Butylazo-2-hydroxy-5-methylhexane (BHMH, Lucel-7) Seven employees of plastic-bathtub manufacturing plant
experience serious neurologic problems after exposure to BHMH
(Horan et al., 1985)
1980s United States 1-Methyl-4-pentyl-1,2,3,6-tetrahydropyridine (MPTP) Impurity in synthesis of illicit drug is found to cause symptoms
identical with those of Parkinson's disease (Kopin and Markey,
1988)
1981 Spain Toxic oil 20,000 persons poisoned by toxic substance in oil; more than 500
die; many suffer severe neuropathy (Altenkirch et al., 1988)
1985 United States and Canada Aldicarb More than 1,000 people in California, other western states, and
British Columbia experience neuromuscular and cardiac
problems after ingestion of melons contaminated with the
pesticide (MMWR, 1986)
1987 Canada Domoic acid Ingestion of mussels contaminated with domoic acid causes 107
illnesses and three deaths; form of marine vegetation found in
estuaries off Prince Edward Island proves to be apparent source
INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY

of contaminant (Perl et al., 1990)

1988 India TOCP Ingestion of adulterated rapeseed oil causes about 600 cases of
polyneuritis (Srivastava et al., 1990)
1989 United States L-trytophan-containing products Ingestion of a chemical contaminant associated with the
manufacture of containing products L-tryptophan by one
company results in outbreak of eosinophilia-myalgia syndrome,
primarily in the western United States; by 1990, over 1,500 cases
have been reported nationwide (Swygert et al., 1990; Belongia et
al., 1990)

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14
Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 15

ingestion of Lathyrus species during famines (Denny-Brown, 1947); paralysis following consumption of
products contaminated with tri-o-cresylphosphate (TOCP), such as the patent medicine Ginger Jake in the United
States (Smith et al., 1930) and cooking oil in Morocco (Smith and Spalding, 1959); toxic psychoses in people
who inhale tetraethyl lead (Cassells and Dodds, 1946); and erethism (a syndrome with such neurologic features
as tremor and such behavioral symptoms as anxiety, irritability, and pathologic shyness) in people exposed to
elemental mercury (Bidstrup, 1964).
Numerous other associations between neurologic impairment and environmental exposures have been noted
more recently: Exposure to low concentrations of environmental lead is correlated with reduced scores on tests
of mental development (Bellinger et al., 1987a,b; Needleman, 1989); the pesticide Kepone (chlordecone) is
associated with nervousness, tremors, and other signs of nervous system dysfunction (Cannon et al., 1978); 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of illicit synthetic-heroin production, causes an
irreversible syndrome that closely resembles parkinsonism (Langston et al., 1983); and exposure to manganese
can produce parkinsonism and dyskinesia (Cook et al., 1974). In the realm of developmental neurotoxicity, early
gestational exposure to ionizing radiation is associated with microcephaly and mental retardation (Otake and
Schull, 1984); infants born to women who use cocaine during pregnancy display significant depression of
nervous system response to environmental stimuli and other congenital anomalies (Shepard, 1989); heavy
ingestion of alcohol by pregnant women produces a syndrome of craniofacial abnormalities and mental
retardation in their children (Jones and Smith, 1973), and it is possible that there are persistent adverse effects on
mental and motor development in children whose mothers drink more moderately during pregnancy and lactation
(Little et al., 1989; Streissguth et al., 1989; West, 1990).
It has been suggested that exposures to environmental chemicals can contribute to clinical
neurodegenerative disorders seen later in life. Calne et al. (1986) have hypothesized that various environmental
agents contribute to Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS) by depleting
neuronal reserves to an extent that becomes observable in the context of natural aging. For example, a syndrome
combining the symptoms of ALS and of parkinsonian dementia is prevalent among people from Guam and has
been postulated to result from earlier ingestion of one or more chemicals found in the seed of the false sago
palm, or cycad (Cycas circinalis L.) (Kurland, 1963; Spencer et al., 1987). Some epidemiologic studies have
found evidence that various environmental factors are involved in the etiology of ALS, Parkinson's disease, and
Alzheimer's syndrome (e.g., Barbeau et al., 1987; Freed and Kandel, 1988; Lilienfeld et al., 1989; Yokel et al.,
1988), but other investigations have not detected such relationships (e.g., French et al., 1985; Shalat et al., 1988).
The extent to which environmental neurotoxicants contribute to chronic neurologic and psychiatric disease is not
yet known.
The nervous system is composed of cells of several types, each with its own functions and characteristic
vulnerabilities. Several unique features of the nervous system influence its reactions to toxic agents, including its
poor regenerative capacity, its unusual anatomy (especially, long axons), its multiple functions, the extensive
interconnections among its cells, its dependence on glucose as an energy source, the existence of highly
specialized cellular subsystems, and the wide variety of highly localized neurotransmitter and neuromodulation
systems. In addition, environmental toxicants can disturb the complex interactions between the nervous system
and other organs. (Chapter 2 discusses the

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 16

structure and function of the nervous system In some detail, including consideration of the complex neural
arrangements involved in information processing and storage.)
The accessibility of a particular part of the nervous system to a specific chemical is a function of both the
tissue and the chemical. Many chemicals are kept from entering the brain by the ''blood-brain barrier''—the tight
junctions formed by endothelial cells surrounding capillaries that supply brain tissue and by endothelial cell-
astrocyte interactions. That barrier can, however, be crossed by lipophilic substances and has a series of specific
transport mechanisms through which required nutrients, hormones, amino acids, peptides, proteins, fatty acids,
etc., reach the brain (Pardridge, 1988). Thus, toxicants can gain entry to the brain if they are lipid-soluble or if
they structurally resemble substances that are normally taken up by the nervous system. Moreover, the blood-
brain barrier might be less effective in immature than in mature organisms (Klaassen, 1986). It is absent in some
parts of the brain, such as in the circumventricular area, and around the olfactory nerve, which runs directly from
the nose to the frontal cortex (Broadwell, 1989).
Some cells in the nervous system cannot reproduce themselves; once damaged, they cannot be replaced.
Many of those cells ordinarily are present in excess, so there is a buffer against damage, and substantial loss need
not affect function or behavior. However, the degree of redundancy, particularly in specific regions, is not known
and might vary with age. The presence of such excess can result in a threshold or nonlinear dose-response
relationship. Also, the interaction between exposures to neurotoxicants and age-related cell loss might explain
delays in the manifestations of toxic consequences. If several processes (e.g., chemical damage, normal aging,
and death of cells) are proceeding simultaneously, it might be impossible to isolate a single cause of functional
impairment. A long latent period before toxicity is manifested makes it difficult to associate an exposure and a
response causally and even harder to derive a quantitative dose-response relationship.
Manifestations of neurotoxic response can be progressive, with small functional deficits becoming more
serious. In those cases, it is almost impossible to define the onset of impairment. For many biologic variables
that respond to exogenous agents, the demarcation between an unimportant change and a health-damaging
change is unclear. A small change might be a marker of exposure, a moderate change might signal preclinical
disease, and a large change might indicate advanced disease.
Some materials, particularly pharmaceuticals, produce different responses in the nervous system at different
doses or have adverse side effects at therapeutic doses. For example, the tricyclic antidepressants have a desired
therapeutic activity at a low dose, but produce life-threatening anticholinergic effects at higher doses; the
antineoplastic drug cis-platinum is a valuable chemotherapeutic agent, but can cause toxic neuropathies;
antipsychotic drugs can produce disabling movement disorders; and some antibacterial agents can trigger loss of
hearing and balance (Sterman and Schaumburg, 1980). Some substances valued for their relatively selective
neuroactivity, such as ethanol, are particularly likely to have simultaneous neurotoxicity (Goldstein and Kalant,
1990).
Exposure to combinations of chemicals can produce interactive effects. Examples include exacerbated
hearing loss in persons exposed to some antibiotics (Bhattacharyya and Dayal, 1984; Lim, 1986; Boettcher et al.,
1987) and cumulative toxic effects of occupational and environmental exposures to mixtures of solvents
(Cranmer and Goldberg, 1986). The general population is exposed to chemicals with neurotoxic properties in
foods, cosmetics, household products, air, water, and drugs used therapeutically or

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 17

recreationally. Exposure to naturally occurring neurotoxins, such as some fish and plant toxins, is yet another
aspect of the problem. The multitude of voluntary and unintentional exposures to neuroactive substances that
characterize the daily life of ordinary people complicates the task of identifying undesirable neurologic outcomes
and attributing them to their proper causes.

MAGNITUDE OF THE PROBLEM OF NEUROTOXICITY

The number of people with neurotoxic disorders and the extent of neurologic disease and dysfunction that
result from exposure to toxic chemicals in the environment are not known. However, it is known that an
enormous number of people are exposed to environmental materials that in sufficient doses could pose a
neurotoxic hazard. The National Institute for Occupational Safety and Health (NIOSH, 1977) estimated that in
1972–1974 there were 197 chemicals to which a million or more American workers were exposed in the
occupational setting for all or part of each working day. Anger (1986), in a review of secondary sources in the
research literature, found that more than one-third of those 197 chemicals had demonstrated the potential, if the
doses were large enough, to produce adverse effects on the nervous system.
The data needed to estimate the overall magnitude of the problem of environmental neurotoxicity do not
exist. A National Research Council committee found a few years ago that few of the 60,000–70,000 chemicals in
commercial use had been tested for neurotoxicity (NRC, 1984); no information was available on any aspect of
the toxicity of approximately 80% of the chemical substances in commercial use. Even among the most
extensively regulated classes of chemicals (pesticides, drugs, and food additives), the information needed for a
thorough health-hazard assessment was available on only about 5–18%. Few compounds have been assessed for
selective toxicity to vulnerable groups within the population, such as the very young and very old. Moreover,
there is little information on the nature and extent of human exposure to even the materials that have been tested
and identified as neurotoxic. Finally, not all neurotoxic effects are equivalently damaging or debilitating; scales
of relative injury are needed, if the effects of exposures to different materials are to be evaluated in comparable
terms.
Given the overall low proportion of substances tested for any type of toxicity and the even smaller fraction
that have been assessed for possible neurotoxicity, it is difficult to estimate how many commercial and industrial
chemicals are neurotoxic. However, there is evidence that many toxic chemicals in wide use have neurotoxic
potential. Of 91 criteria documents produced by NIOSH, 36 (40%) cite neurotoxicity as a reason for
recommending limits on occupational exposure (Anger, 1989). Likewise, of 588 chemicals listed by the
American Conference of Governmental Industrial Hygienists (ACGIH) in 1982 as both widely used in industry
and having toxicologic significance, 167 (28%) had neurologic effects as one basis for recommendations on
maximal exposure concentrations (Anger, 1984). However, O'Donoghue (1986) calculated, on the basis of tests
of a small, unselected sample of chemicals, that 5% of all industrial chemicals, excluding pesticides, are likely to
be neurotoxic. Diener (1987) noted that O'Donoghue's calculation could be an underestimate and should not be
considered a firm basis for extrapolation of risk until broader and more systematic surveys of the neurotoxicity
of chemicals in commercial use have been undertaken.
In addition to unwanted contact with pollutants or contaminants and inadvertent exposures to industrial
products, voluntary exposures to many legal and illegal materials can cause neurotoxic effects (OTA, 1984).
Overuse of the legal stimulant caffeine can lead to tremors, irritability, and sleeplessness. Exposure to the
depressant ethanol is

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 18

responsible for many accidents on the highway and elsewhere, fetal injuries, chronic disease, and antisocial
behavior. The devastating personal and societal consequences of "substance abuse," engaged in at least initially
for psychoactive effects, have become obvious. The list of self-administered neuroactive substances is extensive,
and exposure to them makes epidemiologic identification of additional neurotoxicants difficult.
The Health Care Financing Administration of the U.S. Department of Health and Human Services has
estimated that $23 billion was spent in 1980 for the care of people with diagnosed neurologic diseases; in many
cases, these illnesses are due to accidental exposures to neurotoxicants or to use of legal drugs, rather than to use
of illegal substances (Rice et al., 1985). As has been suggested regarding lead toxicity (Provenzano, 1980; EPA,
1985), it would not be surprising if the direct and indirect societal costs of subclinical neurologic losses or deficits—
such as reduction in intelligence, diminution in achievement, and waste of opportunity—might equal or exceed
those of neurotoxic effects that are clinically recognized. Modifications of behavior induced by neurotoxic
substances can also adversely affect people other than those directly experiencing the toxic effects, as is the case
for victims of drunken drivers.

DETECTION AND CONTROL OF EXPOSURE TO NEUROTOXICANTS

Prevention is the key to dealing with neurologic diseases of toxic environmental origin. Such diseases result
from exposures to synthetic and naturally occurring chemical toxicants encountered in the ambient environment,
ingested in foods, or administered as pharmaceutical agents. Many of the diseases are not curable, so they must
be prevented. They can be prevented by eliminating or reducing exposures at the source (primary prevention), or
they can be controlled by early detection and diagnosis of neurotoxic effects while they are still reversible or
while they are at a relatively early stage of evolution when their progression can still be halted (secondary
prevention).
In light of the difficulty of regulating potentially neurotoxic compounds found in illicit substances of abuse
or in certain foods, particularly in plants, the most effective approach to preventing neurotoxic disease of
environmental origin consists of identification of neurotoxicity through routine pre-market testing of all new
chemicals before they are released and before human exposure has occurred. Disease is prevented by restricting
or banning the use of chemicals found to be neurotoxic or by instituting engineering controls and imposing
protective devices at points of environmental release and potential human exposure.
Given the current absence of data on neurotoxicity of most chemicals in commerce, an extensive program
of primary prevention through toxicologic evaluation is needed. The Environmental Protection Agency (EPA)
has regulatory authority to screen chemicals coming into commerce, but some have expressed the opinion that
the authority is insufficiently exercised and inadequately supported. Therefore, people might be overexposed to
environmental neurotoxicants and might develop neurotoxic responses to chemicals that slip through the
regulatory net. For example, important side effects of pharmaceutical agents continue to surface, such as the 3
million cases of tardive dyskinesia in patients on chronic regimens of antipsychotic drugs. Prudence dictates that
all chemical substances—both those already on the market and new ones—be considered potential
neurotoxicants; a chemical cannot be regarded as free of neurotoxicity in the absence of data on its toxicity.
Prudent public policy therefore dictates that all chemicals, both old and new, be subjected to at least basic
screening for neurotoxicity when use and exposure warrant.
Neurotoxicants can be identified by several means, ranging from clinical case reports and observations by
alert physicians to formal

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Environmental Neurotoxicology

INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 19

in vivo and in vitro screening. Analysis of structure-activity relationships (SARs) has been used extensively to
predict neurotoxicity, but SARs as currently used can provide only minimal guidance for hazard identification.
EPA, however, uses SARs as a basis for decisions on testing under the Toxic Substances Control Act. Testing
procedures designed for in vivo neurotoxicologic evaluation of new chemicals have been developed and are
reasonably effective, but they are tedious and labor-intensive. Additional mechanistically based in vitro test
systems are badly needed, and they must be correlated with in vivo test systems. In summary, a rational and
efficient strategy for neurotoxicity testing is needed to close the gap in neurotoxicity testing. Development of a
blueprint for such a strategy was a major objective of the study reported here.

SCOPE OF THIS REPORT

The Committee on Neurotoxicology and Models for Assessing Risk was formed at the request of the
Agency for Toxic Substances and Disease Registry to review the biologic principles and mechanisms of
neurotoxic action for the purpose of using them in risk assessment. This report deals with environmental
neurotoxicology—with the current state of the field, its implications for human health, and the prospects for its
rational future development. The committee first reviewed current knowledge of environmental neurotoxicity,
paying special attention to the types of neurologic injury caused by chemicals in the environment, to determine
the overall extent of the problem of environmental neurotoxicity. We investigated ways in which improved
biologic markers of neurotoxicity (markers of subtle and subclinical effects, of exposure to neurotoxicants, and
of susceptibility to their effects) could be developed, could improve laboratory tests, and could facilitate the early
recognition of neurotoxic injury in exposed human populations. We then considered available methods of testing
for neurotoxicity and evaluated strategies for recognizing and characterizing the neurotoxic potential of
chemicals and for detecting neurotoxic effects among environmentally or occupationally exposed humans.
Finally, we examined approaches to neurotoxicologic risk assessment.
This chapter has provided an overview of the problem of environmental neurotoxicity, described the level
of existing knowledge, and suggested broad issues for scientific research. Chapter 2 illustrates the biologic basis
of neurotoxicity by reviewing the aspects of the structure and function of the nervous system that are subject to
disruption by exogenous agents. Chapter 3 discusses biologic markers, which provide a conceptual thread
connecting human, animal, and in vitro studies. Chapter 4 considers approaches to identifying neurotoxicants
and describes available in vitro and in vivo tests that could be used to assess toxicity before human exposure has
occurred. It develops a blueprint for a multitiered program to test chemical substances for neurotoxicity, and it
lays out a plan for systematic evaluation of the results of neurotoxicity testing. Chapter 5 describes approaches to
the epidemiologic surveillance of human populations at risk of environmental neurotoxicity and outlines the uses
to which human data can be put in bringing neurotoxic exposures under control. And Chapter 6 describes current
approaches to risk assessment and discusses modifications in the risk-assessment paradigm that will be needed to
accommodate neurotoxic end points. And Chapter 7 presents the committee's conclusions and recommendations.

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INTRODUCTION: DEFINING THE PROBLEM OF NEUROTOXICITY 20

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BIOLOGIC BASIS OF NEUROTOXICITY 21

2
Biologic Basis of Neurotoxicity

The nervous system is the communication and information-storage system of the body. In addition to
coordinating functions that are commonly associated with the brain, such as thinking, the nervous system plays a
major role in physiologic regulation. Virtually all physiologic functions are influenced or controlled by the
nervous system. Other systems, such as the circulatory and reproductive systems, provide information to the
nervous system, which in turn controls their functioning. The nervous system also integrates the functions of the
various organ systems. Thus, neurologic dysfunction has a major impact that goes beyond the nervous system
itself. Conversely, abnormal functioning in other systems can modify neurologic functioning.
Our abilities to act, to feel, to learn, and to remember all depend on the complex anatomic arrangements and
proper integrative functioning of the nervous system, of which we have only a rudimentary understanding. The
ultimate goal of neuroscience is to understand how this system functions normally and how subtle perturbations
can lead to disease and dysfunction. Observation of the response of experimentally exposed animals or
inadvertently exposed humans to neurotoxic chemicals advances our understanding of neurobiology while
generating information about the consequences of exposure to environmental pollutants.
The first two sections of this chapter summarize our current understanding of the nervous system. The next
section discusses several aspects of the nervous system that make it especially vulnerable to chemically induced
damage. The fourth section describes examples of neurotoxicant-induced damage of several types. A final
section summarizes the problems arising from the nature of neurobiology that must be addressed when a
substance is tested for neurotoxicity, the subject of Chapter 4.

CELLULAR ANATOMY AND PHYSIOLOGY

The nervous system is composed of two general types of cells, nerve cells (neurons) and glial cells, which
are present in approximately equal numbers. Neurons are responsible for the reception, integration, transmission,
and storage of information. They can be classified as interneurons and motor, sensory, and neuroendocrine
neurons. Motor neurons innervate and elicit contraction of muscle fibers. Sensory neurons

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BIOLOGIC BASIS OF NEUROTOXICITY 22

monitor the organism's external and internal environments. Specialized receptors on sensory nerves convey
information regarding light, sound, muscle position, taste, and smell to the central nervous system. In many
cases, sensory neurons form the input (afferent) side of information loops in the central nervous system; the
output (efferent) side is mediated by motor fibers or by the endocrine system. Neuroendocrine neurons elicit
secretions from glandular cells. Interneurons facilitate or modulate interactions between other neurons, often in
complex feedback loops. Glial cells differ from neurons in their ability to divide throughout the organism's life,
particularly in response to injury, and in their functions (Table 2-1), which are less well understood than those of
neurons, but are believed to be largely restricted to structuring and regulating the environment surrounding the
neurons. Glial cells also play a major role in myelination.

Structure of the Neuron

A neuron (Figure 2-1) has a more or less round cell body (soma), which contains its nucleus and organelles
for protein synthesis. The diameter of the cell body of a vertebrate animal is about 10–100 µm (the largest would
be roughly the size of the period at the end of this sentence). The neuron gets its distinctive appearance from its
dendrites and axon, elongated processes that emanate from the cell body. The length and branching patterns of
the dendrites vary from one type of neuron to another. Dendrites and their branches greatly increase a neuron's
surface area, and this increases the number of messages that the neuron can receive and the number of sources
from which they can come. The arrangement is analogous to the branching of a tree, which increases its
reception of sunlight by increasing its total leaf surface area.
In most neurons, the axon is a thin, particularly elongated cellular process emanating from the neuronal
soma and is specialized for the conduction of nerve signals away from the cell body and toward its synapse with
other cells (other neurons, muscle cells, or gland cells). One neuron communicates with another by releasing
chemical signals onto a small gap, or the synapse, between the tip of an axon and the dendrites or soma of its
target cell. Axons are shorter or longer, depending on the type of neuron, but an axon's length is generally tens or
thousands of times greater than the diameter of the cell body. An extreme example is the axon of a motor neuron
that innervates the
TABLE 2-1 Nonneuronal (Glial) Cells of the Nervous System and their Function
Glial Cell Type Functions
Astrocyte Repair, structural support, transport
Oligodendrocyte
Satellite Neuronal maintenance
Intermediate Potential for maintenance or myelination
Intrafasicular Myelination
Microglia Phagocytosis
Ependymal cells Service buffer between brain and cerebrospinal fluid
Schwann cells Myelination of peripheral nerves

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BIOLOGIC BASIS OF NEUROTOXICITY 23

Figure 2-1 Diagrammatic representation of neuronal structure. A) The drawing illustrates the gross structure of a
neuron with its many dendrites, myelin sheath, and synaptic contact with another neuron. Source: Liebman, (1991).
B) The drawing shows structures visible at the electron microscope level. The nucleus, nucleolus, chromatin, and
nuclear pores are represented. In the cytoplasm are mitochondria, rough endoplasmic reticulum, the Golgi
apparatus, neurofilaments, and microtubles. Along the surface membrane are such associated structures as synaptic
endings, astrocytic processes, and a capillary containing a red blood corpuscle.
Source: Willis and Grossman, (1973).

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BIOLOGIC BASIS OF NEUROTOXICITY 24

muscles in the human toe. Its cell body (a fairly large one) lies in the spinal cord at the level of the middle
of the back; its axon is more than a meter long. Near its target, an axon divides into many small branches called
telodendria. Telodendria end in small enlargements called synaptic boutons, which contain the chemical
messengers that will be released to the target cell. A single axon can end in thousands or hundreds of thousands
of synaptic boutons, by which a nerve can communicate with a few or many thousands of target cells.
Many axons are insulated by myelin, the extended and modified plasma membrane of specialized glial cells.
During early development, myelin is deposited in a spiral fashion to form a sheath around the axon. Myelin is
richer in lipid than are other membranes, and several unusual lipids are found in the myelin sheath; some well-
characterized proteins are peculiar to myelin. Myelin of the central nervous system is made by oligodendrocytes
and differs from the myelin of the peripheral nervous system, which is made by Schwann cells.
The myelin sheath formed by one oligodendrocyte or Schwann cell covers only a short length of the axon;
adjacent lengths of axon are ensheathed by adjacent myelinating cells. Very short lengths of bare axon between
myelinated areas are called nodes of Ranvier. Within myelinated axons, a nerve impulse travels by jumping from
one node to the next. Compared with conduction in axons that are not myelinated, this mode of travel, called
saltation, increases the speed of impulse conduction by a factor of up to 100. In a poorly understood metabolic
dependence between a myelinated axon and its ensheathing cells, some types of injury to one cause dysfunction
in the other.

The Nerve Impulse

Two processes are involved in communication by neurons: electric (discussed here) and chemical
(discussed in the next section). Within a neuron, transmission is electric and analogous to that in a battery. The
axonal membrane is semipermeable to the positively and negatively charged ions (chiefly, potassium, sodium,
and chloride) that are in solution inside and outside the cell. In the absence of active and directed metabolic
transport, those ions would be distributed equally on each side of the axonal membrane, and the inner and outer
electrochemical potentials would be the same. However, several enzyme systems normally transport ions
actively from the inside of the axon to the outside or from the outside to the inside. As a result, the axonal
membrane separates areas of unequal electrochemical potential; i.e., it is polarized and generates a resting
potential (Davies, 1968). The situation is analogous to a flashlight battery, where an impermeable membrane (a
strip of cardboard) separates two fluids (pastes in a dry-cell battery) that contain ions of positive charge on one
side and negative charge on the other. The neuronal "battery" must be maintained by continuous active transport
of ions across the membrane.
The action potential is generated as a result of opening and closing of various ion channels in the nerve
membrane (Hille, 1984). Several types of ion channels are known, but the most common are sodium and
potassium channels, which are tiny pores that allow sodium and potassium ions, respectively, to pass through.
The action potential is propagated regeneratively along the nerve fiber without loss of amplitude. In the resting
state without stimulation, the potassium channels open and close occasionally, and the sodium channels remain
mostly closed. Therefore, the resting nerve membrane is permeable mainly to potassium, but only sparingly to
sodium. That results in the resting potential, with the cell's interior being negative with respect to the outside by
70 mV or so. On electric stimulation, however, many sodium channels start opening,

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BIOLOGIC BASIS OF NEUROTOXICITY 25

and that makes the membrane permeable predominantly to sodium. Electric stimulation thus generates an action
potential with a peak some 50 mV more positive on the interior than the exterior of the nerve. Soon the sodium
channels start closing and the potassium channels start opening at a high frequency; this brings the membrane
potential back to the resting level. During the action potential, sodium ions enter the cell through open sodium
channels and potassium ions leave the cell through open potassium channels; the result is a slight imbalance of
the internal sodium and potassium concentrations, which is corrected quickly by a metabolic pump that extrudes
extra sodium and absorbs potassium. Thus, the cell is capable of generating many action potentials in rapid
succession.
The ion channels are the primary target sites of some toxicants and therapeutic drugs. For example,
tetrodotoxin (a puffer fish poison) and local anesthetics block the sodium channel, thereby impairing the impulse
conduction. The insecticidal pyrethroids and DDT keep the sodium channels open for unusually long periods,
making the nerve generate impulses in a hyperactive fashion.

Synaptic Transmission
When a nerve impulse reaches a terminal branch of an axon, it depolarizes the synaptic boutons. That
initiates the second type of transmission by means of the cellular release of special messenger chemicals
(neurotransmitters) that are stored in the boutons. The amount of secretion is related to the degree of
depolarization, which, in turn, is a function of the number of nerve impulses that reach a synaptic bouton. A very
narrow gap, the synapse, separates the bouton from its target cell (Figure 2-2). The released chemicals diffuse
across the synapse and attach themselves to specific receptor molecules embedded in the membrane of the target
cell. Attachment (binding) initiates one of several responses, usually involving changes in ion flux, depending on
the type of target cell and the type of messenger-receptor pair involved. For example, when the target cell is a
muscle cell, the neurotransmitter-receptor interaction leads to contraction of the muscle; when the target is a
gland cell, the interaction leads to secretion.
Synaptic transmission between neurons is slightly more complicated. Messenger molecules from the bouton
of a presynaptic (releasing) cell bind to receptors on the surface of the postsynaptic (receiving) cell; that can lead
to either excitation or inhibition of the target neuron. Neuronal cell bodies and dendrites are polarized in the
same way as the axon. At an excitatory synapse, the neurotransmitter-receptor interaction leads to a momentary
opening in ion-specific channels that produces depolarization (excitatory postsynaptic potential). At an inhibitory
synapse, a different neurotransmitter-receptor interaction leads to a momentary opening of different ion-specific
channels that produces an increase in polarization (hyperpolarization) or a small depolarization (inhibitory
postsynaptic potential). Unlike the membrane of the axon, the membrane of the cell body and dendrites does not
generate nerve impulses. Instead, the sum of all the excitatory and inhibitory postsynaptic potentials determines a
moment-by-moment average polarization that is monitored by the portion of the cell's axon adjacent to the cell
body. When the running average exceeds a threshold depolarization at the axon's initial segment, a nerve impulse
is generated and begins its electrochemical journey down the axon.
The duration of neurotransmitter action is a function primarily of the time that it remains in the synapse.
Under normal circumstances, that is a very short time (a few thousandths of a second), because specialized
enzymes quickly degrade the neurotransmitter, and, in some cases, metabolically driven re-uptake systems
rapidly scavenge

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BIOLOGIC BASIS OF NEUROTOXICITY 26

the synapse for neurotransmitter and transport it back into the synaptic bouton of the releasing cell.

Figure 2-2 Events in chemical synaptic transmission. A, action potential shown approaching axon terminal
containing transmitter stored in synaptic boutons. B, arrival of nerve impulse triggers release of transmitter into
synaptic gap. Transmitter diffuses across synapse and reacts with receptor molecules associated with postsynaptic
membrane. Resulting change in membrane permeability to ions, such as sodium and potassium, causes flow of
synaptic current. In this case, current flows inward through postsynaptic membrane and results in depolarization
called excitatory postsynaptic potential. Later transmitter action is terminated by enzymatic destruction, diffusion,
or re-uptake. Source: Willis and Grossman (1973).

Synaptic Messengers
Several dozen neurotransmitters are known, and more are discovered every year. (A partial list is given in
Table 2-2.) A given neuron can synthesize and release a single neurotransmitter or, in the case of peptidergic
neurons, several neurotransmitters. Similarly, a neuron can contain receptors for and be influenced by several
neurotransmitters.

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BIOLOGIC BASIS OF NEUROTOXICITY 27

TABLE 2-2 Neuron Type Classified by Neurochemical Released for Synaptic Transmission
Neuron Type Neurotransmitter
Cholinergic Acetylcholine
Excitatory amino acid Glutamate, aspartate
Inhibitory amino add γ-Aminobutyric acid (GABA), glycine
Catecholaminergic Dopamine, epinephrine, norepinephrine
Sertoninergic Sertonin
Peptidergic Substance P, enkephalins, endorphins, neurotensin, carnosine, angiotensin II, pituitary
hormones and their hypothalmic controlling factors
Other Histamine, taurine, proline, N-acetyl-L-aspartate, serine, octop amine, p-tyramine, N-acetyl
serotonin, tryptamine, purine, 5-methoxytryptamine, pyrimidine derivatives
Research on the functions of the multitude of synaptic messengers is one of the most active fields in
neuroscience today. Although not all the details are known, one conclusion is clear: neurons communicate by a
complex code that involves a large number of chemicals and exquisitely specific chemical interactions.

GENERAL ASPECTS OF NERVOUS SYSTEM STRUCTURE AND FUNCTION

The nervous system has two parts: the central nervous system (CNS) and the peripheral nervous system
(PNS). The CNS comprises the brain and the spinal cord. The PNS is composed of nerve cells and their
processes that lie outside the skull and vertebral column. The nerve cells of the PNS are generally found in
aggregates called ganglia. The neurons of the CNS are often segregated into aggregates called nuclei. Various
nuclei are functionally related to one another to constitute levels of organization called systems; thus, we speak
of the motor system, the visual system, and the limbic system.
The cells of the nervous system are organized in networks of increasing evolutionary complexity. In
anatomic terms, the most primitive level is the spinal cord, wherein responses to sensory signals are virtually
instantaneous; such responses are called reflexes. Spinal-cord functions tend to have been conserved in
evolution, so the system in lower vertebrates resembles that in humans. The oldest regions of the brain regulate
involuntary activities of the body, such as breathing and blood circulation, and exert much of their influence via
the reflexes of the spinal-cord. The brain stem—which comprises the medulla, pons, and mid-brain—has
functions related primarily to motor and sensory activities of the head and neck, including hearing, balance, and
taste. The diencephalon is composed of the thalamus and the hypothalamus. The thalamus processes most
information from the rest of the nervous system on its way to the cerebral cortex, and the hypothalamus regulates
autonomic, endocrine, and visceral integration. The basal ganglia assist the cerebral cortex

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BIOLOGIC BASIS OF NEUROTOXICITY 28

with motor functions. The cerebral cortex, the most highly evolved level, is a vast information-storage and-
retrieval area.
In addition to its role in such functions as perception and learning, the nervous system has a critical role in
controlling the body's endocrine glands. That control is concentrated in several small nuclei at the base of the
brain that are known collectively as the hypothalamus. Nerve cells in the hypothalamus secrete chemical
messengers into a short loop of blood vessels that carries them to the pituitary, the body's master gland. The
pituitary, in turn, releases chemical messengers into the general circulation. The pituitary messengers regulate
other glands, such as the thyroid, the adrenals, and the gonads. The entire system maintains homeostasis, i.e., a
regulated state of optimal physiologic functioning or regular cycling throughout the body.

Metabolism
The CNS has a very high rate of metabolism. In an average adult human, the weight of the brain (about 3
lb) is approximately 2% of total body weight. But its energy demands are so great that it receives about 14% of
the heart's output and consumes about 18% of the oxygen absorbed by the lungs (Milnor, 1968). Unlike other
organs, the brain depends almost entirely on glucose as a source of energy and as a raw material for the synthesis
of other molecules (Damstra and Bondy, 1980). The CNS depends critically on an uninterrupted supply of
oxygen and on the proper functioning of enzymes that metabolize glucose. Other tissues (muscle, for example)
have alternative sources of energy and can survive for a relatively long time when the circulation is
compromised, but nerve cells usually begin to die within minutes.

Blood-Brain Barrier
With few exceptions (e.g., proteins), substances that are carried in the bloodstream can pass from capillaries
to surrounding extracellular fluid (in nearly all regions of the body). That is not true in the central nervous
system (Betz et al., 1989). The cells that form the walls of capillaries in the CNS abut one another with unusually
tight junctions. Those tight junctions, and perhaps other cells that line the outside of the capillaries, form a
barrier that prevents the free passage of most bloodborne substances and thereby helps to create fine control of
the extracellular environment of nerve cells. (Peripheral nerves and the retina have a similar barrier.) Without
this blood-brain barrier, the brain would be even more vulnerable to chemical damage.
Chemical access to cells varies in different regions of the nervous system and at different stages of
development (Klaassen, 1986). The area postrema and circumventricular areas have no blood-brain barrier. In
developing animals, monosodium glutamate selectively kills cells in the arcuate nucleus and the retina, where the
barrier is insufficient to exclude it. Other areas have incomplete barriers; e.g., the dorsal root ganglion is
vulnerable to penetration and resulting cell-killing by chemicals (such as doxorubicin) that are excluded from the
nearby spinal cord by a more complete barrier (Cho et al., 1980), and the olfactory nerve directly connects the
outside environment via the nose to the frontal cortex (Broadwell, 1989).
Most nuclei in the nervous system, particularly in the CNS, have relatively intact barriers and thus are
protected from some classes of chemicals. Although those barriers exclude many chemicals from the brain, some
neurotoxic chemicals can gain access to the CNS via normal uptake mechanisms, if they are structurally similar
to endogenous substances normally taken into the brain.

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Such variable protection places some anatomic regions at particular risk and gives rise to different
expressions of neurotoxicity in response to various agents.
As will be discussed in more detail in Chapter 3, the selectivity of the blood-brain barrier results in fairly
specific biologic markers that correspond to particular substances. Because the CNS is enclosed in a fairly
isolated compartment defined by the blood-brain barrier, monitoring of more readily sampled tissues, such as
blood, is not usually sufficient for detecting biologic markers of exposure or effect in the CNS itself; for that
purpose, it might be necessary to sample spinal fluid.

Development
The organizational complexity of the adult human nervous system is staggering. Indeed, the complexity is
so great that we have only a rough idea of the number of neurons in the brain. The most widely cited estimate is
30 billion. It is reasonable to assume that an average neuron receives input from about 1,000 other neurons and,
in turn, sends its messages to another 1,000 neurons.
Details of the development of the human nervous system are poorly understood, but the broad outline is
known. It begins early in embryonic development and is not complete until about the time of puberty. It proceeds
as a carefully timed multistage process guided by hormones and other chemical messengers. Some of the initial
organization acts as scaffolding, i.e, a temporary framework for later development. The temporal and spatial
organization of the developmental process is precise and complex, and the deleterious effects of a relatively
minor disturbance—one that perturbs developmental interactions among just a few cells and for only a brief time—
might be amplified enormously through the cascade of developmental steps. Developmental stage is important in
determining the limits of plasticity, i.e., the relocation of cellular resources to overcome the effects of injury; this
ability to adapt to damage is thought to be greatest early in development. For example, a lesion that might be
compensated for in a developing nervous system might produce irreversible deficits in an adult. In contrast,
interference with a particular cell-cell interaction can have qualitatively different effects in developing and
mature nervous systems. For example, dopamine receptors respond to a decrease in dopamine input during
development with a permanent decrease in their number, but in adulthood with an increase in their number
(Rosengarten and Friedhoff, 1979; Miller and Friedhoff, 1986). Thus, the nervous system's developmental period
is a time of both vulnerability and ability to compensate.
Disuse of a particular synaptic connection can result in its inactivation and eventual dissolution. When more
than a critical number of synaptic connections on a particular neuron are inactivated, the neuron itself might
become superfluous and be deleted. That phenomenon occurs extensively during development: excess neurons
and synaptic connections are established, many of which apparently disappear during the modeling of the mature
nervous system. The converse also happens: synaptic connections used repeatedly become fixed, and
transmission at these locations is facilitated through long-lasting changes in how ion channels or receptors are
linked to second messenger systems. It appears that the shape of a given synapse is plastic and that the
arrangement of structural proteins within a synapse can be modified to alter the efficiency of information
transmission.
Neuron production does not cease at birth in the human (or in any common laboratory species), but
continues through most of the first year of postnatal life. However, neurogenesis for most regions is completed
during embryonic stages or fetal stages. Thereafter,

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neurons that die are not replaced, although supporting cells continue to divide. Later development, maturation, or
repair of the nervous system involves extension of the population of neuronal processes (but not production of
new neurons) and modification of interconnections. That is in marked contrast with most other tissues, in which
cell replacement is continuous. The absence of neurogenesis in the adult places severe constraints on the nervous
system's ability to recover from damage.

Plasticity
Although the general pattern of interconnections in the nervous system is fixed according to the genetic
program that guides development, the fine structure of the pattern of connections in at least some regions
probably responds to exogenous factors and retains a considerable degree of adaptability throughout life. This
characteristic of plasticity involves rearrangement of the details of synaptic connections. Plasticity confers on the
CNS the capacity to respond to environmental signals by learning and memory formation. Damage to the
nervous system can also evoke rearrangement of connections among neurons that survive the insult;
rearrangement permits a limited, but important, functional adjustment to compensate for neuronal damage.
The clearest example of neuronal plasticity is seen after damage to axons in peripheral nerves. Neurons
supplying peripheral nerves usually survive when their axons have been damaged. Such a neuron is able to
regenerate its axon, which then locates its original target. That is the basis, for example, of the eventual partial
return of sensation and muscle control in a surgically reattached limb. Neurons in the CNS are also able to
regenerate damaged axons, but it is far more difficult for them to reacquire their targets, partly because of the
barrier formed by proliferating glia, partly because of the inherently more complex interconnections of the CNS,
and partly because the CNS lacks a pathway for regenerating axons to follow, such as is formed in the PNS by
basal laminae and collagen and reticular fibers of the endoneurium (the tissue that separates the fibers in a
peripheral nerve).

Trophic Interactions
The functioning of individual neurons is determined partly by a continuous supply of trophic factors,
chemicals that originate in the cell's targets. For example, if the axons of motor neurons are severed, both the
motor neuron and the muscle cells atrophy because of the lack of the chemicals that they normally exchange. A
target provides trophic feedback to the neuron that innervates it, so compromise of neuronal function can be
secondary to injury to nonneuronal cells. Such interdependence is also seen among neurons. Some neurons
atrophy when the neurons that normally communicate with them die (Kelly, 1985). Thus, it is possible that
damage can spread from a small number of inactivated neurons.

VULNERABILITY OF THE NERVOUS SYSTEM TO CHEMICAL TOXICANTS

In general terms, neurotoxic injury involves adverse functional or structural change. But the different
vulnerabilities of various cells to injury by a given toxicant and the normal roles of susceptible cells determine
the biologic markers of effect or clinical manifestations of exposure to a particular neurotoxic chemical. For
example, myelin degeneration after exposure to triethyltin or hexachlorophene is manifested as spasticity; effects
on neurons in the hippocampus due to exposure to trimethyltin, triethyllead, or methylmercury are associated
with learning and memory deficits; degeneration of dopamine

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neurons in the substantia nigra due to MPTP exposure leads to the tremor and rigidity of parkinsonism; and
axonal degeneration in the PNS due to γ-diketones or acrylamide is reflected in muscular weakness and sensory
deficits. Such effects can often be detected in either exposed humans or corresponding experimental animal
models with clinical examination or other diagnostic means, such as electrophysiology, computed axial
tomography and positron-emission tomography (CAT and PET scanning), and magnetic resonance imaging. In
contrast, some of the acute effects of toxic chemicals are manifested as dysfunction of the nervous system
without any observable structural changes; examples are intoxication by insecticide pyrethroids, DDT, and
malathion. Several factors render the nervous system especially vulnerable to chemical assault; they are
described briefly in the next few pages.

Complexity of Structural and Functional Integration

The proper functioning of the nervous system depends critically on complex interactions among different
cell types in many anatomic locations that communicate via a variety of electric and chemical signals. The sheer
complexity of the system makes it vulnerable, just as the functioning of a computer is more vulnerable to
damage than is the functioning of an abacus. Other organs also are complex and have many cells, but most have
a high degree of functional redundancy. For example, the loss of a few liver cells creates little disturbance in the
organ's function. In contrast, although the nervous system is able to compensate extensively after injury, its
functional redundancy is nonetheless circumscribed. A small lesion might initially result in a functional deficit
(for example, a small stroke might impair speech or motor coordination), but eventually be compensated for as
new synaptic pathways are established. If a lesion is widespread, the corresponding functional capacity might be
lost; for example, a relatively small loss of neurons that use acetylcholine as their neurotransmitter might
produce a disturbance of memory, whereas a relatively minor insult concentrated in a subsystem that relies on
dopamine as its neurotransmitter might have no effect, and a larger insult might impair motor coordination.

Limitations on Repair
To understand neurotoxicity, one must remember that adults have no neurogenesis. Nerve cells that are
destroyed by chemical insult are not replaced; the lesion is permanent. However, the nervous system has
remarkable compensatory mechanisms. Such compensation is evident, for example, in the development of
tolerance to morphine and alcohol, the long-term potentiation phenomenon documented in experimental animals,
functional recovery after stroke or brain surgery, and neuronal sprouting after axonotomy. However, the
scantiness of functional redundancy and the lack of neurogenesis limit (although they do not preclude)
compensatory ability, particularly after extensive neuronal death. And the limits are exacerbated by normal aging
processes: Neurons die throughout adult life, and additional cell loss due to exposure to toxicants may cause a
further deficit in the functional reserve that is ordinarily drawn on in aging.

Accessibility to Lipid-Soluble Toxicants

All cell membranes are composed chiefly of lipid molecules. However, the lipid content of the nervous
system is especially high—the myelin in axonal sheaths, for example, constitutes approximately 25% of the
brain's dry weight. Many classes of toxicants, such as chlorinated hydrocarbon

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pesticides and industrial solvents, dissolve readily in lipids. Molecules dissolved in membrane lipids are
relatively protected from enzymatic degradation and sheltered from the general circulation. Thus, lipids act as
depots where various toxicants can accumulate and reach high local concentrations. Toxicants can gradually
leach out of lipid depots, such as liver and adipose tissue, to such an extent that the exposure of the nervous
system itself to a toxic chemical lasts much longer than the external exposure of the organism (Boylan et al.,
1978). Toxicant concentrations in adipose tissue can reflect exposure of other lipid-rich tissue, such as is found
in the nervous system, but determination of the temporal course of exposure or of tissue doses in the CNS is
more problematic.

Dependence on Glucose
The nervous system depends almost exclusively on glucose for energy and as a starting material for the
synthesis of other molecules (Sokoloff, 1989). Its energy requirements, e.g., for maintaining electric potentials
for meeting the bioenergetic demands of neurotransmitter reuptake, and for performing axonal transport are high.
The system is therefore particularly vulnerable to chemicals that inhibit the enzymes that metabolize glucose.

Axonal Transport
All types of cells must transport proteins and other molecular components from their site of production near
the nucleus to other sites in the cell. Intracellular transport is relatively simple in most cells, because the greatest
distance to be traversed is only a small fraction of a millimeter. Neurons are unique among cells, in that the cell
body not only must maintain the functions normally associated with its own metabolic support, but also must
provide support to dendrites and axons, often over relatively vast distances. The axon and synaptic boutons, have
little ability to synthesize the materials needed to sustain their structure and function, so they depend on delivery
of the materials from their parent-cell body (Hammerschlag and Brady, 1989). Delivery of such substances by
intracellular transport down the axon (axonal transport) is highly vulnerable to interruption by toxic chemicals.
In addition, the functional integrity of the neuronal cell body often depends on a reciprocal supply of trophic
factors from the cells that it innervates, and these trophic factors must also be supplied via axonal transport over
long distances with very high energy requirements.
Axonal transport has several forms. Fast anterograde (forward) transport carries macromolecular assemblies
of primarily membrane-associated glycosylated or sulfated proteins along structural tracks of microtubules from
cell body to distal axon at a rate of about 400 mm/day. From the cell body, slow anterograde transport carries
soluble enzymes involved in metabolism and neurotransmission at 1–2 mm/day in conjunction with microtubules
and neurofilaments or at 5–10 mm/day in association with microfilaments. Fast retrograde (backward) transport
carries a variety of materials up the axon to the cell body at about 250 mm/day; its cargo might be endogenous
molecules (largely glycoproteins) destined for recycling, trophic signals, or exogenous materials (e.g., tetanus
toxin, lead, and doxorubucin, which might circumvent the blood-brain barrier by traveling up the axon from the
PNS).
It has been hypothesized that the great demand of axonal transport for energy derived from oxidative
metabolism is a point of neural vulnerability to some toxicants. Toxicants that interfere with that metabolism or
that disrupt the spatial arrangement or manufacture of neurofilaments block axonal transport. Many chemicals
that produce distal axonopathies probably act by such mechanisms. For example, γ-diketones

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BIOLOGIC BASIS OF NEUROTOXICITY 33

(metabolites of n-hexane and methyl n-butyl ketone) appear to exert their toxic effect via the mechanism of
neurofilament cross-linking (St. Clair et al., 1989), whereas several biochemical mechanisms (e.g., reaction of
sulfhydryl moieties and altered glycolysis) might contribute to acrylamide's impairment of axonal transport
(Miller and Spencer, 1985).

Synaptic Transmission
Neurons communicate with one another and with muscle and gland cells via a large number of chemical
messengers that interact in precisely defined ways with specialized receptor molecules. Chemically mediated
communication is vulnerable to disruption by exogenous chemicals.
Disruption can occur in several ways. For example, it is functionally important for synaptically released
neurotransmitters to have brief effects. Some neurotoxicants, such as the organophosphate pesticides, inhibit the
enzyme that terminates the effect of the neurotransmitter acetylcholine on its target. The result is an injurious
overstimulation of the target cell. Other neurotoxicants, particularly toxins and their chemical relatives, mimic
the action of a neurotransmitter by interacting with its receptor molecule. For example, LSD (lysergic acid
diethylamide), a synthetic relative of a natural toxin (ergot) produced by a fungus that infects cereal grain,
interacts with the receptor for the neurotransmitter serotonin (Aghajanian, 1972).
Other neurotoxic chemicals act by deranging other aspects of chemical neurotransmission. Some chemicals
interfere with the synthesis of particular neurotransmitters, and others block a neurotransmitter's access to its
receptor molecule (Marwaha and Anderson, 1984). Neuroactive pharmaceuticals commonly have those types of
mechanisms of action. Some neurotoxicants are metabolized by neuronal enzymes and produce damaging
metabolites. For example, 6-hydroxydopamine is taken up by the reuptake system at synapses that use dopamine
as a neurotransmitter. The metabolism of 6-hydroxydopamine within the synaptic bouton yields hydrogen
peroxide and other toxic species, which kill the cell (Graham et al., 1978).

Ion Channels
A nerve impulse depends on the proper functioning of ion-specific channels in the membrane. During the
rising phase of the action potential, sodium ions enter the neurons through open sodium channels. During the
falling phase, potassium ions leave neurons through open potassium channels. Thus, the internal sodium
concentration increases and the internal potassium concentration decreases during an action potential. Although
the resulting ionic imbalance is small (around 0.1% for an axon 1 µm in diameter), it must be restored. That is
accomplished by the sodium-potassium pump, which extrudes sodium and absorbs potassium. The ion pump is
operated by metabolic energy, so substances that inhibit metabolic enzymes cause sodium to accumulate in the
neurons and potassium to be lost; that leads to membrane depolarization and, eventually, the loss of excitability.
Several kinds of neurotoxic chemicals—including such local anesthetics as lidocaine and such natural toxins as
tetrodotoxin and saxitoxin—block the sodium and potassium channels and thereby halt the electric conduction of
nerve impulses.

EXAMPLES OF NEUROTOXIC MECHANISMS

In some kinds of neurotoxicity, understanding of the pathogenetic progression from molecular lesion to
clinical manifestation is well developed. Pyrethroids cause acute, but reversible, neurotoxic effects. Chronic, but
not acute, exposure to n-hexane results in

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BIOLOGIC BASIS OF NEUROTOXICITY 34

degeneration of motor and sensory axons. MPTP, in sufficiently high doses, causes acute degeneration of
dopamine neurons, which results in a severe parkinsonian syndrome; however, it is suspected that the
progressive reduction in dopamine neurons that occurs with age will result in additional cases of parkinsonism in
people exposed to smaller amounts of MPTP (Calne et al., 1985). The possibility of long latency between
exposure to a toxicant and clinical manifestation of disease has profound implications both for demonstrating a
toxic etiology in degenerative CNS disease and for testing chemicals for neurotoxicity.

Pyrethroids
Pyrethroids are synthetic analogues of pyrethrins, the active substances in the flowers of Chrysanthemum
cinerariaefolium. The pyrethrum insecticides (made from the dried flowers) had been used extensively until the
end of World War II, but a variety of potent synthetic insecticides developed after the war—such as DDT,
lindane, dieldrin, parathion, and malathion—made pyrethrum almost obsolete. The pendulum swung in the
1960s, when the persistence of pesticides became a major issue in their use. Many pyrethrin derivatives were
then synthesized and tested as insecticides, and some of them have proved extremely useful and safe. The
pyrethroid insecticides now in use are characterized generally by potent insecticidal activity, relatively low acute
mammalian toxicity, little known chronic mammalian toxicity (including mutagenicity and carcinogenicity,
although information is incomplete), and—what is very important—environmental biodegradability.
Among insecticides, pyrethroids and organophosphate-carbamate anticholinesterases have been studied
most thoroughly for their mechanisms of action. Pyrethroids exert primarily acute toxicity, so studies have
focused on how the signs of poisoning are produced. The major aim of the studies has been to generate
information for improving pyrethroids as insecticides.
Pyrethroids can be classified into two large groups (Figure 2-3). Type I pyrethroids do not contain a cyano
group in their molecules and include allethrin, tetramethrin, permethrin, and phenothrin. Type II pyrethroids
contain a cyano group at the α-carbon position and include newer compounds, such as deltamethrin,
cyphenothrin, cypermethrin, and fenvalerate. The two types of pyrethroids cause somewhat different symptoms
of mammalian poisoning. Poisoning with type I pyrethroids is characterized by hyperexcitation, ataxia,
convulsions, and eventual paralysis; poisoning with type II pyrethroids, by hypersensitivity, choreoathetosis,
tremors, and paralysis. Despite differences in the symptoms, both types of pyrethroids have the same major
target site: the sodium channel of nerve membrane, i.e., the channel directly responsible for generating action
potentials.
Pyrethroids cause essentially similar signs

Figure 2-3 Structures of type I and type II pyrethroids. Source: Narahashi (1985).

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BIOLOGIC BASIS OF NEUROTOXICITY 35

of poisoning in mammals and invertebrate animals. In vitro studies of their mechanism of action on the nervous
system have used nerve preparations isolated from insects, crayfish, and squid and cultured mouse
neuroblastoma cells (Narahashi, 1971, 1985, 1989; Ruigt, 1984; Wouters and van den Bercken, 1978).
Pyrethroid poisoning is associated with an increase in electric activity of the central and peripheral nervous
systems: a single presynaptic stimulation in the presence of pyrethroids causes repetitive excitation of
presynaptic nerve and nerve terminals, thereby evoking repetitive postsynaptic discharges. This can be
demonstrated by exposing isolated nerve fiber preparations, such as crayfish giant axons and frog myelinated
nerve fibers, to low concentrations of pyrethroids (especially type I pyrethroids). Intracellular potential recording
experiments reveal that the depolarizing after-potential is gradually increased in amplitude and prolonged after
application of pyrethroids, until it finally generates repetitive after-discharges (Lund and Narahashi, 1983).
How the depolarizing after-potential is increased by pyrethroids is best studied by voltage-clamp
techniques, whereby membrane ionic currents can be recorded. The sodium current that generates action
potentials is markedly prolonged after the preparation is exposed to pyrethroids, and that causes a sustained
depolarization after an action potential (Narahashi and Lund, 1980). However, the sodium current thus recorded
represents an algebraic sum of currents passing through a large number of open sodium channels. The activity of
individual ion channels can be studied by patch-clamp techniques, which allow measurements of ionic currents
flowing through individual open channels. Pyrethroids have been found usually long periods (Yamamoto et al.,
1983). A sodium channel normally is kept open during a depolarizing step for only a few milliseconds, whereas a
sodium channel exposed to pyrethroids can remain open much longer—even up to several seconds, depending on
the pyrethroid in question. Type I and type II pyrethroids have similar effects, but the increase in open time is
more pronounced with type II pyrethroids.
Pyrethroids exert another effect on sodium channels. A pyrethroid-exposed sodium channel can be opened
at negative membrane potentials near the resting potential. Prolonged opening of many sodium channels near the
resting membrane potential leads to the nerve-membrane depolarization observed in the presence of pyrethroids.
Membrane depolarization produces several changes in nervous function: depolarization in sensory neurons sends
massive discharges to the central nervous system, causing hypersensitivity to external stimuli and paresthesia or
a tingling sensation in the facial skin; depolarization of presynaptic terminals increases transmitter release,
thereby disturbing synaptic transmission; and depolarization beyond some magnitude blocks nerve conduction
and results in paralysis. In addition to the modified electric behavior of the sodium channels, all the more readily
detected changes are biologic markers of exposure to pyrethroids.
During the last several years, it has been hypothesized that the γ-aminobutyric acid (GABA) receptor-
channel complex, rather than the sodium channels, might be the target site of type II pyrethroids. That was based
on several observations, including pyrethroid inhibition of ligand binding to the GABA receptor channel
(Lawrence and Casida, 1983). However, recent patch-clamp experiments with rat dorsal root ganglion neurons in
culture have unequivocally demonstrated that, whereas sodium channel current undergoes drastic and
characteristic prolongation during exposure to the type II pyrethroid deltamethrin, the GABA-induced chloride
channel current remains unaffected (Ogata et al., 1988). The GABA receptor-channel system thus plays a
negligible role in poisoning with type II pyrethroids.
Pyrethroids thus seem to modify the

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BIOLOGIC BASIS OF NEUROTOXICITY 36

gating kinetics of sodium channels, causing prolonged opening of individual channels, which leads to a wide
range of toxic signs. Only a very small fraction of sodium channels need to be modified by pyrethroids to cause
severe signs of poisoning (Lund and Narahashi, 1982). That is why pyrethroids are such potent insecticides.
Although the mechanism of action on nerve membrane sodium channels is generally the same in both
mammals and insects, pyrethroids are less toxic to mammals than to insects. The selective toxicity to insects
might be due to at least three major factors: pyrethroids are more effective on nerves at low temperature
(Narahashi, 1971), so insects at room temperature would be affected more severely than mammals at 37°C;
pyrethroids are detoxified by enzymes, and enzymatic reactions are slower at low temperature; and, because
insects are smaller than mammals, pyrethroid molecules are likely to reach the target site before being
inactivated metabolically.
In summary, pyrethroids primarily modify gating kinetics of sodium channels, and this action can account
for the various symptoms of poisoning in animals. Future studies should be directed toward the molecular
mechanisms whereby the pyrethroid molecule interacts with sodium channels, including the identification of the
subunit of a channel to which pyrethroids bind.

γ-Diketones
At high concentrations, the simple alkane n-hexane (a glue solvent) can be acutely toxic; e.g., it can cause
dizziness and eye, throat, and skin irritations, all of which are nonspecific biologic markers of exposure and
effect. Those manifestations, however, are reversible. The progressive weakness and sensory loss that arise from
chronic exposure to n-hexane are the major human health hazards that motivated its regulation.
After it was reported that workers exposed to n-hexane at high concentrations developed a motor-sensory
peripheral neuropathy (Yamamura, 1969; Herskowitz et al., 1971), Schaumberg and Spencer (1976) showed that
rats develop an identical neuropathy after inhaling n-hexane. The finding that humans exposed to a solvent
mixture containing methyl butyl butane developed peripheral neuropathy identical with that found after n-hexane
exposure (Billmaier et al., 1974) demonstrated the peripheral neuropathy as a biologic marker characteristic of
the two chemicals.
Biopsies of humans and careful morphologic studies of exposed rats showed that exposure to n-hexane and
exposure to methyl n-butyl ketone each resulted in large neurofilament-filled swellings of axons, with axonal
degeneration distal to the swellings developing over time. Thus, a biologic marker was established that was
useful across species, as well as for the chemical class. Cavanagh and Bennetts (1981) showed that the
neurofilament-filled swellings in the CNS and the PNS uniformly developed along the distal portions of long
axons; however, accompanying axonal degeneration was much less common in the CNS (Griffin and Price,
1980). Spencer and Schaumberg (1977a,b) classified the neuropathy as a central-peripheral distal axonopathy
and observed that myelinated axons were more vulnerable than nonmyelinated axons. Swelling and distal
degeneration were most prominent in myelinated axons of the largest diameter.
The common morphologic findings after n-hexane and methyl n-butyl ketone exposure suggested a
metabolic relationship between the two toxicants, which was elucidated by Krasavage et al. (1980) and Couri
and Nachtman (1979). Studies disclosed a common metabolite, 2,5-hexanedione (HD), a biologic marker of
exposure that itself caused an identical neuropathy. The Krasavage group coined the term ''γ-diketone''

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BIOLOGIC BASIS OF NEUROTOXICITY 37

neuropathy to emphasize the importance of the gamma spacing of the two ketone groups (separated by two
carbons; sec Figure 2-4). γ-Diketones and γ-diketone precursors produce a specific neurotoxic response not
shared by diketones with α, β, or γ spacing (with zero, one, or three intervening carbons, respectively) (Spencer
et al., 1978; Katz et al., 1980). However, 2,4-pentanedione, an α-diketone, produces a different form of
neurotoxicity (Misumi and Nagano, 1984). The seminal observation regarding the significance of gamma
spacing began the process of understanding the neuropathy at the molecular level.
The search for the fundamental mechanism focused on two concepts: inhibition of energy metabolism
(Spencer et al., 1979) and neurofilament cross-linking (Graham, 1980). The key observation, however, was that
lysyl amino groups of proteins are the primary target of γ-diketones and that pyrroles are the product of the
reaction (DeCaprio et al., 1982; Graham et al., 1982).
A number of studies have tested the hypothesis that pyrrole formation is the initial step in the sequence that
leads to neurofilament accumulation. Anthony et al. (1983a,b) showed that 3,4-dimethyl-2,5-hexanedione
(DMHD) was a γ-diketone that

Figure 2-4 Metabolism of hexane. Both n-hexane and 2-hexanone (methyl n-butyl ketone) are neurotoxic, and both
are activated through ω-1 oxidation to the ultimate toxic metabolite, 2,5-hexanedione. The toxicity of g-diketones
derives from the ability of these diketones to react with protein amino groups (RNH2) to form an amine in an initial
reversible step, and then to cyclize irreversibly to form a pyrrole. Source: Anthony and Graham (1991). Reprinted
with permission from Amdur et al. (1991).

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BIOLOGIC BASIS OF NEUROTOXICITY 38

formed pyrroles more rapidly than HD and was correspondingly more neurotoxic. Larger substituents—such as
ethyl, isopropyl, and phenyl groups on the 3 and 4 carbons—impeded pyrrole formation by steric hindrance
(Szakal-Quin et al., 1986) and were not neurotoxic (Genter et al., 1987). Thus, although the gamma spacing is
critical, not all γ-diketones are neurotoxic. Genter et al. (1987) clarified the role of pyrrole formation with the
observation that the dl diastereomer of DMHD forms pyrroles more readily and is more toxic than the meso
diastereomer. Given that dl-[14C]DMHD and meso-[14C]DMHD disperse comparably in the nervous system, as
shown by Rosenberg et al. (1987), and that the tetramethylpyrroles formed are the same, the conclusion that
pyrrole formation is the first step in the pathogenetic sequence was virtually ensured. The conclusion was
strongly supported by the finding that 3,3-dimethyl-2,5-hexanedione, which cannot form a pyrrole, is not
neurotoxic (Sayre et al., 1986) and by the reduced rate of pyrrole formation and neurotoxicity of deuterium-
substituted HD (DeCaprio et al., 1988).
The significance of pyrrole formation for neurofilament function, however, has been a point of controversy.
DeCaprio (1985) and Sayre et al. (1985) have proposed that conversion of the hydrophilic lysyl amino groups to
hydrophobic pyrrole derivatives alters neurofilament transport through impairment of interactions between
neurofilaments and other cytoplasmic components in the axon. The alternative hypothesis (Graham et al., 1982)
has been that pyrrole autoxidation leads to covalent cross-linking of neurofilaments. Progressive cross-linking of
neurofilaments during chronic intoxication would lead eventually to the formation of masses of neurofilaments
too large to pass through the constrictions in axonal diameter that occur at every node of Ranvier. Indeed, the
greater vulnerability of large-diameter myelinated axons is apparently explained by the greater proportional
reduction in diameter at their nodes. Thus, continued transport up to the point of occlusion could account for the
large neurofilament-filled swellings of the axon that most often occur proximal to nodes of Ranvier (Spencer et
al., 1977a; Jones and Cavanaugh, 1983).
To test whether pyrrole derivatization itself is sufficient to account for the neurofilament-filled swelling or
whether neurofilament cross-linking is necessary, St. Clair et al. (1988) used another γ-diketone, 3-acetyl-2,5-
hexanedione (AcHD). The acetyl group of AcHD is small enough not to hinder pyrrole formation; in fact, the
rate is nearly as great as that for dl-DMHD and greater than that for meso-DMHD. However, whereas the
electron-donating methyl groups of the tetramethylpyrrole derived from DMHD result in a reduced oxidation
potential (362 mV), compared with that of the dimethylpyrrole derived from HD (620 mV), the oxidation
potential of the pyrrole formed by AcHD is much greater (975 mV), because of the electron-withdrawing
properties of the acetyl group. Thus, the pyrrole formed from AcHD is relatively resistant to oxidation and so
provides a probe for discriminating between the competing hypotheses. If pyrrole derivatization itself is
sufficient, AcHD should be highly neurotoxic. Conversely, if pyrrole autoxidation and neurofilament cross-
linking are required, AcHD would not result in neurotoxicity. When rats were given AcHD, massive pyrrole
formation occurred, but neither clinical neurotoxicity nor neurofilament accumulation was observed; the results
point strongly to the role of neurofilament cross-linking in the pathogenesis of n-hexane neurotoxicity.
Thus, in n-hexane neuropathy, it appears established that the γ-diketone HD is the toxic metabolite.
Reaction with amino groups yields pyrrole adducts, which undergo autoxidation that results in covalent cross-
linking of proteins. It is the extreme stability of the neurofilaments that results in the localization of toxic injury
in the nervous

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BIOLOGIC BASIS OF NEUROTOXICITY 39

system, and it is the particular anatomy of the myelinated axon at nodes of Ranvier that makes these axons most
vulnerable.

MPTP
A particularly unfortunate byproduct of the so-called designer drugs produced in the 1980s was 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Figure 2-5). Instead of the desired opiate-like characteristics of the
intended meperidine derivative (which was to be sold illicitly as "synthetic heroin"), a synthetic drug that
contained MPTP caused a profound, acute-onset neurologic disorder with extreme muscular rigidity and tremor
that was indistinguishable from idiopathic Parkinson's disease (Langston et al., 1983). Animal studies showed
that MPTP itself is not neurotoxic, but requires metabolism by monoamine oxidase B, a process that appears to
take place in astrocytes, a type of glial cell (Jenner, 1989). The product of MPTP oxidation is the pyridinium ion
MPP+ (Figure 2-5); this putative toxic metabolite of MPTP leaves the astrocytes and is preferentially
concentrated by catecholaminergic neurons via their own catecholamine uptake system. Whether the mechanism
of MPP+ cytotoxicity is related to oxidation-reduction cycling, interference with mitochondrial energy
production, or some other process, the end result is cell death, particularly death of the dopaminergic neurons of
the substantia nigra (Langston and Irwin, 1986). The marked depletion of dopamine seen in the brain of MPTP-
treated

Figure 2-5 Diagram of MPTP toxicity. MPP+, either formed elsewhere in the body following exposure to MPTP or
injected directly, is unable to cross the blood-brain barrier. In contrast, MPTP gains access and is oxidized in situ to
MPDP+ and MPP+. The same transport system that carries dopamine into the dopaminergic neurons also transports
the cytotoxic MPP+. Source: Anthony and Graham (1991). Reprinted with permission from Amdur et al. (1991).

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BIOLOGIC BASIS OF NEUROTOXICITY 40

animals is also the central neuropathologic feature of Parkinson's disease in humans.

Not all people exposed to MPTP have developed parkinsonism, ostensibly because the dose administered
was sometimes insufficient to cause death of a large enough proportion of dopaminergic neurons to produce the
80% or greater depletion of dopamine in the brain that is required to produce a clinical syndrome (Hornykiewicz,
1986). Recently, a number of exposed people have been studied with positron-emission tomographic (PET)
scanning (Calne et al., 1985). Through the use of positron-emitting fluorodopa, which is sequestered in the
striatum (where the axonal projections of the substantia nigra form synapses), a deficiency of striatal dopamine
was demonstrated. Although the exposed people were asymptomatic, the loss of dopaminergic neurons with
normal aging (McGeer et al., 1977) is likely to reduce the population of these cells, and thus brain dopamine
concentrations, below the 80% required for normal function; at that time, the signs and symptoms of Parkinson's
disease would be manifested (Calne and Langston, 1983).
This is a critically important concept: An acute toxic injury can be separated from its clinical manifestations
by years. If MPTP-exposed people who are now asymptomatic (and there might be over 500 such cases)
eventually develop parkinsonism, it will open up the possibility that some patients who develop progressive
degenerative CNS disease later in life (such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral
sclerosis) might have suffered neuronal injury from a toxicant years earlier. If that turns out to have been the
case, tests for neurotoxicity will have to take into account the possibility that effects develop after a long latency.

SUMMARY
The nervous system has several unique aspects, e.g., poor regenerative capacity, unusual anatomy,
specialized metabolic requirements and subcellular systems, and a wide variety of neurotransmitter and
neuromodulator systems. As the discussions of pyrethroids, γ-diketones, and MPTP illustrate, the complexity of
the nervous system and its tremendous structural and functional heterogeneity might make it particularly
susceptible to disruption by toxic agents, and neuronal dysfunction can have impacts on a number of organ
systems. Even minor changes in the structure and function of the nervous system can have profound effects on
neurologic, behavioral, and other body functions.
Many foreign substances can alter the normal activity of the nervous system via a variety of mechanisms.
Some produce immediate short-term effects; others cause neurotoxic effects only after long-term, repeated
exposures. Some substances can cause permanent damage after a single exposure; others can cause subtle and
reversible damage. This diversity in nervous system response increases the challenge to toxicologists to develop
biologically appropriate and empirically reliable test systems.
Considerable progress has been made in recent decades in understanding the nervous system on an
integrated and comprehensive basis. Some mechanisms underlying behavior, such as sensorimotor function and
information-processing, are being elucidated from the translation of molecular and cellular activities into organ-
level events and from the translation of organ-level events into whole-organism behavior. Only by understanding
the basic mechanisms involved in neurobiologic processes can we develop adequate neurotoxicity test methods.
Our present understanding of some expressions of nervous system function permits such an integrated approach—
e.g., much is known about the roles of molecules, cells, organs, and the whole organism in visual processing and
proprioceptive control of movement. For most other neurologic functions, although

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BIOLOGIC BASIS OF NEUROTOXICITY 41

gaps in our knowledge still restrict integrative analyses, we can examine their components at specific levels of
integration-with in vivo and in vitro approaches. An understanding of the nervous system guides the search for
useful biologic markers, as is discussed in Chapter 3. This information is developed further in Chapter 4 to
assess available in vivo and in vitro testing methods and address the need for efficient means for identifying
neurotoxic substances.

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 43

3
Biologic Markers in Neurotoxicology

The purpose of this chapter is to form a bridge between the issues of nervous system structure and function
that are the principal subjects of contemporary neuroscience (Chapter 2) and the goals and emerging methods of
quantitative risk assessment (Chapter 6). Identification of biologic markers and understanding of their role in cell
response and damage can provide information that will be essential for predicting, and therefore preventing,
diseases and disability. This chapter applies to the nervous system the important conceptual framework
developed recently by the National Research Council's Committee on Biologic Markers and applied in
pulmonary toxicology (NRC, 1989a), reproductive and neurodevelopmental toxicology (NRC, 1989b), and
immunotoxicology (NRC, 1991a). We believe that the concepts and definitions of the Committee on Biologic
Markers constitute a valuable tool that can be applied to neurotoxicology, as described in the following sections.

CONCEPTS AND DEFINITIONS

Biologic markers, broadly defined, are indicators of change or variation in cellular or biochemical
components or processes, structures, or functions that are measurable in organisms or samples from those
organisms. Understanding the processes, structures, and functions of the nervous system is the starting point for
identifying biologic markers. Current interest in the biologic markers of the nervous system stems from a desire
to identify the early stages of system impairment and damage and to understand the dynamics of exposure and
the basic mechanisms of response to toxic exposure.
There is growing interest in the use of biologic markers in clinical medicine, epidemiology, toxicology, and
related biomedical fields to study the human neurologic effects of exposures to environmental toxicants (NRC,
1989a,b). Clinical medicine uses markers to allow earlier detection and treatment of disease; epidemiology uses
them as indicators of internal dose or of health effects and for surveillance; and toxicology uses them to help to
determine underlying mechanisms of diseases, develop better estimates of dose-response relationships, and
improve the technical bases for assessing risks associated with low levels of exposure (see Chapter 6).
The rapid growth of new molecular and biochemical tools in the practice of medicine has resulted in the
development of markers

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for understanding disease, predicting outcome, and directing treatment. Over the next few years, many of the
tools will be used to estimate chemical exposure. Even now, many diseases are defined, not by overt signs and
symptoms, but by the detection of biologic markers at the subcellular, molecular, or functional level after
exposure to a toxic agent. The identification, validation, and use of markers in neurobiology and medicine
depend fundamentally on increased understanding of mechanisms of action and of molecular and biochemical
processes. Biologic markers are allowing investigators to gain a better understanding of chemical-receptor
interactions. As noted in Chapter 2, for example, some toxicants interfere with ion channels and thus enhance or
suppress impulse conduction along nerves. Tetrodotoxin, a puffer fish poison, blocks the sodium ion channel,
thereby impairing impulse conduction. However, the insecticidal pyrethroids keep the sodium ion channels open
too long, thereby making the nerve hyperactive.
As we use the term here, markers can be signals or indicators of normal physiology, forerunners of nervous
system damage, or clinical signs and symptoms of neurologic disease. A specific biologic marker can serve
several purposes and is best characterized by the use to which it is put in a particular context. Markers can
indicate susceptibility, exposure to an exogenous agent, internal dose, biologically effective dose (dose at a
receptor site), early biologic effect, structural or functional alteration, physiologic status, system damage, or
disease. Thus, the choice of a marker and its interpretation depend on the purpose of its use, and its intended use
depends on characteristics peculiar to a particular exogenous agent, to an individual organism, or to a particular
target organ or tissue (see Table 3-1). If the goal is prevention, the major emphasis will be on monitoring
markers that
TABLE 3-1 Examples of Characteristics of Exogenous Agents, Organisms, or Targets That Influence Choice of Biologic
Marker
Agent-specific characteristics Organ- or tissue-specific characteristics
Physiochemical properties Blood flow
Interactions Membrane permeability
Routes of exposure Transport
Duration of exposure Receptors
Exposure concentration Function
Pattern of exposure Homeostasis
Metabolism Structure
Activation Physiologic state
Detoxification
Organism-specific characteristics
Species
Age
Sex
Physiologic state
Pharmacokinetic characteristics
Genetic factors
Lifestyle factors

Source: NRC (1989b).

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identify biologic changes in the nervous system that are predictive of health impairment or overt disease, such as
the various biochemicals, metabolites, responses, and characteristics listed in Table 3-2.
Markers represent points in a continuum between health and disease. How they are applied to the
neurologic sciences might change as our knowledge of the fundamental processes of disease of the nervous
system increases. What are perceived at first to be early signals of exposure and of a system's response to a
toxicant could come to be considered health impairments themselves, because of their strong predictive
relationship to disease. By the time early changes (markers) are detected, it might be difficult to prevent
neuropathy from occurring in response to exposure to a toxicant whose effects are progressive and irreversible.
But many neurotoxicants produce damage that is reversible if exposure is stopped (e.g., agents that cause CNS
depression). Thus, biologic markers related to the nervous system can be valuable in prevention, in estimation of
exposure, in early detection of damage, and in early treatment of disease.
The Committee on Biologic Markers found it useful to define three general categories of biologic markers:
those of exposure to chemical or physical agents, those of effects of exposure, and those of susceptibility to the
effects of exposure (NRC, 1989a,b). In the context of the nervous system, a biologic marker of exposure is an
exogenous substance or its metabolite or the product of an interaction between a substance and some target
molecule or other nervous system receptor. A biologic marker of effect is a detectable alteration of an
endogenous component within the nervous system that, depending on magnitude, can be recognized as a
potential or established health impairment or disease. A biologic marker of susceptibility is an indicator of an
inherent or acquired limitation of an organism's nervous system's ability to respond to the challenge of exposure
to a specific toxic substance. Organisms might be particularly susceptible to a toxic substance for several
reasons, including a genetic predisposition, existing disease, unique metabolic characteristics, or even the
consequences of increased stress (see discussion on markers of susceptibility).
TABLE 3-2 Selected Markers of Neurotoxicity in Nervous System
General neuronal measures
Cell number
Tetanus-toxin binding
Neurofilament protein
Neuronal structure
General glial measures
Glial fibrillary acidic protein
Oligodendrocyte probe
Transmitter systems
Amino Acid
Excitatory
Inhibitory
Cholinergic
Choline acetyltransferase
Muscarinic and nicotinic receptors
Aminergic
Norepinephrine
Serotonin
Dopamine
Peptidergic
Vasoactive intestinal peptide
Substance P
Enkephalin
Cell biologic responses
Second messengers
Cyclic nucleotide
Phosphorylation
Calcium-dependent transmitter release
Voltage-dependent NA+ or Ca2+ uptake

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 46

Biologic Markers of Exposure

External exposure is the amount or concentration of a substance in the environment of an organism; internal
dose is the amount that is transferred or absorbed into the organism (see Figure 3-1). Biologically effective dose,
in general terms, is the internal dose that is quantitatively correlated with an identifiable biologic effect;
however, it is more precisely considered to be the amount of a substance that has interacted with a critical
cellular or tissue receptor or target where the biologic effect is initiated. Because many such receptor sites in the
nervous system are not known or not accessible, it is often necessary to use a surrogate site. For example, the
neurofilament cross-linking that follows exposure to γ-diketones is accompanied by dimerization of erythrocyte
spectrin; blood samples can reveal that a biologically effective dose has been received by the organism (St. Clair
et al., 1988) in that these compounds penetrate to the nervous system sites in proportion to concentrations in
blood samples.
Markers of exposure can be based on steady-state or pharmacokinetic measures, such as peak circulating
concentration, cumulative dose, or plasma half-life. Individual variations in physiologic characteristics—such as
sex, age, blood flow, membrane permeability, and respiratory rate—can affect the absorption and distribution of
a chemical and its metabolites (Doull, 1980; NRC, 1986). Age and health status, such as disease, can alter the
respiratory rate and thus the pulmonary dose of a toxicant (NRC, 1989a). Exposure concentration, size of
delivered dose, and dose rate also affect internal dose. When absorption capacities are exceeded, alternate
pathways of clearance come into play. High vapor concentrations (e.g., high concentrations of organic solvents)
might be exhaled, rather than absorbed. Species and individual differences in metabolism can drastically alter
internal doses of reactive metabolites.
The internal dose of a substance can vary with route of exposure, chemical species, and physical form. To
make qualitative or quantitative estimates of exposure with biologic markers, the concentration, duration and
pattern of exposure, and physicochemical nature of a toxicant must be considered in the selection of an
appropriate marker of exposure (Gibaldi and Perrier, 1982). Other environmental factors, such as temperature,
can affect exposure by changing amounts of water consumption and thus of waterborne pollutants ingested. Diet
alters intestinal motility and gastric emptying time, as well as the transport of specific substances across barriers
in the gastrointestinal system. The

Figure 3-1. Simplified classification of biologic markers (indicated by boxes). Solid lines indicate progression, if it
occurs, to the next class of marker. Dashed lines indicate that individual susceptibility influences the rates of
progression, as do other variables described in the text. Biologic markers represent a continuum of changes, and the
classification of change might not always be distinct. Source: NRC Committee on Biologic Markers, 1987.

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 47

formulation or type of mixture that contains a toxicant can affect its transport across protective barriers and
membranes in the nervous system.
The presence of active mechanisms of transport into an organ or tissue and the density of nervous system
receptor sites can all influence internal dose and biologically effective dose. Metabolism critically affects
biologically effective doses of many compounds; the tissue distribution of metabolizing enzymes is an important
determinant of effective dose. Many nervous system toxicants act by interfering with neurotransmitter-receptor
interactions. Thus, the interpretation of dosimetric data involves an understanding of the role of the receptor in
overall cell, organ, or organism function, of coexisting or pre-existing stresses on the organism, and of the
existence and availability of repair or compensation during and after exposure (Doull, 1980). These interactive
aspects of exposure and response are especially important for the nervous system, which participates in the
physiologic regulation of other body systems.
An example of a marker of exposure is the inhibition of blood acetylcholinesterase by organophosphorus
pesticides. However, although moderate levels of inhibition—i.e., up to 30%—are not associated with
physiologic effects (NRC, 1986), inhibition of blood acetylcholinesterase greater than 30% can be used as a
marker of both exposure and effect. As another example, urinary concentrations of 2,5-hexanedione have been
suggested as markers of exposure to n-hexane.

Biologic Markers of Effect

For purposes of environmental health research, biologic markers of effects in the nervous system of an
organism after exposure to a toxicant are considered in the context of their relationship to health status—normal
health, early health impairment and damage, or overt disease (see Figure 3-1). In that context, an effect is defined
as any of the following:
• An alteration in a tissue or organ.
• An early event in a biologic process that is predictive of the development of a health impairment.
• A change consistent with early damage to an organ or tissue.
• A health impairment or clinically recognized disease.
• A response peripheral or parallel to a disease process, but correlated with it and thus usable in predicting
development of a health impairment.
Thus, a biologic marker of a nervous system effect can be any qualitative or quantitative change that is
predictive of system impairment or damage resulting from exposure to a substance, e.g., in brainwave
recordings, in release of a neurotransmitter (such as dopamine), or in the presence of amyloid in Alzheimer's
disease or of neuron-specific enolase in some CNS neoplasms. Markers of early biologic effects include
alterations in the functions of target tissues of the nervous system after exposure. As early-warning signals, such
markers can be useful dosimeters to guide intervention aimed at reducing or preventing further exposure. Such
early-warning signals might also be observed in organs or tissues other than the sites that are critical for toxic
action, in that effects on the nervous system can themselves affect the functioning of other organ systems—e.g.,
caffeine can stimulate the CNS, which can then affect the heart and kidneys.
A tissue affected by a toxicant might exhibit altered function even if the exposed organism has no overt
manifestations. Such alteration can in some cases be determined by testing, particularly with the new
biochemical probes (e.g., molecular adducts) or

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 48

imaging techniques (e.g., magnetic resonance imaging). Biologic markers of functional alterations are most
useful if related to specific functions—e.g., acetylcholinesterase for a cholinergic response and epinephrine for a
catecholaminergic response.
If the internal dose of a toxicant is great enough, disease will develop, because the biologically effective
dose will be sufficient to affect some function irreversibly or produce an effect for a substantial period, as in the
case of ethanol. Disease that occurs soon after exposure might be easy to link directly to the toxicant. Disease
that occurs long after exposure might be difficult to relate to a specific toxicant, as is postulated for some cases
of Alzheimer's disease and other neurodegenerative disorders (Calne et al., 1986), unless the findings are
pathognomonic, i.e., are relatively specific to a particular type of exposure (such as γ-diketones and methyl n-
butylketone) or are rare in unexposed persons (such as distal axonopathy).
The transition to overt disease can depend on properties of the toxicant, the nature of exposure, the disease
process itself, or individual susceptibilities. Because people respond differently to toxicants, it is not surprising
that only some members of a population similarly exposed to a given environmental agent will develop a given
disease. That is especially true of the nervous system, in which large observed variations in population response
to chemical toxicants have been attributed to differences in metabolism, pharmacokinetics, protective
mechanisms and barriers, and system complexity.
Although scientists tend to divide biologic markers into groups, there is a continuum between health and
disease, and advances in toxicology have demonstrated a continuum between dose and response. Biologic
markers are best divided operationally, depending on how they are assessed and how they can be used, but the
divisions should not be interpreted to imply mechanistic distinctions (Figure 3-1).

Biologic Markers of Susceptibility

Some biologic markers indicate individual or population factors that can affect response to environmental
agents. Those factors are independent of whether exposure has occurred, although exposure sometimes increases
susceptibility to the effects of later exposures (e.g., in sensitization to formaldehyde). An intrinsic characteristic
or preexisting disease state that increases the internal dose or the biologically effective dose or that amplifies the
effect at the target tissue can be a biologic marker of increased susceptibility (NIEHS, 1985; Omenn, 1986).
Such markers can include inborn differences in metabolism, variations in immunoglobulin concentrations, low
organ reserve capacity, or other identifiable genetically or environmentally induced factors that influence
absorption, metabolism, detoxification, and effect of environmental agents. These types of biologic markers are
not extensively considered in this report.

VALIDATION OF BIOLOGIC MARKERS

To validate the use of a biologic measurement as a marker, it is necessary to understand the relationship
between the marker and the event or condition of interest, e.g., between muscular weakness and exposure to
acrylamide. Sensitivity and specificity are critical components of validity (MacMahon and Pugh, 1970).
Sensitivity is the ability of a marker to identify correctly those who have been exposed or have a disease or
condition of interest; specificity is the ability to identify correctly those who have not been exposed or do not
have the disease or condition.

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 49

Animal models are useful for understanding the basic mechanistic processes of the expression of markers
and the relationships among exposure, early effects, and disease and thus are useful in validating biologic
markers (see Chapter 4). If a disease can be satisfactorily induced in experimental animals (e.g., axonopathy in
rats exposed to n-hexane), then potential biologic markers for predicting eventual disease can be explored, and
early indicators of the disease might be identified for use in human studies (e.g., peripheral neuropathy). A major
goal of research with biologic markers is to develop markers that reliably indicate an early stage in the
development of a disease process in humans when effective intervention is still possible. That is especially
important for the nervous system, where repair is limited and early intervention might offer the only chance of
recovery.
One of the most serious challenges for the use of animal models in neurotoxicology is that many
expressions in the human central nervous system are quite different from those which can be assessed directly in
animal models. Moreover, even for events that appear to be similar, humans might differ from specific
experimental animals in their functional reserve capability. Therefore, the degree of impairment required to
cause a particular functional deficit could differ for apparently equivalent levels of damage. Use of appropriate
biologic markers for changes in neurologic functions that are similar between animals and humans could help
determine functionally equivalent amounts of damage. For example, changes in neurotransmitter turnover might
provide a basis for interspecies comparability. This application of markers has been useful in
neuropharmacology and should find increased application in the development of neurotoxicologic markers. Until
recently, such markers were not directly measurable in humans. However, new scanning techniques of positron-
emission tomography and magnetic resonance imaging can provide information in vivo on the neurochemistry of
humans and experimental animals.
The principal purpose of markers in health research is to identify exposed people, so that risk can be
estimated and disease prevented. Particularly critical for marker validation is the strength of biologic plausibility
that supports an association between a change in a specified signal (a marker) and the occurrence of a specific
exposure or of a change in a specific effect.
Validation of a specific marker depends on its expected use in monitored populations. Biologic markers
observed well before the onset of disease might have low predictive value for the disease itself, but function
acceptably as criteria for defining exposed populations and thus be useful for long-term followup. For example,
heroin addiction in California might be a useful clue of potential exposure to the neurotoxicant MPTP for
epidemiologic research, even though its relationship to a parkinsonian syndrome is indirect. Conversely, an
effect marker that is expressed long after exposure could be of relatively little use in exposure assessment, but
very important in predicting progression of disease or calculating risk. For example, neurobehavioral
manifestation of prenatal exposure to methylmercury can persist well into adulthood.
Quality assurance and quality control are fundamental to the objective development and application of
accurate and verifiable biologic markers. The objective of laboratory quality-assurance practices is to ensure that
findings reported by one laboratory are in fact verifiable and within acceptable limits of measurement error, that
they accurately indicate the concentrations or presence of the biologic changes reported to have been found, and
that they are objective and free from sources of bias introduced through the analytic process. General issues of
quality assurance and quality control have been addressed by documents produced by the

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 50

Food and Drug Administration (FDA), the U.S. Environmental Protection Agency, the Organization for
Economic Cooperation and Development, and other regulatory organizations. FDA developed a set of guidelines
known as good laboratory practices, or GLPs (FDA, 1988), which are now incorporated into the standard
procedures of most testing and analytic laboratories for well-established assays. GLPs are intended to reduce the
chance of contamination (particularly important in the measurement of biologic markers of exposure) or of
changes in biologic variables introduced by sample storage, processing, or measurement (Zeisler et al., 1983).
The application of GLPs to analysis of biologic samples, especially human tissue, has been reviewed by
operational units of the Centers for Disease Control, the National Institute of Standards and Technology
(formerly named the National Bureau of Standards), and various clinical laboratories (ACS, 1980; NCCLS,
1981, 1985). Because most of the biologic markers discussed in this report are still at a research stage,
standardized GLP practices regarding them remain to be established for laboratories.

USE OF BIOLOGIC MARKERS IN RISK ASSESSMENT

Cellular and molecular markers can serve as powerful new tools for the assessment of risks associated with
exposure to environmental toxicants. Markers that indicate the delivery of an internal dose or a biologically
effective dose or the induction of a disease process can be useful in hazard identification—i.e., in the qualitative
step by which an environmental agent is causally associated with an adverse effect. Biologic markers can also be
used to determine dose-response relationships, especially at the low doses relevant to exposure to many
environmental chemicals. In addition, they can be used to quantify actual exposures.
Biologic markers in environmental-health studies offer an opportunity to determine the shape of the lower
end of the dose-response curve in humans—an opportunity that has not yet been used extensively in standard
human epidemiologic or laboratory animal studies. Nevertheless, it is likely to remain uncommon to have
information on concentrations of a toxicant at the site of action within the nervous system.
Many neurotoxic effects can be studied in experiments conducted with doses at which enzymatic
detoxification or activation and other biochemical reactions along the causal chain are not saturated—i.e., at
which the dose-response relationship is still linear. When dose-response relationships are not linear, measures of
dose of active forms of toxicants at relevant sites in the nervous system are important for understanding those
relationships. In addition, the use of biologic markers can help to sort out potential nonlinearities with respect to
dose and can facilitate approaches to four problems critical in predicting neurotoxicity:
• Differences in sensitivity of the individual to neurotoxicants at different stages of life. (The previous report
on biologic markers in reproductive toxicology contains an extensive discussion of neurologic markers of
developmental effects [NRC, 1989b].)
• Protective mechanisms and barriers that vary with respect to developmental age, location in the nervous
system, and degree of metabolic integrity.
• Repair and compensation processes for types of damage that appear to be at least partly reversible under
some conditions of exposure.
• Differences in pharmacokinetics (absorption, distribution, metabolism, and excretion) between chemicals
and between animals and humans.
It has long been recognized that sensitivity to adverse effects during development can differ dramatically
over short periods—especially during gestation. Developmental signals might be present over short periods at
the intensity required to induce axons to find their targets and for other key physiologic

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 51

and biochemical events to occur; these signals might be disrupted by toxic chemicals. Understanding the
physiologic basis of temporal differences in sensitivity will be a key to determining over what periods exposures
should be aggregated for best experimental and epidemiologic quantification of risks. For example, Marsh et al.
(1987) have shown the relationship between the incidence of a variety of fetal methylmercury effects and the
time of maximal exposures during gestation, as indicated by sequential mercury concentrations in the hair of the
mothers in the Iraqi mass-poisoning incident (see Chapter 1).
It is important to note that, because of the metabolism and the pharmacodynamics of chemical toxicants,
there is a continuing need for the development of better and more accurate markers of internal dose that can be
used to estimate biologically effective doses. That refinement of dose-response modeling is fundamental to risk
assessment.
Neurons do not regenerate in adult life, and some important neurologic conditions (including Alzheimer's
disease, Parkinson's disease, and amyotrophic lateral sclerosis) are associated with progressive cumulative loss
of specific types of neurons. However, considerable numbers of cells can be lost before clinical signs of
impairment are detectable. Thus, exposure to agents causing such loss might proceed without apparent effect for
years. Even after symptoms appear, identifying the potentially relevant exposures among those of a lifetime is
nearly impossible.
Epidemiologic research on neurodegenerative diseases would be greatly facilitated if either or both of the
following types of biologic markers were available:
• Measures of the past accumulation of relevant damage in people who have not yet developed clinical illness.
For example, can our new tools for imaging the brain be applied to develop a census of the relevant cells left
in people of different ages with different past exposures? Imaging methods are being applied to studies of
people exposed to doses of MPTP that have not yet produced clinical effects. It is analogous to the use of
chest x-rays, FEV1, (forced expiratory volume in 1 second), and FVC (forced vital capacity) to assess the
accumulation of chronic respiratory damage (NRC, 1989a).
• Measures of the current rate of loss of the relevant cells. When cells die, do they release measurable
amounts of a distinctive form of an enzyme or some other cellular component that could be used as a
biologic marker? Distinctive patterns of electric activity in discrete brain regions can be monitored, as can
the integrity of neurochemical systems, e.g., receptor binding, synthetic enzyme activity, and glucose
utilization.

SUMMARY
In toxicology and epidemiology, biologic markers have several major advantages as means of exploring the
relationship between exposures and effects:
• A more complete scientific understanding than a simple input-output analysis based on external dose and the
incidence of disease, by virtue of the incorporation of more relevant information about critical events and
molecular mechanisms.
• The eventual prospect of improving mechanism-based estimations of risk.
• Improved sensitivity, specificity, and predictive value of detection and quantification of adverse effects at
low dose and early in exposure at the cellular or subcellular level.
• The prospect of confirming exposure-related effects and narrowing the search for exposures.
• A better understanding of unique susceptibilities in various population groups.
• A better understanding of disease processes in the nervous system.
Neuroscience and neurotoxicology can be enhanced by directing more research attention

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BIOLOGIC MARKERS IN NEUROTOXICOLOGY 52

to quantitative questions—''How much?'' "How fast?" "According to what quantitative functional relationship?"—
among the series of causal intermediate processes involved. The role of biologic markers is to measure the steps
of disease processes at as many different points as is feasible and to aid the development of means to stop and
reverse the processes.

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TESTING FOR NEUROTOXICITY 53

4
Testing for Neurotoxicity

Diseases of environmental origin result from exposures to synthetic and naturally occurring chemical
toxicants encountered in the environment, ingested with foods, or administered as pharmaceutical agents. They
are, by definition, preventable: they can be prevented by eliminating or reducing exposures to toxicants. The
fundamental purpose of testing chemical substances for neurotoxicity is to prevent disease by identifying toxic
hazards before humans are exposed. That approach to disease prevention is termed "primary prevention." In
contrast, "secondary prevention" consists of the early detection of disease or dysfunction in exposed persons and
populations followed by prevention of additional exposure. (Secondary prevention of neurotoxic effects in
humans is discussed in Chapter 5.)
In the most effective approach to primary prevention of neurotoxic disease of environmental origin, a
potential hazard is identified through premarket testing of new chemicals before they are released into commerce
and the environment. Identifying potential neurotoxicity caused by the use of illicit substances of abuse or by the
consumption of foods that contain naturally occurring toxins is less likely. Disease is prevented by restricting or
banning the use of chemicals found to be neurotoxic or by instituting engineering controls and imposing the use
of protective devices at points of environmental release.
Each year, 1,200–1,500 new substances are considered for premarket review by the Environmental
Protection Agency (EPA) (Reiter, 1980), and several hundred compounds are added to the 70,000 distinct
chemicals and the more than 4 million mixtures, formulations, and blends already in commerce. The proportion
of the new chemicals that could be neurotoxic if exposure were sufficient is not known (NRC, 1984) and cannot
be estimated on the basis of existing information (see Chapter 1). However, of the 588 chemicals used in
substantial quantities by American industry in 1982 and judged to be of toxicologic importance by the American
Conference of Governmental Industrial Hygienists (ACGIH), 28% were recognized to have adverse effects on
the nervous system; information on the effects was part of the basis of the exposure limits recommended by
ACGIH (Anger, 1984).
Given the absence of data on neurotoxicity of most chemicals, particularly industrial chemicals, it is clear
that comprehensive primary prevention would require an extensive program of toxicologic evaluation. EPA

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TESTING FOR NEUROTOXICITY 54

has regulatory mechanisms to screen chemicals coming into commerce, but overexposures continue to occur, and
episodes of neurotoxic illness have been induced by chemicals, such as Lucel-7 (Horan et al., 1985), that have
slipped through the regulatory net. Serious side effects of pharmaceutical agents also continue to surface, such as
the 3 million cases of tardive dyskinesia that developed in patients on chronic regimens of antipsychotic drugs
(Sterman and Schaumburg, 1980). It is now possible to identify only a small fraction of neurotoxicants solely on
the basis of chemical structure through analysis of structure-activity relationships (SARs), so in vivo and in vitro
tests will be needed for premarket evaluation until greater understanding of SARs permits them to be used with
confidence.
If neurotoxic disease is to be prevented, public policy must be formulated as though all chemicals are
potential neurotoxicants; a chemical cannot be regarded as free of neurotoxicity merely because data on its
toxicity are lacking. Prudence dictates that all chemical substances, both old and new, be subjected to at least
basic screening for neurotoxicity in the light of expected use and exposure. However, the sheer number of
untested chemicals constitutes a practical problem of daunting magnitude for neurotoxicology. Given the number
of untested chemicals and current limitations on resources, they cannot all be tested for neurotoxicity in the near
future. Testing procedures designed for neurotoxicologic evaluation that have been developed so far might be
reasonably effective, but are so resource-intensive that they could not be applied to all untested chemicals.
A rational approach to neurotoxicity testing must contain the following elements:
• Sensitive, replicable, and cost-effective neurotoxicity tests with explicit guidelines for evaluating and
interpreting their results.
• A logical and efficient combination of tests for screening and confirmation.
• Procedures for validating a neurotoxicologic screen and for guiding appropriate confirmatory tests.
• A system for setting priorities for testing.
This chapter discusses systematic assessment of chemicals for neurotoxic hazards. It begins by describing
biologic and public-health issues that are peculiar to neurotoxicology. It then presents a review of current
techniques for neurotoxicologic assessment to address the question: "How effective are current test procedures
for identify neurotoxic hazards?" Concomitantly, the review seeks to identify gaps and unmet needs in current
neurotoxicity test procedures. Finally, the chapter describes how the regulatory agencies currently evaluate
potential neurotoxicity and how the tests reviewed could be combined in an improved neurotoxicity testing
strategy.
A byproduct of the testing strategy outlined in this chapter will be the development of an array of biologic
markers of neurotoxicity. These markers can be used in future studies of experimental animals, as well as in
clinical and epidemiologic studies of humans exposed to neurotoxicants, as was proposed in Chapter 3.

APPROACH TO NEUROTOXICITY TESTING

Difficulties in Neurotoxicity Testing

Neurotoxicity testing is relatively new. Although its rapid development is noteworthy, its progress has been
constrained by several factors that complicate neurotoxicologic assessment. Some of the complexities, such as
sex- or age-related variability in response, are common to all branches of toxicology. Neurotoxicology, however,
faces unique difficulties, because of several characteristics

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that make the nervous system particularly vulnerable to chemically induced damage (Chapter 2). Those
characteristics include the limited ability of the nervous system to repair damage, because of the absence of
neurogenesis in adults; the precarious dependence of axons and synaptic boutons on long-distance intracellular
transport; the system's distinct metabolic requirements; the system's highly specialized cellular subsystems; the
use of large numbers of chemical messengers for interneuronal communication; and the complexity of the
nervous system's structural and functional integration. The nervous system exhibits a greater degree of cellular,
structural, and chemical heterogeneity than other organ systems. Toxic chemicals potentially can affect any
functional or structural component of the nervous system—they can affect sensory and motor functions, disrupt
memory processes, and cause behavioral and neurologic abnormalities. The large number of unique functional
subsystems suggests that a great diversity in test methods is needed to ensure assessment of the broad range of
functions susceptible to toxic impairment. The special vulnerability of the nervous system during its long period
of development is also a critical issue for neurotoxicology.
Despite the inherent difficulties of neurotoxicity testing, some validated tests have been developed and
implemented. Testing strategies must take those facets of the nervous system into account, and they must
consider a number of variables known to modify responses to neurotoxic agents, such as the developmental stage
at which exposure occurs and the age at which the response is evaluated. The issue of timing is complex. During
brain development, limited damage to cell function—even reversible inhibition of transmitter synthesis, for
instance—can have serious, long-lasting effects, because of the trophic functions of neurotransmitters during
neuronal development and synaptogenesis (Rodier, 1986). Kellogg (1985) and others have shown the striking
differences between the effects of perinatal exposure of animals to some neuroactive drugs (e.g., diazepam) and
the effects of exposure of adult animals to the same drugs. Other agents (e.g., methylmercury and lead) are toxic
at every age, but are toxic at lower doses in developing organisms. In addition, in developing animals, the blood-
brain barrier might not be sufficiently developed to exclude toxicants. Stresses later in development might lead
to the expression of relatively sensitive effects that were latent or unchallenged at earlier stages of development.
During senescence, the CNS undergoes further change, including a loss of nerve cells in some regions. CNS
function in senescence could be vulnerable to cytotoxic agents that, if encountered earlier in development, might
have been protected against by redundant networks or compensated for by "rewiring" of networks (Edelman,
1987). To address those complex issues, testing paradigms that incorporate both exposures and observations
during development and during aging need to be considered. The disorders might be acute and reversible or
might lead to progressive disorders over the course of chronic exposure. More sensitive biologic markers as early
indicators of neurotoxicity are urgently needed. Opportunities should be exploited for detecting neurotoxicity
when chronic lifetime bioassays are conducted for general toxicity or carcinogenicity.
In the design of neurotoxicity screens, no test can be used to examine all aspects of the nervous system. The
occurrence of an effect of a chemical on one function of the nervous system will not necessarily predict an effect
on another function. Therefore, it is important to use a variety of initial tests that measure different chemical,
structural, and functional changes to maximize the probability of detecting neurotoxicity or to use tests that
sample many functions in an integrated fashion.

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TESTING FOR NEUROTOXICITY 56

The Testing Strategy

Efficient identification of potential hazards warrants a tiered testing strategy. The first tier of testing (the
screen) need not necessarily be specifically predictive of the neurotoxicity likely to occur in humans, unless
regulatory agencies are to use the results for direct risk-management decisions. The tests in later tiers are
essential to assess specificity and confirm screening results and are appropriate for defining dose-response
relationships and mechanism of action (Tilson, 1990a). Screening tests would be followed, as appropriate, by
more specific assessments of particular functions. Such an approach permits a decision to be made about whether
to continue testing at each step of the progression. In the case of chemicals already in use (in which case people
are already being exposed and financial consequences might be considerable), detailed testing to determine
mechanisms of toxicity would be pursued when screening tests revealed neurotoxic effects; positive findings on
a chemical undergoing commercial development might trigger its abandonment with no further testing.
Testing at the first tier is intended to determine whether a chemical has the potential to produce any
neurotoxic effects—i.e., to permit hazard identification, the first step of the NRC (1983) risk-assessment
paradigm. The next tier is concerned with characterization of neurotoxicity, such as the type of structural or
functional damage produced and the degree and location of neuronal loss. During hazard characterization (the
second step of the NRC paradigm), tests are used to study quantitative relationships between exposure (applied
dose) and the dose at the target site of toxic action (delivered dose) and between dose and biologic response. The
third and final tier of neurotoxicity testing is the study of mechanism(s) of action of chemical agents.
The decision to characterize a chemical through second-tier testing might be motivated by structure-activity
relationships, existing data that suggest a chemical is neurotoxic, or reports of neurotoxic effects in humans
exposed to the chemical, in addition to the results of first-tier testing. Testing at this second tier can help to
resolve several issues, including whether the nervous system is the primary target for the chemical and what the
dose-effect and time-effect relationships are for relatively sensitive end points. Such tests can also be useful in
the determination of a no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level
(LOAEL). Experiments done at the third tier can also examine the mechanism of action associated with a
neurotoxic agent; they will often involve neurobehavioral, neurochemical, neurophysiologic, or neuropathologic
measures. They might also suggest biologic markers of neurotoxicity for validation and use in new toxicity tests
and in epidemiologic studies.

Characteristics of Tests Useful for Screening

Like any other toxicity test, screening tests for neurotoxicity should be sensitive, specific, and valid.
Sensitivity is a test's ability to detect an effect when it is produced (the ability to register early or subtle effects is
especially desirable). Sensitivity depends on inherent properties of the test and on study design factors, such as
the numbers of animals studied and the amount and duration of exposure. Specificity is a test's ability to respond
positively only when the toxic end point of interest is present. Specificity and sensitivity are aspects of accuracy.
An inaccurate test fails to identify the hazardous potential of some substances and incorrectly identifies as
hazardous other substances that are not. In statistical terms, the failure to identify a hazard is a false-negative
result, and the mistaken classification of a safe substance as hazardous is a false-positive result. Increasing test
specificity reduces the

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incidence of false positives, but often has the unwanted consequence of increasing the incidence of false
negatives (decreasing sensitivity); increasing test sensitivity reduces the incidence of false negatives, but often
increases the incidence of false positives.
An ideal screen would have broad specificity, so that it could detect all aspects of nervous system
dysfunction. In practice, no screening procedure is likely to provide the desired coverage without producing false
positives. The sensitivity of the first tier might be maximized by a battery of screening tests that are individually
quite specific. Several durations of exposure, long postexposure observation periods, and lifetime tests might all
be necessary to cover the possible manifestations of neurotoxicity. In a tiered test system, very high sensitivity of
the screening tier is usually considered essential; the loss of specificity must be compensated for in later tiers, to
reveal the false positives. If, however, product development will be aborted on any indication of neurotoxicity,
false positives could have high social costs. In the later tiers, narrower specificity is appropriate to characterize a
suspected toxicant or to establish its mechanism of action.
Validation is the process by which the credibility of a test is established for a specific, purpose (Frazier,
1990). It entails demonstrating the reliability of the test's performance in giving reproducible results within a
laboratory and in different laboratories and giving appropriate results for a control panel of substances of known
toxicity. The usefulness of the results of a neurotoxicity screening test depends on a positive outcome's being
strongly correlated with a neurotoxic effect that is actually caused by exposure to the test substance. Direct
mechanistic causality is not essential for their interpretation, although direct insight into mechanism would be
valuable, as is sought when developing a biologic marker of effect.
Validation of a test system should also include demonstration that positive test results indicate that
neurotoxic effects would occur if humans were exposed to the substance. Predicting an influence on human
affect or cognition with nonhuman test systems is challenging, but possible. Many animal testing models do
produce effects that correspond to those seen in humans exposed to the same substance, but a result would also
be considered valid if it correctly predicted any neurotoxic effect in the human population. For example, consider
a screening test for an effect produced only at high doses that is consistently correlated with a milder,
presumably precursor effect that occurs at lower doses. The easily detectable effect occurring at high doses in
experimental animals might never be observed in humans, whose exposure would never be extreme, but its
occurrence in the animal model might indicate that low-level exposure in humans could produce a more subtle
toxic effect. O'Donoghue (1986) has noted that chemicals that damage axons are often metabolic poisons that
produce a retardation of weight gain without decreasing food intake—a more easily observed end point. It is
important to recognize that chemicals can adversely affect multiple organ systems, and an effect in other organs
might influence some measures of neurotoxicity. In vitro assay systems often measure end points that appear
unrelated to neurologic functioning in whole animals, but detect alterations to crucial underlying mechanistic
processes.

Application of the Screen and Later Tiers

Given the enormous number of substances that have not been tested for neurotoxicity (or any form of
toxicity), some characteristics of chemicals, such as their structure or production volume, must contribute to the
determination of priority for screening. Confidence in the combined sensitivity of tests composing a screening
battery should

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be high enough for negative findings to be reliably regarded as acceptable evidence that a substance is unlikely
to have neurotoxic activity without the need for additional testing. If a new chemical gives positive results, there
might nevertheless be academic or commercial interest in pursuing a risk assessment, or its development for use
might be abandoned without further testing. Detailed testing to characterize neurotoxicity revealed by screening
procedures will be more common for existing substances with commercial value or wide exposure. In either
case, the path followed through the tiers of testing would be contingent on a chemical's unfolding toxicity
profile, including types of toxicity other than neurotoxicity. A feasible screening system must strike a balance
among the amount of time and expense that society is willing to expend in testing, its desire for certainty that
neurotoxic substances are being kept out of or removed from the environment, and its interest in gaining benefits
from various types of chemicals.

CURRENT METHODS BASED ON STRUCTURE-ACTIVITY RELATIONSHIPS

Given the overwhelming lack of epidemiologic or toxicologic data on most chemical substances and the
need to develop rational strategies to prevent adverse health effects of both new and existing chemicals, attempts
have been made in many subfields of toxicology to generate predictive strategies based primarily on chemical
structure. The basis for inference from structure-activity relationships (SARs) can be either comparison with
structures known to have biologic activity or knowledge of structural requirements of a receptor or
macromolecular site of action. However, given the complexity of the nervous system and the lack of information
on biologic mechanisms of neurotoxic action, SARs should be regarded as, at best, providing information that
might be useful to identify potentially neuroactive substances. SARs are clearly insufficient to rule out all
neurotoxic activity; it is not prudent to use them as a basis for excluding potential neurotoxicity. Caution is
warranted in interpreting SARs in anything other than the most preliminary analyses. An intelligent use of SARs
requires detailed knowledge not only of structure, but also of each critical step in the pathogenetic sequence of
neurotoxic injury. Such knowledge is still generally unavailable. SAR evaluations form a major basis for EPA
and FDA decisions on whether to pursue full neurotoxicity assessments, so it must be concluded that the
approach of these agencies cannot be expected to provide an adequate screen for identifying neurotoxicants.
SAR approaches are more successful when the range of possible sites of action or mechanisms of action is
narrow. Thus, SARs have had more use in relation to carcinogenicity and mutagenicity than in other kinds of
toxicity. The SAR approaches used in the development of novel neuropharmacologic structures deserve
consideration in neurotoxicology, but they must rest on fuller understanding of neurotoxic mechanisms.
SARs have not been used extensively in neurotoxicology, for several reasons. Many agents are neurotoxic—
from elements, such as lead and mercury, to complex molecules, such as MPTP and neuroactive drugs.
Neurotoxicants have so many potential targets that it is difficult to rule out any chemical a priori as not likely to
be neurotoxic. Finally, for most neurotoxicants, even those which are well characterized, data on the mechanism
of action at the target site are insufficient for the elucidation of useful SARs.
SARs have proved useful in some cases—usually, within particular classes of compounds on which some
mechanistic data are available. Extensive studies of anticholinesterase organophosphates and carbamates, for
example, have led to a mechanistic

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model that requires a structure that binds to a specific site on the cholinesterase molecule and to the
establishment of an SAR for cholinesterase inhibition. The SAR is relatively straightforward, in that enzyme
inhibition is the primary action that leads to various symptoms of poisoning. Another series of studies has been
conducted in an attempt to develop an SAR for the pyrethroid insecticides. Although the nerve membrane
sodium channel has been identified as the critical target site of pyrethroids, studies have failed to establish a
clear-cut SAR, despite the large number of pyrethroids synthesized and tested. However, the importance of the d-
cyano group in distinguishing two behavioral manifestations (choreoathetosis and salivation versus tremor) and
the importance of the ester moiety in pyrethroid SAR are well documented (Aldridge, 1990). A more
comprehensive SAR for pyrethroids is lacking partly became most studies were performed with insecticidal
action as a measure of activity, not the interaction with the sodium channel. Action at the target site must be
known to establish an SAR. However, insecticidal potency involves factors other than recognition at the target
site, including absorption and metabolism. As the mechanism of action of n-hexane and methyl n-butylketone
neurotoxicity becomes more clear, so does the capacity to look at an alkane or ketone and predict its neurotoxic
potential. The critical issue in the neurotoxicity of such a chemical is whether it will be metabolized to a γ-
diketone; α-, β-, and δ-diketones are less likely to be neurotoxic (Krasavage et al., 1980; Spencer et al., 1978),
but most γ-diketones and γ-diketone precursors cause a specific type of neurotoxicity, a motor-sensory peripheral
neuropathy caused by neurofilament abnormalities. The gamma spacing of the two ketone groups allows
formation of the five-membered heterocyclic ring, the pyrrole, except where steric hindrance is present (Genter
et al., 1987).

CURRENT IN VITRO PROCEDURES

In vitro procedures for testing have practical advantages, but studies must be done to correlate their results
with responses in whole animals. One advantage of validated in vitro tests is that they minimize the use of live
animals. Some of the more developed in vitro tests might be simple and might not have to be conducted by
highly trained personnel, but, as with many in vivo tests, the analysis and interpretation of results is likely to
require expertise. Some in vitro tests lend themselves to computer-controlled automated operation, as do some
well-developed, highly sophisticated behavioral tests (see, for example, Evans, 1989), and that results in savings
in time and expense and allows testing of large numbers of substances. Experience with the Ames test for
mutagenesis confirms the advantages of in vitro procedures, but also illustrates the problems that arise when an
assay is used to predict an end point that is not exactly what it measures (e.g., carcinogenicity, rather than
specific sorts of genotoxicity).

Biochemical Assays
Biochemical assays epitomize the advantages of in vitro tests. Their current usefulness, however, is limited
to substances in a very few chemical classes. Their usefulness will no doubt increase as the molecular basis of
action of other classes of neurotoxicants becomes known.
Neurotoxic chemicals exert their effects via specific molecular interactions with biologic targets. For a few
toxicants, the molecule that is affected is well established, and it is possible to investigate the potential toxicity of
other substances in the same class with a biochemical assay. An example of this approach is the enzyme-
inhibition assay of organophosphorus (OP) esters. Some OP esters cause a distal polyneuropathy that is

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not evident until several days after exposure (OP-induced delayed neuropathy, or OPIDN). There is now good
evidence (Abou-Donia, 1981) that the capacity of a given OP ester to cause OPIDN correlates strongly with the
relative inhibition of a CNS enzyme activity called neurotoxic esterase. The enzyme activity is easily measured
in a test-tube assay, and the addition of OP esters and OP-like compounds to the assay allows one to screen for
ability to inhibit the enzyme and thus for its potential to produce OPIDN (Johnson, 1977). The ease of this assay
is complicated, however, by the precautions necessary to protect the technicians performing the assay from
exposure to potentially highly neurotoxic substances. Many OP compounds also produce an immediate toxic
effect via inhibition of another enzyme, acetylcholinesterase. A test-tube assay of that enzyme's activity can
serve as a screen for such acute neurotoxicity.

Tissue Culture
The study of in vitro systems has provided much fundamental information of value in understanding the
nervous system in vivo, and in vitro investigations have sometimes been invaluable in guiding in vivo
neurotoxicologic research. The physiologically excitatory actions of some amino acids (for example, glutamate
and aspartate) on neurons can become pathologic, and brain lesions can be produced by amino acid analogues,
such as kainate or quisqualate. In vitro systems are being used extensively to study the mechanisms of the
neuronal injury produced by those agents and to identify antagonists that might be useful in reducing excitotoxic
brain damage. In vitro studies can also identify compounds with a high neurotoxic potential that might merit
study in intact organisms. An example is β-N-methylamino-L-alanine (BMAA), a constituent of a dietary plant,
the false sago palm. BMAA was initially identified as an excitotoxic amino acid in cultures of tissue from spinal
cord and cerebral cortex. That led to experiments with primates that showed that BMAA produces motoneuronal
lesions in the cortex and spinal cord.
A broad range of tissue-culture systems are available for assessing the neurologic impact of environmental
agents. Although those systems are not now used for hazard detection, they can be used to characterize chemical-
induced effects. They can be classified according to their increasing complexity, from cell lines to organ cultures.
• Cell lines. Neuronal and glial cell lines have been used in many neurobiologic studies and are valuable in
neurotoxicology. They consist of populations of continuously dividing cells that, when treated appropriately,
stop dividing and exhibit differentiated neuronal or glial properties. Various neuronal lines, for instance,
develop electric excitability, chemosensitivity, axon formation, transmitter synthesis and secretion, and
synapse formation. Large quantities of cells can be generated routinely to develop extensive dose-response
or other quantitative data. For example, the dose range over which a group of chemicals affect cell
differentiation and proliferation was established in neuroblastoma cells (Stark et al., 1989). Those are tumor
cells, however, so the interpretation of such data with regard to toxicity in the intact nervous system must be
guarded. A culture system for nontransformed neural cells was recently announced (Ronnett et al., 1990).
• Dissociated cell cultures. When neural tissue, typically from fetal animals, is dissociated into a suspension
of single cells, and the suspension is inoculated into tissue-culture dishes, the neurons and glia survive,
grow, and establish functional neuronal networks. Such preparations have been made from most regions of
the CNS and exhibit highly differentiated, site-specific properties that constitute an in vitro model of
different portions of the CNS. Most of

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the neuronal transmitter and receptor phenotypes can be demonstrated, and a variety of synaptic interactions
can be studied. Glial cells are also present, and neuroglial interactions are a prominent feature of the
cultures. A substantial battery of assays (neurochemical and neurophysiologic) is now available to assess the
development of the cultures and to indicate toxic effects of test agents added to the culture medium.
Relatively pure populations of different cell types can be isolated and cultured, so that effects on specific
cell types can be assessed independently. Pure glial cells or neurons, or even specific neural categories, can
be prepared in this way and studied separately, or interaction between neurons and glial cells can be studied
at high resolution. The neurobiologic measures used to assess the effect of any agent can be very specific
(for example, activity of a neurotransmitter-related enzyme or binding of a receptor ligand) or global (for
example, neuron survival or concentration of glial fibrillary acidic protein). The two-dimensional character
of the preparations makes them particularly suited for morphologic evaluation, and detailed
electrophysiologic studies are readily performed. The toxic effects and mechanisms of anticonvulsants,
excitatory amino acids, and various metals and divalent cations have been assessed with these preparations.
The cerebellar granule cell culture system, for example, has been exploited recently in studies of the
mechanism of alkyllead toxicity (Verity et al., 1990).
• Reaggregate cultures. A related but distinct preparation made from single-cell suspensions of neural tissue is
the reaggregate culture. Instead of being placed in culture dishes and allowed to settle onto the surface of the
dishes, the cells are kept in suspension by agitation; under appropriate conditions, they stick to one another
and form aggregates of controllable size and composition. Typically, the cells in an aggregate organize
themselves and exhibit intercellular relations that are a function of and bear some resemblance to the brain
region that was the source of the cells. The cells establish a three-dimensional, often laminated structure,
perhaps approximating the in vivo nervous system more closely than do the dissociated cultures grown on
the surface of a dish. Reaggregate cultures lend themselves to large-scale, quantitative experiments in which
neurobiologic variables can be examined, although morphologic and ligand-binding studies are performed
less readily than with surface cultures.
• Explant cultures. Organotypic explant cultures are even more closely related to the intact nervous system.
Small pieces or slices of neural tissue are placed in culture and can be maintained for long periods with
substantial maintenance of structural and cell-cell relations of intact tissue. Specific synaptic relations
develop and can be maintained and evaluated, both morphologically and electro-physiologically. Because all
regions of the nervous system are amenable to this sort of preparation, it is possible to analyze toxic agents
that are active only in specific regions of the central or peripheral nervous system. Explants can be made
from relatively thin slices of neural tissue, so detailed morphologic and intracellular electrophysiologic
studies are possible. Their anatomic integrity is such that they capture many of the cell-cell interactions
characteristic of the intact nervous system while allowing a direct, continuing evaluation of the effects of a
potentially neurotoxic compound added to the culture medium. The process of myelination has been studied
extensively in explant cultures, and considerable neurotoxicologic information has been gained. As noted
above, the pathogenetic actions of excitatory amino acids normally active in the nervous system, as well as
such analogues as the neurotoxin BMAA, have been revealed by experiments with organotypic cultures.
• Organ Cultures. A preparation similar to an explant culture is the organ culture, in which an entire organ,
such as the inner ear or a ganglion, rather than slices or fragments,

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is grown in vitro. Obviously, only structures so small that their viability is not compromised can be treated
in this way.
The advantages of the various types of in vitro systems are summarized in Table 4-1. Most in vitro
preparations are made from young, usually prenatal animals. (But cultures derived from human neural tissue
have been the object of a number of studies.) Typically, a period of rapid change and development occurs
immediately after the cultures are initiated, and conditions become much more stable if the cultures are
maintained for weeks or months. Thus, the preparations can be used to study neurotoxic effects that might be
specific to developing nervous tissue and to compare the effects of agents in developing stable tissue.
In general, the technical ease of maintaining a culture varies inversely with the degree to which it captures a
spectrum of in vivo characteristics of nervous system behavior. The problem of biotransformation of potentially
neurotoxic compounds is shared by all, although the more complete systems (explant or organ cultures) might
alleviate this problem in specific instances. In many culture systems, complex and ill-defined additives—such as
fetal calf serum, horse serum, and human placental serum—are used to promote cell survival. A number of
thoroughly described synthetic media are now available, however, and such fully defined culture systems can be
used where necessary. Indicators of neuronal and glial function, and hence indicators of neurotoxicity, are
outlined in Table 4-2.

Plausibility of an In Vitro Screening Battery

A broad range of in vitro systems are now available for studying development of the nervous system and the
normal function of neurons and glial cells. The possible neurotoxic impact of any chemical on any specific
neurobiologic variable could, in principle, be screened with an appropriate set of in vitro tests. In practice, of
course, because the number of potential neurobiologic end points to be measured is so large, screening for the
effects of any agent on all of them would be prohibitively expensive in time and money. The question, then, is
whether a feasible battery of tests will pick up an acceptably large percentage of toxic chemicals (while
generating an acceptably low percentage of false positives). Ideally, the screen would
TABLE 4-1 In Vitro Neurobiologic Test Systems
Culture Type Comparablity to In Vivo Systema Mechanistic Analysisa Features
Cell line + ++++ Large quantities of material
Dissociate cell culture ++ +++ Good accessibility for study
Reaggregate culture +++ ++ Three-dimensional structure
Explant culture ++++ ++ Good approximation of intact
sytems
Organ culture ++++ + Less long-term survival than
other models

aNumber of pluses reflects estimated relative advantage and represents the committee's judgment.

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TABLE 4-2 Markers for Assessing Neurotoxicity in In Vitro Systems

Degree of Difficultya
General neuronal measures
Cell number 1
Tetanus-toxin binding 2
Neurofilament protein 3
Neuronal structure 2
General glial measures
Glial fibrillary acidic protein 2
Oligodendrocyte probe 3
Transmitter systems
Amino Acid
Excitatory 3
Inhibitory 1
Cholinergic
Choline acetyltransferase 1
Muscarinic and nicotinic receptors 3
Aminergic
Norepinephrine 2
Serotonin 2
Dopamine 2
Peptidergic
Vasoactive intestinal peptide 3
Substance P 3
Enkephalin 3
Cell biologic responses
Second messengers
Cyclic nucleotide 3
Phosphoinositide turnover 3
Phosphorylation 3
Calcium-dependent transmitter release 3
Voltage-dependent NA+ or Ca2+ uptake 2

aThe higher the number, the more difficult to measure. This represents the committees judgment.

provide direction for the more intensive study of substances that it identifies as having neurotoxic potential.
To what extent could an in vitro system provide such a screening instrument? Two basic questions are
associated with the use of in vitro tests for that purpose:
• What indicators of neuronal (or glial) damage would be sufficiently general to be useful?
• What specific test systems or combinations would be adequate to cover a number of different and
differentially site-specific neurotoxic agents?
The first question might be answered by a combination of assays that would include general indicators of
neuronal and glial survival and a few more specific indicators. Counts of numbers of surviving neurons (or glia)
and biochemical measurements of tetanus-toxin-specific or sodium-channel-specific ligand binding could be
used as general indicators. Uptake of γ-aminobutyric acid (GABA), benzodiazepine binding, and cholinergic and
aminergic markers could be used, depending on the neural system chosen, to get some indication of
neurotransmitter-related functions. If a chemical has a single very specific neurologic target, this would in
general be missed, but it might be anticipated that such a specific effect would be accompanied by more general
secondary neuropathologic consequences. For instance, if spinal-cord or cerebral cortical cell cultures are
exposed to the specific voltage-dependent sodium channel blocker tetrodotoxin for 4–5 days, the decrease in
electric activity kills about half the neurons.
As to the second question, the choice of culture systems to be used is difficult. It is axiomatic that no cell
culture represents a normal nervous system. Even in cocultured explants, the normal connections among cells are
disrupted. Specific neurobiologic properties have been shown not to be expressed in various in vitro preparations
where they

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might be expected. For instance, in dissociated hippocampal pyramidal cells, serotonergic responses cannot be
demonstrated; the responses appear during development in vivo, but appropriate signals that induce their
expression are evidently lacking in vitro. It is impossible to predict how such departures from normality will
influence the screen's ability to detect the effects of a test substance. Some compromise between
comprehensiveness and fiscal feasibility would have to be made. Neural and glial cell lines are available and
relatively straightforward technically. Robust versions of cerebral-cortex, spinal-cord, and subcortical systems
are available either as dissociated preparations or in explant form. Indeed, essentially all regions of the nervous
system are being grown in vitro in one form or another. If a candidate neurotoxic material has properties that
suggest site-specific activity, obviously one would include its putative target in a test battery. If no such
information is available, then some arbitrary panel of test systems would have to be used.
Even if these two questions are answered, cell-culture techniques have several remaining disadvantages
when used as screens. A given toxicant may require metabolism outside the nervous system to produce a toxic
metabolite, so exposure to the toxicant in vitro may give a false-negative result; conversely, the chemical might
be detoxified before reaching the nervous system. A related disadvantage might be low solubility of a given
toxicant in an aqueous culture medium, which could limit the quantity of toxicant to which the cells are actually
exposed. A related problem concerns the lack of a blood-brain barrier in in vitro experiments. Toxicity could be
attributed to a compound that would not reach the brain, either at all or in sufficient concentration to cause
toxicity, in in vivo exposure. Last, and most important, the more complex functions of the nervous system are
properties of assemblies of neurons. Learning, memory, emotions, the coordination of movement, and
homeostatic regulation, for instance, cannot be studied in vitro.
Despite those disadvantages, some applications of cell cultures are nearly ready for use in neurotoxicity
screening. Several indicators of glial and neuronal function in vitro can be used as biologic markers of effect
(Table 4-2). In vitro systems have been extremely useful in identifying and analyzing excitotoxic materials. Also,
in vitro demonstrations of neurotoxicity of anticonvulsants based on both general and specific indicators have
been corroborated by clinical studies of IQ decrements related to phenobarbital treatment in children. Many
neurotoxicants attack the glial cells that form myelin sheaths around axons, and cell cultures can demonstrate
chemically induced myelin abnormalities; this has been shown with the myelin abnormalities produced by
methylmercury (Kim, 1971), thallium. (Spencer et al., 1985), triethyltin (Graham et al., 1975), and several other
substances. Tests on cultures can also detect toxic damage to neurons themselves. For example, aluminum
neuronopathy is reproduced in cultures of dorsal root ganglion cells (Seil et al., 1969), and the axonal
degeneration characteristic of γ-diketone toxicity is reproduced in organotypic cocultures (Veronesi et al., 1978).
Furthermore, as noted in Chapter 2, the cells of the CNS are uniquely dependent on a high rate of glucose
metabolism; many neurotoxicants impair neural glucose metabolism (Damstra and Bondy, 1980), and this toxic
mechanism could be reflected in a decrease in the viability of cultured cells.
A somewhat arbitrary, but specific, protocol for developing a set of in vitro tests for screening is presented,
for illustrative purposes, in Table 4-3. It is an empirical question whether such a battery of in vitro tests could
contribute importantly to neurotoxicologic screening of a broad range of environmental agents. Research needs
to be done with known neurotoxic agents and related compounds to see whether culture systems can reliably
identify dangerous

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TABLE 4-3 Proposed Protocol for Developing an In Vitro Neurotoxicity Screening System
1. Test candidates representing various types of potential neurotoxicants, e.g., anticholinesterases, excitatory amino
acids, trimethyl tin.
2. Use three concentrations, e.g., less than, equal to, and greater than in vivo toxic concentrations.
3. Use three types of preparation, e.g., PC12 cells, dissociated neurons and glia of cerebral cortex, and explants of
dorsal root ganglia and spinal cord.
4. Study developing (0–2 weeks in vitro) and mature (> 2 weeks in vitro) preparations.
5. Evaluate appropriate end points, such as cell counts, glial fibrillary acid protein, structure, choline acetyltransferase,
and glutamic acid decarboxylase.
6. Use inactive isomers or analogues as negative control agents.
substances and distinguish them from related, but inactive or less active, substances. Analyses of the
contributions of short-term in vitro tests to neurotoxicity are for less developed and for fewer in number than
analyses of in vitro tests in other fields, e.g., carcinogenesis (Lave and Omenn, 1986).
It should be possible to define the goals of such research to be finite and realizable. The question may be
framed as follows: What is the smallest set of tests that gives positive indicators of neurotoxicity for all of a
control panel of compounds known to have diverse neurotoxic properties? Of equal importance, and perhaps
more difficult, would be the evaluation of presumably nonneurotoxic substances to preclude a high rate of false
positives. One might start with a fairly inclusive set of test cultures, such as of cerebral cortex, brain stem,
cerebellum, spinal cord, sympathetic ganglion neurons, and a continuous cell line, such as PC12 cells. Global
assays, such as cell counts and tetanus-toxin binding, would be initial indicators, and specific neurotransmitter-
related probes could be used as appropriate. The essential feature of such an effort is the reliability of the culture
systems. Reliability typically is achieved only after a fairly long period of use in a given laboratory.
It will be difficult to cover all end points of concern with a modest number of in vitro assays. Whole-animal
tests have the potential of exploiting the integrative nature of behavior, thereby covering a diversity of adverse
end points that might result from exposure to neurotoxic agents. It might be more efficient to screen with in vivo
tests and use an in vitro approach when concentrating on specific mechanisms of action. A goal of research
directed at development of adequate neurotoxicologic methods should be to combine behavioral testing on intact
animals with selected in vitro tests. Can some behavioral tests be replaced by less expensive in vitro tests without
loss of diagnostic power? Can individual behavioral tests function as well as or better than groups of in vitro
tests? One might start with the study of Tilson (1990a) as a base and complement it with in vitro study of the
same chemicals. The results might lead to a choice of tests that will yield an optimal combination of broad
coverage and low cost.

CURRENT IN VIVO PROCEDURES

Neurotoxic effects on complex integrative functions—such as motor performance, sensory

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acuity, memory, and cognitive processes—can be detected only in vivo. Moreover, the neural activities that
mediate integrative function involve large numbers of neurons relatively distant from each other. Integration may
be disrupted by the removal and isolation of neural tissue that is necessary for some in vitro techniques, but
neurotoxic effects of relatively low exposures involving integrative functions can be detected in whole animals.
Species differ in their susceptibility to various toxic agents and the degree to which their nervous systems
resemble that of humans. Tests using species closest to humans would logically yield the data about which we
could feel most confident; however, various considerations—including cost, ethics, and the extent of pre-existing
data bases—favor the use of small laboratory rodents, such as mice and rats, for in vivo hazard identification and
characterization. Table 4-4 lists commonly reported neurotoxic effects of several classes of toxic chemicals in
humans and animals. It is apparent from the list that motor disturbances, mood alteration, and sensory
abnormalities are especially common in humans, but that the findings in animals are only sometimes
comparable. Species differences might account for some of these discrepancies; however, it is not clear that
animal tests always are intended to be or can be exactly comparable with human tests.
Testing for neurotoxicity in humans implies that exposure has occurred. Such testing is therefore considered
a means of secondary prevention, so the testing methods specifically for humans are discussed in Chapter 5.

Behavioral Assessment
Chemical-induced functional alterations of the nervous system are often assessed with behavioral
techniques. Perhaps the greatest scientific challenge to neurotoxicology is to integrate observations of behavior
with other aspects of neurobiology—such as morphology, neurochemistry, and neurophysiology—to develop a
unified theory not only of toxicity, but also of the nervous system. By itself, behavior is an important end point,
even if its biologic substrates have not been clearly identified. Numerous behavioral techniques are available to
measure chemical-induced alterations in sensory, motor, autonomic, and cognitive function (Table 4-5).
Behavioral methods differ greatly in their complexity and specificity. At one extreme, some methods (e.g.,
some observational tests) can be applied broadly and routinely to assess the neurotoxicity of a wide array of
chemicals and chemical exposures; that is, they are useful in hazard identification. Such tests typically
incorporate responses already well established in an organism. Other methods require instrumentation or training
of the animals before chemical exposure; although they might not be appropriate for the routine screening (tier 1)
of chemicals for neurotoxicity, they could be used for characterization of toxicant-induced effects (tier 2).

Functional Observational Batteries

Functional observational batteries (FOBs) are designed to detect and measure major overt neurotoxic
effects. Several have been used, each consisting of tests generally intended to evaluate various aspects of
sensorimotor function (EPA, 1985; Haggerty, 1989; Kulig, 1989; Moser, 1989; O'Donoghue, 1989). FOB tests
are essentially clinical examinations that detect the presence or absence, and in some cases the relative degree, of
specific neurologic signs.
Screening neuroactive chemicals with an FOB is well established. Irwin (1968), for example, described a
series of tests for evaluating the effects of drugs in mice and showed how different drugs produced different
patterns of effects that could be easily recognized. Gad (1982) described a battery

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 TABLE 4-4 Neurotoxic Effects of Representative Agents in Humans and Animals
Chemical Class Representative Agents Neurotoxic Effects in Humans Neurotoxic Effects in Animals
Solvents Hexane, acrylamide Ataxia, tremor, paresthesia, hypersomnia, slurring of Loss of fine motor control, weakness, sensory system
Environmental Neurotoxicology

speech, delirium and hallucinations disturbance

Carbon disulfide Anosmia, paresthesia, depression, anxiety, psychoses Sensory system disturbance
Organochlorine insecticides Chlordecone, DDT Ataxia, tremor, slurring of speech, euphoria and Tremor, hyperreflexia, impaired acquisition
excitement, nervousness and irritability, depression and
anxiety, mental confusion, memory disorders
Organophosphate esters Parathion, paraoxon Ataxia, paresthesia, insomnia, slurring of speech, tinnitus, Weakness, sensory system disturbance, autonomic
amblyopia, nystagmus, abnormal pupil reactions, dysfunction, impaired acquisition
nervousness and irritability, depression and anxiety,
TESTING FOR NEUROTOXICITY

psychoses, memory disorders

Organometals Methylmercury Ataxia, myoclonus, paresthesia, insomnia, slurring of Weakness, sensory system disturbance, visual deficits,
speech, hearing loss, abnormal pupil reactions, mental learning deficits
confusion

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67
 Chemical Class Representative Agents Neurotoxic Effects in Humans Neurotoxic Effects in Animals
Heavy metals Inorganic lead Ataxia, tremor, pathologic reflexes, paresthesia, hearing loss, abnormal pupil Motor disturbances, sensory system disturbance
reactions, depression and anxiety
Mercury vapor Facial tic, tremor, insomnia, amblyopia, depression and anxiety Motor disturbances, tremor
Environmental Neurotoxicology

Manganese Tremor, paresthesia, hypersomnia, euphoria and excitement, delirium and Tremor, motor disturbances
hallucinations, memory disorders
Cadmium Anosmia Anosmia
Arsenic Hyperesthesia Hyperesthesia
Source: Modified from Tilson and Mitchell (1984).
TESTING FOR NEUROTOXICITY

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68
 TABLE 4-5 Examples of Behavioral Measures of Functional Neurotoxicity
Tier of Testing
Function Affected Signs and Symptoms Animal Testa 1: Hazard Identification 2: Characterization
Environmental Neurotoxicology

Sensory Abnormalities of smell, vision, taste, hearing FOB X

Reflex modification X
Conditioned discrimination X
Motor Muscle weakness FOB X
Grip strength X
Hindlimb splay X
Motor discrimination X
Swimming endurance X
TESTING FOR NEUROTOXICITY

Suspension from bar X

Tremor FOB X
Spectral analysis X X
Convulsions FOB X
Incoordination FOB X
Negative geotaxis X
Rotorod X
Inclined screen X
Motor discrimination X
Hypoactivity or hyperactivity Motor activity X

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69
 Tier of Testing
Function Affected Signs and Symptoms Animal Testa 1: Hazard Identification 2: Characterization
Autonomic Abnormalities of sweating, temperature control, FOB X
gastrointestinal function
Environmental Neurotoxicology

Cognitive Disruption of learned behavior Schedule-controlled operant behavior (SCOB) X

Learning and memory Habituation X
Classical X
Instrumental X
aFOB = functional observational battery
TESTING FOR NEUROTOXICITY

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70
Environmental Neurotoxicology

TESTING FOR NEUROTOXICITY 71

of tests for assessing the neuromuscular effects of industrial chemicals in rats. More recently, Moser and
colleagues (Moser et al., 1988; Moser, 1989) developed a similar battery for assessing the neurobehavioral
effects of a broad range of industrial and pesticidal chemicals in rats.
FOBs are sets of observations and tests each made on individual experimental animals; e.g., the FOB
suggested by Moser (1989) is presented in Table 4-6. It is assumed that many of the individual observational
components overlap in the neurologic functions that they assess (autonomic function, motor function,
equilibrium, excitability, and sensorimotor reflexes). Therefore, if several unrelated observed end points in an
entire FOB were affected, there would be little concern about a chemical's neurotoxicity. If several unrelated
neurologic functions were affected, but only at high doses and in conjunction with other overt signs of toxicity,
including death or debilitation, there would be more concern. If several related functions were affected and the
effects appeared to be dose- and time-dependent, there would be still more concern. As the number of chemicals
tested for neurotoxic potential increases, many different combinations of affected functions will emerge.
Deciding which combinations of positive findings indicate the need to continue testing will not be trivial.
From many standpoints, FOBs have shortcomings. Most of their observations are semiquantitative. The
sensitivity, reliability, and reproducibility of some have not been well documented. This deficiency can often be
overcome by using more quantitative methods. For example, extensor thrust and grip strength can be measured
with simple devices that use a strain gauge (Cabe and Tilson, 1978; Meyer et al., 1979). Hindlimb splay can be
measured by inking the animal's paws, dropping it onto a sheet of paper, and measuring the distance between the
footprints (Edwards and Parker, 1977). Equilibrium and muscle coordination can be measured by the length of
time a rat can maintain itself on a rotating rod (Bogo et al.,
TABLE 4-6 End Points That Might be Included in a Functional Observational Batterya
In Home Cage and Open Field Manipulative Physiologic
Postureb(D) Ease of removal(R) Body temperature (I)
Convulsions and tremorsb(D) Ease of handling(R) Body weight (I)
Palpebral closureb(D) Palpebral closureb(R)
Lacrimationb(R) Approach responseb(R)
Piloerectionb(Q) Click responseb(R)
Salivationb(R) Tail-pinch responseb(R)
Vocalizationsb(Q) Righting reflex(R)
Rearingb(C) Landing foot splay(I)
Urinationb(C) Forelimb grip strengthb(I)
Defecationb(C) Hindlimb grip strength(I)
Gaitb(R) Pupil response(Q)
Arousalb(R)
Mobility(R)
Stereotypy(D)
Bizarre behavior

aType of data yielded: D, descriptive; R, rank-order, scalar, Q, quantal data; I, interval, continuous; C, count.
bSpecifiedin EPA guidelines (1986).
Source: Moser (1989).

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TESTING FOR NEUROTOXICITY 72

1981). Tremor can be characterized with automated devices (Gerhart et al., 1985; Newland, 1988). Many studies
have shown that the observations commonly included in FOBs can detect the effects of known neurotoxicants
(Haggerty, 1989; Kulig, 1989; Moser, 1989; O'Donoghue, 1989).

Motor Activity
Motor activity includes a broad class of behaviors involving coordinated participation of sensory, motor,
and integrative processes (MacPhail et al., 1989). Motor activity has several advantages for testing: it is
noninvasive; motivational procedures, such as food deprivation, are not needed to produce it; and its recording is
usually automated, and that reduces experimenter-animal interactions (Maurissen and Mattsson, 1989). Many
studies have shown that motor activity can be affected by psychoactive and neuroactive chemicals (Reiter and
MacPhail, 1979; Tilson, 1987; MacPhail et al., 1989). MacPhail et al. (1989) evaluated motor-activity measures
and found them highly consistent across replications. The sensitivity of motor-activity measures in detecting
reproducible neurotoxic effects is generally comparable with that of more sophisticated measures of
neurobehavioral function.
Although motor-activity measures are often used to identify neurotoxic chemicals, they have disadvantages,
particularly with regard to specificity of adverse effects on the nervous system. It has been argued that the results
of motor-activity tests alone lack specificity and do not often provide information useful for later testing or
characterization (Maurissen and Mattsson, 1989). However, specificity might not be as important in hazard
identification as consistency and sensitivity. Motor-activity measurements are commonly used in conjunction
with FOBs.

Schedule-Controlled Operant Behavior

Schedule-controlled operant behavior (SCOB) involves the maintenance of behavior (performance) by
intermittent reinforcement. Different patterns of behavior and response rates are controlled by the relationship
between response and later reinforcement. SCOB affords a measure of learned behavior and is useful for
studying chemical-induced effects on motor, sensory, and cognitive function.
The primary end points for evaluation are agent-induced changes in response rate or frequency and the
temporal pattern of responding. Response rate is usually related to an objective response, such as a lever press or
key peck, and differs according to the schedule of reinforcement. Response rates are expressed per unit of time.
For some classes of chemicals, the direction of an effect on response rate can differ between low and high doses.
Agent-induced changes in temporal pattern of responding can occur independently of changes in rate and require
analysis of the distribution of responses relative to the reinforcement schedule.
SCOB has been used to study the effects of psychoactive drugs on behavior and is sensitive to many
neurotoxicants, including methylmercury, solvents, pesticides, acrylamides, carbon monoxide, and organic and
inorganic lead (see, for example, MacPhail, 1985; Tilson, 1987; and Rice, 1988). The experimental animal often
serves as its own control, and the procedure provides an opportunity to study a few animals extensively over a
relatively long period. SCOB typically requires motivational procedures, such as food deprivation; and training
sessions are usually required to establish a stable baseline of responding. Because of its sensitivity to neuroactive
chemicals, SCOB has greater potential for use in hazard characterization than in hazard identification.
Many of the behavioral tests, and particularly

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TESTING FOR NEUROTOXICITY 73

the FOB, have been developed and validated with well-characterized neurotoxicants. It is much easier to
interpret the results of behavioral tests when a large body of information already exists on a particular substance;
one can use this information to help interpret the results. It is much more difficult to interpret the results of
screening tests on a new product on which very little information is available.

Specialized Tests of Neurologic Function

Neurotoxicants produce a wide array of functional deficits, including motor, sensory, and learning-memory
dysfunction. Many procedures have been devised to assess relatively subtle changes in those functions; hence
their applicability to hazard characterization. Specialized tests and agents that affect them have been reviewed
recently (WHO, 1986; Tilson, 1987) and are discussed only briefly below.

Motor Function
Motor dysfunction is a common neurotoxic effect, and many different types of tests have been devised to
measure time-and dose-dependent effects. Anger (1984) reported 14 motor effects of 89 substances, which could
be classified into four categories: tremor, convulsions, weakness, and incoordination. Chemical-induced changes
in motor function can be determined with relatively simple techniques and may be used as a component of an
FOB.
Several procedures have been used to characterize chemical-induced motor dysfunction. An example has
been described by Newland (1988), who trained squirrel monkeys to hold a bar within specified limits (i.e,
displacement) to receive positive reinforcement. The bar was also attached to a rotary device, which allowed
measurement of chemical-induced tremor. Spectral analysis was used to characterize the tremor, which was
found to be similar to those seen in humans exposed to neurotoxicants or with such neurologic diseases as
Parkinson's disease.
Incoordination and performance changes can be assessed with procedures that measure chemical-induced
alterations in force (Fowler, 1987). Animal are trained to receive positive reinforcement by applying force to a
fixed lever. Training can also include maintenance of an appropriate force for a specified period. The accuracy of
performance is sensitive to many psychoactive drugs (Walker et al., 1981; Newland, 1988). Gait has been
measured in rats under standardized conditions and can be a sensitive indication of specific damage to the basal
ganglia and motor cortex (Hruska et al., 1979) as well as damage to the spinal cord and peripheral nervous system.
Procedures to characterize chemical-induced motor dysfunction have not been used extensively in
neurotoxicology. Most of them require pre-exposure training (including alterations of motivational state) of
experimental animals. However, such tests might be useful, inasmuch as similar procedures are often used in
assessing humans.

Sensory Function
Alterations in sensory processes (e.g., paresthesias and visual or auditory impairments) are frequently
reported signs or symptoms in humans exposed to toxicants (Anger, 1984). Several approaches have been
devised to measure sensory deficits. Data from tests of sensory function must be interpreted within the context of
changes in body weight and body temperature. Furthermore, many tests assess the behavioral response of an
animal to a specific stimulus; the response is usually a motor movement

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TESTING FOR NEUROTOXICITY 74

that could be directly affected by chemical exposure. Thus, care must be taken to determine whether proper
controls were included to eliminate the possibility that changes in response to a stimulus were related to agent-
induced motor dysfunction.
Several testing procedures have been devised to screen for sensory deficits. Many rely on orientation or the
response of an animal to a stimulus. Such tests are usually included in the FOB used in screening (e.g., tail-pinch
or click responses). Responses are usually recorded as being either present, absent, or changed in magnitude
(Moser, 1989; O'Donoghue, 1989). The tests would not be suitable to characterize chemical-induced changes in
acuity or fields of perception. Sensory deficits are usually characterized with psychophysical methods, which
study the relationship between the physical dimensions of a stimulus and the behavioral response it generates
(Maurissen, 1988).
One approach to the characterization of sensory function involves the use of reflex-modification techniques
(Crofton and Sheets, 1989). A stimulus of varying intensity is presented before a stimulus that elicits a defined
sensorimotor reflex. If the time between the two stimuli is appropriate, the response to the eliciting stimulus can
be significantly inhibited (i.e., prepulse inhibition). The observation of inhibition is contingent on the ability of
the animal to perceive the presence of the first stimulus. Agent-induced changes in the frequency or threshold
required to inhibit the reflex are taken as possible agent-induced changes in sensory function. Changes in the
ability of the first stimulus to inhibit the reflex must be interpreted within the context of changes in the response
to the eliciting stimulus, i.e., a sensory change is inferred primarily on the basis of an agent's ability to alter the
degree of inhibition of the reflex in the absence of any related change in sensorimotor function. Control runs
should be performed to determine the basal response without the initial stimulus. Prepulse inhibition has been
used only recently in neurotoxicology (Fechter and Young, 1983) and can be used to assess sensory function in
humans, as well as in experimental animals.
Various behavioral procedures require that a learned response occur only in the presence of a specific
stimulus. Chemical-induced changes in sensory function are determined by altering the physical characteristics
of the stimulus (e.g., magnitude or frequency) and measuring the alteration in response rate or accuracy. In an
example of the use of a discriminated conditional response to assess chemical-induced sensory dysfunction,
Maurissen et al. (1983) trained monkeys to report the presence of a vibratory or electric stimulus applied to the
fingertip. Repeated dosing with acrylamide produced a persistent decrease in vibration sensitivity; sensitivity to
electric stimulation was unimpaired. That pattern of sensory dysfunction corresponded well to known sensory
deficits in humans. Discriminated conditional response procedures have been used to assess the ototoxicity
produced by toluene (Pryor et al., 1983) and the visual toxicity produced by acrylamide (Merigan et al., 1982).
Procedures to characterize chemical-induced sensory dysfunction have been used often in neurotoxicology.
As in the case of most procedures designed to characterize toxicity, training and motivational factors can be
confounding factors. Many tests designed for laboratory animals can be applied to humans.

Learning and Memory

Learning and memory disorders are neurotoxic effects of great importance. Impairment of memory is
reported fairly often by adult humans as a consequence of toxic exposure. Behavioral deficits in children have
been caused by lead exposure (Smith et al., 1989). And it is hypothesized (Calne et al., 1986) that chronic low-
level

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Environmental Neurotoxicology

TESTING FOR NEUROTOXICITY 75

exposure to toxic agents can have a role in the pathogenesis of senile dementia.
Learning is defined as a lasting change in behavior, memory as the persistence of learned behavior.
Alterations in learning and memory must be inferred from changes in behavior. However, changes in learning
and memory must be separated from other changes in behavior that do not involve cognitive or associative
processes (e.g., motor function, sensory capabilities, and motivational factors), and an apparent toxicant-induced
change in learning or memory should be demonstrated over a range of stimuli and conditions. Before it is
concluded that a toxicant alters learning and memory, effects should be confirmed in a second learning
procedure. It is well known that lesions in some regions of the brain can facilitate some types of learning by
removing behavioral tendencies (e.g., inhibitory responses due to stress) that moderate the rate of learning under
normal circumstances. A discussion of learning procedures and examples of chemicals that can affect learning
and memory have appeared in recent reviews (Heise, 1984; WHO, 1986; Tilson, 1987; Peele, 1989).
One simple end-point procedure to measure in assessing learning and memory is habituation, which is
defined as a gradual decrease in the magnitude or frequency of a response after repeated presentations of a
stimulus. A toxicant can affect habituation by increasing or decreasing the number of stimulus presentations. An
example of chemical-induced effects on habituation can be found in a study by Overstreet (1977), who reported
that diisopropyl fluorophosphate (DFP), a choline acetyltransferase inhibitor, had no effect on the response to a
novel stimulus; with repeated presentations of the stimulus, however, DFP-treated rats habituated slower than
controls. Habituation is a very simple form of learning and would be perturbed by a number of chemical effects
not related to learning.
A general approach to studying the effects of a chemical on learning and memory involves the pairing of a
novel stimulus with a second stimulus that produces a known, observable, and quantifiable response (i.e.,
classical ''Pavlovian'' conditioning). The novel stimulus is known as the conditioned stimulus, and the second,
eliciting stimulus the unconditioned stimulus. With repeated pairings of the two stimuli, the conditioned stimulus
comes to elicit a response similar to the response to the unconditioned stimulus. The procedure has been used in
behavioral pharmacology and, to a lesser extent, in neurotoxicology. Neurotoxicants that interfere with learning
and memory would alter the number of presentations of the pair of stimuli required to produce conditioning or
learning. Memory would be tested by determining how long after the last presentation of the two stimuli the
conditioned stimulus would still elicit a response. For example, Yokel (1983) dosed rabbits repeatedly with
aluminum and found that exposed rabbits learned the conditioned eyeblink response more slowly than controls;
such effects on establishing the conditioned relationship between the two stimuli were seen in the absence of
aluminum-induced alterations in sensitivity to the unconditioned stimulus or ability to respond. Other classically
conditioned responses known to be affected by psychoactive or neurotoxic agents are the conditioned taste
aversion (Riley and Tuck, 1985) and conditioned suppression (Chiba and Ando, 1976).
Other procedures use instrumental learning, which involves the pairing of a response with a stimulus that
increases the probability of future response through reinforcement. Response rate can be increased by using
positive reinforcement or removing negative reinforcement. Learning is usually assessed by determining the
number of presentations or trials needed to produce a defined frequency of response. Memory can be defined
specifically as the maintenance of a stated frequency of response after initial training. Neurotoxicants may
adversely

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TESTING FOR NEUROTOXICITY 76

affect learning by increasing or decreasing the number of presentations required to achieve the designated
criterion. Decrements in memory may be indicated by a decrease in the probability or frequency of a response at
some time after initial training. Toxicant-induced changes in learning and memory should be interpreted within
the context of possible toxicant-induced changes in sensory, motor, and motivational factors. An example of a
test based on instrumental learning is the repeated-acquisition procedure. It requires that an animal learn how to
solve a series of problems that vary from session to session. The results of the repeated-acquisition procedure are
affected by carbon monoxide and microwave exposures (Schrot et al., 1980, 1984). Other examples of
instrumental learning procedures used in neurotoxicology are passive and active avoidance, Y-image avoidance,
spatial mazes (radial-arm maze), and delayed matching to sample (see Heise, 1984; WHO, 1986; Tilson, 1987).
One problem with almost all measures of learning and memory in animals is that it is sometimes difficult to
extrapolate the procedures to human testing or to predict analogous effects in humans. However, some tasks,
such as the conditioned eyeblink test and delayed matching to sample, can be easily adapted for use with humans.

Neurophysiologic Procedures
Several clinical neurophysiologic tests have been applied to exposed animals for diagnostic or prognostic
evaluation of the nervous system. The tests are noninvasive or only minimally invasive, readily adapted to
longitudinal studies, and capable of detecting and measuring neurologic manifestations of neurotoxicity,
regardless of the initiating mechanism. When properly chosen, they can probe the functional status of
particularly affected portions of neuronal networks (such as reflexes and evoked responses). Their results are
reproducible within the constraints of biologic variation and the skill of the experimenter. Although of
considerable value in risk assessment, they are nonetheless post hoc studies that yield results that reflect varied
and often unknown exposures. The use and limitations of the tests have recently been reviewed by LeQuesne
(1987).
Under conditions of the same locus and mechanism of neurotoxic action (such as demyelination),
neurophysiologic testing in experimental animals appears to produce reliable indicators of what might be
expected after similar exposure of other species, including humans. That can be illustrated by a brief comparison
of data from clinical and experimental studies of acrylamide neurotoxicity. Clinical features of the neuropathy
included ataxia, diminished or lost tendon reflexes, and sensory complaints (LeQuesne, 1980). Nerve-conduction
velocities in affected patients were normal or (in a very few patients) just below normal (Takahashi et al., 1971).
Studies in baboons revealed a similar picture of weakness and ataxia, also with minimal decrements in
conduction velocities (Hopkins and Gilliatt, 1971). Cats with acrylamide intoxication had normal sensory and
motor nerve-conduction velocities (Lowndes and Baker, 1976; Lowndes et al., 1978a), but were markedly ataxic
and hyporeflexic. The close temporal association between onset and severity of signs of intoxication and
impairment of static and dynamic properties of muscle spindles (Lowndes et al., 1978b) revealed that
proprioceptive defects form the basis for the loss of tendon reflexes and almost certainly contribute to the ataxia.

Nerve-Conduction Studies
A number of end points can be recorded, but the critical variables are velocity of nerve conduction (e.g., in
meters per second), response amplitude, and excitability, which is usually a measure of the time required

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before a nerve can generate another impulse (refractory period). Any long-term change in peripheral nerve
function is likely to be accompanied by lesions observable with a light microscope. Although chemical-induced
changes in the velocity of nerve conduction are generally rare, they are most likely to occur with decreases in
body temperature and are associated with severe behavioral dysfunction. Changes in amplitude are often
observed after exposure to neurotoxicants, and can be associated with sensory or motor deficits. Changes in
excitability are likely to cause altered sensory thresholds, altered behavioral reaction times, and altered
susceptibility to seizures. Nerve-conduction responses depend on temperature, and studies should control for this
variable.
The utility of maximal motor or sensory nerve-conduction velocities depends on the neuropathologic
expression of neurotoxicity. Exposure resulting in demyelination markedly reduces maximal conduction
velocity. In carbon disulfide neurotoxicity, sensory conduction velocities declined earlier than motor conduction
velocities (Vasilescu, 1976). Similarly, neuropathies resulting from γ-diketone precursors (n-hexane and methyl-
n-butylketone) and lead produce paranodal or segmental demyelination that is manifest in diminished conduction
velocities (Spencer et al., 1975; Korobkin et al., 1975; Buchthal and Behse, 1979). Toxic neuropathies with
axonal degeneration as hallmarks exhibit mild or no decrease in maximal conduction velocities; neuropathies
resulting from γ-diketone precursors (LeQuesne and McLeod, 1977), organophosphates (Hierons and Johnson,
1978; Shiraishi et al., 1983), or acrylamide (Fullerton, 1969; Takahashi et al., 1971) are in this category, as are
mild neuropathies caused by methylbutylketone (Allen et al., 1975).
Electrodiagnostic tests that rely on amplitudes of compound action potentials (muscle or sensory nerve
action potentials) provide sensitive indexes of peripheral nerve abnormality. The tests depend on the number and
synchrony of conducted impulses, which are reduced when there is a loss of contributing fibers or a dispersion
due to conduction slowing. Sensory nerve action potentials are the more widely studied and are reportedly
decreased in intoxication with acrylamide (Takahashi et al., 1971), Dipterex (Shiraishi et al., 1983), or
methylmercury (Murai et al., 1982).

Sensory Evoked Potentials

Evoked-potential methods assess the neurophysiologic response of a particular sensory system to a
particular stimulus (e.g., light or sound) and can help to detect and characterize neurotoxicity (Mattsson et al.,
1989). The most commonly measured end points are peak latency (time from the onset of the stimulus to the
peak response) and peak amplitude. Changes in latency imply changes in conduction velocity or in synaptic
transmission. Acute exposures are likely to change latencies by changing body temperature. Long-term changes
in latency might actually reflect alterations in conduction, which could be produced by alterations in
myelination. When such changes are interpreted to reflect central, rather than peripheral, nervous system
involvement, they might indicate an irreversible effect.
Changes in amplitude of peaks can imply changes in the number of nerve cells. Several peaks are averaged
across numerous trials (stimulus presentations), and a change in average peak magnitude might be associated
with an increase in variability of measurement. The importance of an amplitude change depends on the particular
peak and sensory system under investigation. Changes in the evoked potential are likely to be associated with
agent-induced sensory changes, which can be confirmed at the behavioral level.
Sensory evoked-potential techniques are used extensively in human neurotoxicity studies and are adapted
easily to laboratory

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animals. The biologic basis of the electro-physiologic response is generally well understood and can be collected
relatively quickly (Mattsson et al., 1989). The utility of sensory evoked potentials in neurotoxicity evaluation has
been demonstrated, for example, in the case of toluene-induced auditory impairment (Rebert et al., 1983;
Mattsson and Albee, 1988) and hexachlorophene-induced somatosensory dysfunction (Mattsson et al., 1989).
However, the time and expertise required to implant electrodes and interpret the data and the recording
equipment required reduce the likelihood that this technique will be used routinely for identification of chemical
hazards.

Electroencephalography
Electroencephalographic (EEG) analysis is used widely in clinical settings for the diagnosis of neurologic
disorders and less often for the detection of subtle toxicity-induced dysfunction. However, it is well known that
dissociation between the EEG pattern and behavior can occur, particularly in chemical-treated animals. Changes
in the pattern of the EEG can be elicited by stimuli that produce arousal (e.g., light and sound), by normal sleep,
and by anesthetic drugs. In studies with toxicants, changes in EEG pattern can precede alterations in other
objective signs of neurotoxicity. Experiments with the EEG must be done under highly controlled conditions,
and the data must be considered case by case. EEG abnormalities have been noted in patients exposed to carbon
disulfide (Seppalainen and Haltia, 1980) and alkylmetals (Cossa et al., 1959; Fortemps et al., 1978).

Neurochemical Procedures
Functions within the nervous system depend on synthesis of, release of, and receptor activation by specific
neurotransmitters among specific groups of neurons. Many neurochemical end points could be measured in
neurotoxicologic studies, including effects on neurotransmitters (e.g., changes in synthesis, transport, storage,
release, re-uptake, or degradation of serotonin, norepinephrine, acetylcholine, or amino acids) and their
receptors; effects on lipids, glycolipids, glycoproteins, or other constituents of neural membranes; effects on ion
channels or membrane-bound enzymes that regulate neuronal activity; and effects on metabolic processes
necessary to maintain neural activity (e.g., changes in glycogen and creatine phosphate concentrations, glucose
availability, and mitochondrial structure and function). The large number of possible neurochemical effects is not
amenable to the development of a battery of neurochemical tests to screen large numbers of chemicals. Results
of in vivo tests at the behavioral or neurophysiologic level can suggest mechanistic hypotheses to test with
biochemical and neurochemical end points (Mailman, 1987).
Neurochemical effects of neurotoxicants have been investigated in many laboratories (WHO, 1986;
Mailman, 1987). Those of lead, for example, have been extensively studied in animals (Silbergeld and Hruska,
1980; Winder and Kitchen, 1984). Attempts have been made to use the results of these studies to further the
understanding of lead-induced neurotoxicity in humans. For example, Silbergeld and Chisolm (1976) studied
monoamine metabolites in urine of children exposed to lead and found a correlation between blood lead content
and 24-hour urinary excretion of the dopamine metabolite homovanillic acid (HVA). HVA was measured before
initiation of chelation therapy and within a week after the children had been removed from lead-contaminated
environments. Over the long term, urinary HVA content was reduced, as was blood lead content, although both
blood lead and urinary HVA remained higher in treated lead-exposed children than in age-matched controls. In
contrast with dopamine, some

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neurotransmitters reported to be altered by lead in animal models—such as GABA and enkephalin—are less
amenable to clinical measurement, because they require an invasive procedure to collect cerebrospinal fluid.
Neurochemical procedures appear to have great potential in development of biologic markers of
neurotoxicant-induced exposure. The organophosphorus (OP) compounds have stimulated development of such
biologic markers; an erythrocyte acetylcholinesterase assay has been used to monitor human exposure to OP
compounds that cause cholinergic poisoning, and assays of lymphocyte neurotoxic esterase (NTE) activity have
been used to detect exposure to OP chemicals that cause delayed neurotoxicity.
Recently, hemoglobin adducts (such as the lysyl amino groups of hemoglobin modified by γ-diketones)
have been evaluated as possible biologic markers of exposure to some neurotoxicants. Because of the long
lifetime of the erythrocyte (120 days in humans) and because erythrocytes become more dense as they age,
density-gradient centrifugation of blood samples allows differentiation between recent and earlier exposure to
the neurotoxicant. The same sample of blood can be used to detect protein cross-linking, because spectrin dimers
eluted from membranes of lysed erythrocytes will be stable if cross-linking occurred in vivo (St. Clair et al.,
1988).
A variety of neurochemical probes of neuronal and glial integrity could be used to evaluate the possible
neurotoxic effects of a candidate chemical in experimental animals (O'Callaghan, 1988). As discussed with
regard to in vitro systems, these neurochemical assessments could deal with very general indicators, such as total
protein or DNA, in a given region of the nervous system. Alternatively, more specific indicators—such as
transmitter-related enzymes, various receptors, or the state of phosphorylation of axonal proteins—could be
compared in control and experimental groups of animals. One much-used assay detects glial fibrillary acidic
protein (GFAP). The immunoassay for GFAP revealed areas of neuronal damage detectable with silver stain but
not with routine neuropathologic studies. Agents and conditions that increase brain GFAP include trimethyltin,
triethyltin, methylmercury, cadmium, MPTP, stab wounds, and aging. The use of GFAP as a sensitive and
specific markers of central neuronal damage is simple and provides quantitative data that can be useful in risk
assessment. The choice of indicators should be made in the light of any structure-activity relationship or other
information that suggests an optimal test strategy for a given agent.

Neuroendocrine Interactions
Neuroendocrine toxicology is an emerging field. Despite the relative paucity of formal studies, suspicion
that toxicants can affect neuroendocrine processes has been voiced since the earliest case studies of poisoning.
For example, reports of lead poisoning in historical times comment on the short stature of children exposed to
lead (Schwartz et al., 1986). Similar observations have been made in cases of infantile methylmercury
intoxication (Matsumoto et al., 1965; Takeuchi, 1977). Anecdotal reports suggesting neuroendocrine toxicity
have appeared in conjunction with pesticides, drugs of abuse, medicinal agents, and industrial residues.
However, there has been no systematic review of neuroendocrine toxicity in conjunction with any of those
classes of chemicals.
Neuroendocrine toxicology should receive greater attention than it does now. Evidence is growing that
endocrine organs are important targets for many toxicants and that deleterious effects have not been recognized,
simply because no one has looked for them. For instance, prenatal exposure to TCDD at very low concentrations
alters sexual behaviors

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in male rats by an endocrine-dependent mechanism related to early hormone imprinting of the CNS (Moore et
al., 1991). It is important to note that, because damage to the neuroendocrine system might be expressed as
dysfunction or injury at the target or end organ, the neural contribution to the effect could be misinterpreted. For
example, some chemicals described as reproductive toxicants exert their primary effect on the pituitary-
reproductive axis (NRC, 1989b). Similarly, growth effects of teratogens can result from injury to the
hypothalamic neurons that control release of growth hormone from the pituitary, rather than from direct effects
of the agents on somatic tissues (Rodier et al., 1991).
Many areas of the brain responsible for regulating hormone production or specific hormonal response are
very small and specialized. Groups of only several thousand or even hundreds of cells can be responsible for
major endocrine functions. Although the size of those specialized regions in the brain makes them difficult to
identify during routine histologic assessments, their dysfunction can have major consequences. The regulation of
the endocrine system has been well characterized, so routine techniques are available to identify endocrine
perturbations and to pinpoint their sites of origin; these should be more widely used.
The ability to detect disturbances in the neuroendocrine axis by provocative challenge, by
radioimmunoassays of plasma from peripheral blood, or by end-organ evaluation has implications for risk
assessment. The techniques are somewhat less invasive than autopsy or brain biopsy, are more quantitative than
many behavioral procedures, and (under optimal circumstances) can pinpoint sites of neurotoxic injury with a
high degree of specificity. The neuroendocrine system is an interactive axis involving many structures, and a
toxicant can act on end organ, brain, or both.

Neuropathology
Neurologic lesions can be classified according to their characteristics or site of action. Lesions can be
classified as neuronopathies (changes in the neuronal body), axonopathies (changes in the axons),
myelinopathies (changes in the myelin sheaths), dendropathies (changes in the dendrites), and peripheral
neuropathies (changes in the peripheral nerves). For axonopathies, a more precise location of the changes should
be described (i.e., proximal or distal; central or peripheral).
In general, chemical effects lead to two general types of primary alterations: the accumulation, proliferation,
or rearrangement of structural elements (e.g., intermediate filaments and microtubules) or organelles (e.g.,
mitochondria); and the breakdown of cells, in whole or in part. Partial cellular breakdown can be associated with
regenerative processes that can follow chemical exposure.
Most neurotoxic damage is evident at the microscopic level, but gross changes in structure can be reflected
in significant changes in the weight of the brain. Weight changes, discoloration, discrete or massive hemorrhage,
and obvious lesions are clear indicators of adverse effects.
Careful histologic analyses form the cornerstone of our knowledge of toxic neuropathies that alter nervous
tissue structurally, and the changes can be unambiguously correlated with clinical outcomes and with aging.
Many structural changes have been identified neurohistologically. Methylmercury produces selective granule
cell loss in the cerebellum (Hunter and Russell, 1954) and axonal degeneration in sensory ganglion cells
(Cavanaugh and Chen, 1971). Degeneration of the long sensory and motor axons in both the central and
peripheral nervous systems is produced by OP compounds, acrylamide, and 2,5-hexanedione (Cavanaugh, 1964;
Spencer and Schaumburg, 1977a,b). Myelin loss has been seen as a result of diphtheria with no axon loss
(Cavanaugh

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and Jacobs, 1964) and as a result of exposure to lead with only slight loss (Fullerton, 1966); myelin
vacuolization in the CNS and PNS has been caused by hexachlorophene (Towfighi et al., 1974) and in central
neurons by triethyltin (Aleu et al., 1963).
A careful examination of properly preserved and prepared tissues (which are generally available only from
experimental animals) establishes or rules out structural damage, identifies the most vulnerable sites within the
nervous system, and traces the temporal evolution of pathologic changes. The nervous system can be
systematically assessed by sampling its various areas or particular tissues, as recommended by different expert
groups (Tables 4-7 and 4-8). Table 4-7 reflects a recommendation by the World Health Organization (1986), and
Table 4-8 contains a somewhat expanded sample of tissues believed adequate to identify most known disease
states and vulnerable areas of the nervous system with light microscopy (Spencer et al., 1980). As the number of
anatomic regions assessed increases, so do the labor and resource requirements of the assays. Sampling all
regions listed in Tables 4-7 and 4-8 could be quite costly. Fuller characterization of tissue damage and
identification of mechanisms of effect at the structural level often require electron microscopy guided by light
microscopy (WHO, 1986).

Imaging Procedures
Available neuroimaging procedures to assess structural changes in the brain can be divided into three major
categories: computed axial tomography (CAT), magnetic resonance imaging (MRI) and nuclear magnetic
resonance spectroscopy (MRS), and positron-emission tomography (PET).
The first two categories of neuroimaging procedures can be carried out with current clinical techniques. The
CAT scan combines computerized imaging techniques with radiologic tomography to develop a detailed image
of the brain. Because the technique is noninvasive, it has gained widespread clinical acceptance in the last 15
years. Contrast agents can be used to enhance visualization of some abnormalities, such as tumors or vascular
malformations.
The newer MRI technique is similar to CAT, but, rather than using ionizing radiation, recreates anatomic
information with nuclear magnetic moments. It requires a high-intensity magnetic field, and it monitors changes
in nuclear magnetic moment relaxation times after the magnetic field has been applied. MRI is unique in that it
uses three kinds of data to characterize anatomic information: nuclear-specific data, i.e., data specific to the
nuclear magnetic moment; imaging-specific data, i.e., data specific to the way MRI is being performed; and
tissue-specific data, i.e., data specific to characteristics of
TABLE 4-7 Areas of the Nervous System to be Used in Neuropathologic Evaluation
Primary Areas Secondary Areas
Cerebellum Olfactory epithelium and tubercles
Brain stem Inner ear and labyrinths
Pituitary gland Plantar nerves and skin receptors
Eye, oculomotor muscles, optic nerve Autonomic ganglia
Spinal cord Nerves and organs of innervations
Sensory ganglia
Sciatic nerve (branches from vertebral column to ankles and selected muscles it
innervates)

Adapted from WHO (1986).

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TABLE 4-8 Tissues of the Nervous System to be Used in Neuropathologic Evaluation

Central Tissues Peripheral Tissues
Motor cortex Gasserian ganglion
Visual cortex Lumbar dorsal root ganglia
Subfornical organ Lumbar dorsal root
Area postrema Lumbar ventral root
Lateral geniculate body Proximal sciatic nerve
Optic tract Tibial nerve at knee
Optic nerve Tibial nerve, calf muscle branches
Retina Plantar nerves at ankle
Cerebellar vermis Gastrocnemius muscle
Gracile nucleus Lumbrical muscle spindles
Cuneate nucleus Lumbrical neuromuscular junctions
Gracile tract (T6, L5)
Ventromedial tract (medulla oblongata, T6, L5)
Dorsal spinocerebellar tract
(medulla oblongata)
Hypoglossal nucleus
Descending tract of V
Lumbar cord, anterior horn
Mammillary bodies
Hypothalamus
Hippocampus
Striatum
Substantia nigra

Adapted from Spencer et al. (1980).

the tissues that are being imaged. The three kinds of data provide some of the unusual power of MRI to
define precisely the pathophysiology of CNS disease. The procedure also provides a detailed image of the brain
and might be even safer than CAT, in that there is no exposure to radiation.
The use of nuclear MRS can provide a more detailed evaluation of the biochemical status of the CNS. The
kinds of biochemical information obtained from nuclear MRS include rates of energy generation and use,
substrate metabolism, blood flow, and tissue integrity. Taken together, the uniqueness of the anatomic and
biochemical data provided by MRI and MRS suggests that the combination of the two techniques will become a
powerful adjunct to neurotoxicology both in humans and in experimental animals.
PET is potentially much more sensitive. It represents a major advance in the ability to monitor some types
of neurotoxicity by labeling compounds with positron-emitting elements (e.g., manganese and fluorine) so that it
is possible to observe alterations in neurochemistry, as well as in receptors in the brain of living subjects. Before
the introduction of PET scanning, such examination was possible only in postmortem material.
The potential value of PET was recently recognized in the study of a group of heroin addicts exposed to
MPTP. A number of addicts mistakenly took synthetic heroin contaminated with the dopaminergic neurotoxicant
MPTP; some became severely parkinsonian, but many did not. By using positron-emitting labeled 6-fluorodopa,
striatal dopamine deficiency was shown in those who were exposed to MPTP, but were not symptomatic (Calne
et al., 1985]. That finding

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has already led to the hope that similar techniques will make it possible to predict idiopathic Parkinson's disease.
Another PET scanning technique involves the evaluation of cerebral glucose use. Scanning revealed
decreased glucose metabolism in the brains of people poisoned by ingesting mussels contaminated with domoic
acid (Teitelbaum et al., 1990). In patients with Alzheimer's disease, some areas of the cerebral cortex use less
glucose (Friedland et al., 1989). Toxicants, whether their effects are selective or diffuse, are likely to decrease
oxygen consumption and glucose use at their target sites. That might provide a way of monitoring the
physiologic consequences of exposure to a wide variety of neurotoxicants.
The potential applications of PET procedures are enormous. If a neurotoxicant damages one or more areas
of the brain, causing a change in neurotransmitter concentrations, it might be possible to measure the effects of
the toxicant early, while they are still subclinical. The techniques can be used in experimental animals for
longitudinal studies, because it is not necessary to kill them at different times to follow the evolution of
neurotoxic effects. Ligands have been developed to image various receptor populations throughout the brain.
Thus, it could eventually be possible to examine the effects of neurotoxicants on a wide variety of
neurotransmitter systems and their corresponding receptor populations in living subjects.
Both CAT and MRI techniques can be used to demonstrate gross structural alterations in the brain. They
might be useful in defining any neurotoxic process that causes substantial atrophy in the CNS. A specific
example is the cerebellar and cortical atrophy that can occur after exposure to organic solvents—glue sniffer's
encephalopathy. The limitation of the procedures is that they require major, if not massive, loss of neuronal
populations before atrophy is visible. Other clinical measures would probably demonstrate abnormalities much
earlier in the course of neurotoxic exposure.
Imaging procedures are not useful for assessing spinal-cord, nerve, and muscle damage. They are also
expensive, and there is only a small data base available on their use in neurotoxicology.

Modifying Variables
Not all people exposed to a given neurotoxicant respond similarly. The determinants of susceptibility are
not always well understood. Some variables, however, are known to modify responses to neurotoxic agents and
must be taken into account in screening and any testing at higher tiers. The importance of age at exposure and at
testing has already been mentioned in Chapter 2. This section indicates how age, sex, and genetic background of
subjects should be considered in neurotoxicity testing.

Age at Exposure
Organisms can differ in sensitivity to neurotoxicants at different ages. Developing organs, for example,
resist damage from low oxygen concentrations (a condition that is normal in utero). But, because of their high
mitotic activity, they are more sensitive to antimitotic agents, such as x rays. Some
TABLE 4-9 Tests Used in NCTR Collaborative Study
Auditory-startle habituation
1-hour activity in figure-8 maze
Diurnal activity in figure-8 maze
Activity before and after amphetamine challenge
Visual-discrimination learning
Negative geotaxis
Olfactory discrimination

Source: Buelke-Sam et al. (1985).

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agents (such as diazepam) cause temporary effects in the mature CNS, but permanent effects in the developing
CNS (Kellogg, 1985); others (such as methylmercury) are toxic at every age, but toxic at lower doses in
developing organisms (Spyker, 1975). Some differential effects can be explained on the basis of
pharmacokinetics, but that is not the case for any of the agents discussed above. Thus, it is never safe to assume
that effects observed in adults can be simply extrapolated to developing subjects. Developing organisms are not
always more sensitive to toxic agents than adults; they are sometimes qualitatively different in their responses,
because they are biologically different.
The difference between developing animals' and mature animals' responses to toxicity in other body
systems is already recognized in regulations that require developmental exposure as a separate part of toxicity
testing. The teratology guidelines focus on malformations and provide no information on the integrity of the
nervous system when injuries are less obvious than anencephaly or exencephaly; greater attention to neurologic
effects after developmental exposures is needed. Guidelines being written for the Environmental Protection
Agency are based on recommendations from several groups and on experience with various test batteries.
Butcher and Vorhees (1979) proposed a neurobehavioral test battery for developing animals. Tests were
selected to reflect the guidelines for assessing reproductive toxicity then in force in Japan, Britain, and France.
The battery was validated with vitamin A, a classic behavioral teratogen (Butcher and Vorhees, 1979). The
Butcher and Vorhees battery was later expanded by Vorhees and others to include tests on pivoting, olfactory
orientation, auditory startle, neonatal T maze, figure-8 activity, M-shaped water maze, open-field grip strength,
midair righting, rotorod, crossing parallel rods, jumping down to home cage, elevated maze learning, and E-
shaped water maze (Adams, 1986).
The National Center for Toxicological Research (NCTR), which serves as a research arm of the Food and
Drug Administration (FDA), conducted a major project known as the NCTR Collaborative Study. It was
designed to demonstrate the reliability of classical behavioral-teratology tests (Table 4-9) when administered in
five laboratories. The tests proved to be highly reliable between and within laboratories, and the effects of
methylmercuric chloride on auditory startle were identified in all laboratories (Buelke-Sam et al., 1985).
Developmental neurotoxicology is a relatively new and important field. Because functional development of
offspring can be influenced by maternal toxicity or chemical-induced growth deficits, care must be taken in the
interpretation of data from developmental-neurotoxicity studies. Whether developmental neurotoxicologic
assessments should be done at the hazard-identification or hazard-detection phase is being debated. Certainly
concerns raised about the ability of a chemical to affect reproduction and development would apply to the
postnatal growth and function of organisms exposed during development.

Age at Testing
Age at testing is an important modifying variable, for several reasons. When injury occurs early in life, it
might damage systems that are not fully mature and thus not be fully expressed until later in life. The classic
demonstrations of that phenomenon are in monkeys with lesions, in which some deficits take as long as 2 years
to express their full effect (Goldman, 1971). It is not a characteristic of the damage itself, but of the organism on
which it is imposed. Thus, just as antimitotic injury from radiation to the hippocampus during gestation will not
lead

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to hyperactivity until after puberty (Rodier et al., 1975), a mechanical lesion of the hippocampus will have a
delayed effect (Isaacson et al., 1968). The decrease in norepinephrine content and turnover in the hypothalamus
that follows fetal exposure to diazepam is minor at 30 days in the rat, when the norepinephrine system is
immature, but greater than 50% at 90 days, when the system is mature (Kellogg, 1985).
A separate phenomenon is the increasing effect of lesions with aging—initially suggested to describe
performance after neonatal x irradiation, which differed from performance in controls in preweaning animals,
was near normal in young adults, and then differed again as animals aged (Wallace et al., 1972). Brain weights
of treated subjects were always low, but the difference from control subjects became more pronounced with
advancing age. The same pattern is suspected to account for the late onset of parkinsonian dementia after early
exposure to cycad products. It is possible that a natural decline in function uncovers a longstanding deficit.
It is not easy to introduce aging as a variable in screening tests, because lifetime studies in animals are
expensive. More basic research is needed to determine how often a toxic insult interacts with aging. In the
meantime, it is reasonable to hold test animals until maturity, rather than doing short-term studies. Although
short-term studies might be appropriate for teratology experiments aimed at gross structural changes, they
preclude evaluation of normal adult function of the nervous system.

Sex
Toxicity to sexually dimorphic structures can be sex-specific, and the brain is in some respects a sexually
dimorphic structure. A few examples of how the effects of some toxic agents on the nervous system might
depend on sex are known, although not necessarily understood. Developmental effects of methylmercury on
motor tests were more pronounced in boys than in girls in studies of a fish-eating population (McKeown-Eyssen
et al., 1983), and neonatal male mice show more mitotic arrest and later cerebellar loss in response to methyl-
mercury than do females, even though they do not have higher concentrations in the brain after being treated
(Sager et al., 1984). Some sex differences might be due to pharmacokinetic differences, rather than differences in
the target tissue, but that does not weaken the point that it is reasonable to test both sexes for effects.

Genetic Differences
Specific genetic differences in response to toxic agents have not been commonly demonstrated, and without
some knowledge of mechanisms it is unlikely that susceptible populations can be identified in the screening of
chemicals. However, when such populations are known, they should be evaluated. The classic human example is
the case of the recessive gene for metabolism of the amino acid phenylalanine. People who are homozygous for
the normal allele can clear excess phenylalanine; those who are homozygous for the mutant are severely brain-
damaged by postnatal exposure to the amounts of phenylalanine found in a normal diet. Heterozygotes fall
between the extremes in their metabolic capacity and should be sensitive to high concentrations of
phenylalanine, but not harmed by the amount in a normal diet. Some toxicologists have been concerned about
exposure of this group to the phenylalanine sweetener aspartame. Because most heterozygotes are unaware of
their status with regard to this gene, they cannot avoid products that might be hazardous to them.

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CURRENT REGULATORY APPROACHES

The Organization for Economic Cooperation and Development (OECD) (Brydon et al., 1990) and other
organizations (Tilson, 1990b) have recommended simple and rapid observations of neurobehavioral function of
experimental animals in their home cages or in large open fields in protocols for many subchronic and chronic
tests. Such observations include those of naturally occurring behaviors (e.g., rearing, stereotypy, and urination)
and procedurally easy and quick assessments (e.g., of click response, landing foot splay, and righting reflex)
(Moser, 1989). Other home-cage testing protocols involve more elaborate 24-hour recording of naturally
occurring behaviors (Evans, 1989). It is not clear how effective such relatively general and nonbinding
instructions have been in fostering the detection of neurotoxicity in the context of more general toxicity testing.
Moreover, such approaches do not provide for the detection of alterations in complex behaviors, such as learning
and motor performance, inasmuch as their testing requires special instruments and, depending on the test,
training of the animals.
No U.S. federal laws are directed specifically at protecting humans from systemic toxicity, much less from
neurotoxic hazards specifically, but regulatory agencies have considerable latitude to emphasize specific health
effects (Fisher, 1980; Reiter, 1987; Sette, 1987; OTA, 1990). With the exception of a test for delayed neuropathy
in hens required by EPA for OP esters (Federal Register, 1978), there are no specific regulatory requirements to
evaluate neurotoxicity in any current federal guidelines or regulations.
The testing requirements for neurotoxicity vary among the regulatory agencies. Pre-market testing is
required for pesticides by EPA under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and for
drugs and food additives by FDA, but not specifically testing for neurotoxicity. The Consumer Product Safety
Commission and EPA may require testing of consumer products and industrial chemicals, respectively, but only
if they can justify the need for such testing. In practice, neurotoxicity testing is rarely required, but is usually left
to the discretion of the manufacturer.
Until passage of the Toxic Substances Control Act (TSCA) in 1976, there was no legal mechanism in the
United States for prospective evaluation of the neurotoxicity of new industrial chemicals (OTA, 1990). Under
TSCA (Section 5), there must be some indication that a ''new'' chemical might be neurotoxic before EPA can
require specific tests in the premanufacturing notice (PMN) program, but then EPA can prohibit the chemical
from entering into commerce until the required data are available. Generally, determination of whether additional
testing is needed is based on a structure-activity analysis, because about 50% of PMNs are submitted with no
toxicity data and the toxicity data that are submitted are rarely more than the results of acute lethality or irritation
tests. Of the 6,120 PMNs submitted in 1984–1987, 1,200 underwent additional review (about half these were
found to be associated with unreasonable risk); of those identified for additional review, 220 were suspected of
being potentially neurotoxic (OTA, 1990). The SAR-based approach is now regarded as generally insensitive
and problematic. Moreover, many thousands of potentially neurotoxic compounds developed before passage of
TSCA remain untested. Except in unusual cases, the chemicals were simply registered with EPA and added to
the TSCA inventory. For "old" chemicals (TSCA, Section 4), EPA can issue a test rule and require testing only if
neurotoxicity is suspected, but little progress has been made in establishing final requirements for testing such
compounds or completing evaluation of what new data have been generated (GAO, 1990a,b). Proposed test rules
or consent decrees for 19 chemicals or chemical classes have included requirements

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for neurotoxicity testing (OTA, 1990). FIFRA also has a provision for the retroactive reassessment of the
toxicity of active ingredients in existing pesticides, but this program is progressing slowly (OTA, 1990).
For assessing suspected neurotoxicants, EPA's Office of Toxic Substances has issued a series of test
guidelines (Federal Register, 1985). They include guidelines for testing motor activity and a functional
observational battery. Schedule-controlled operant behavior, neuropathology, peripheral nerve function, and
organophosphate-induced delayed neuropathy are also addressed. A testing guideline for developmental
neurotoxicity is pending. Those guidelines are generally intended to be generic guides as to how to make
particular measurements, rather than lists of specific techniques. Data generated with the guidelines are used in
conjunction with other toxicity data to establish potential risks and to establish reference doses.
FDA follows a pattern similar to that of EPA for assessing potential neurotoxicity. Therapeutic agents are
assessed in preclinical (animal) and clinical (human) studies requiring careful, but unspecified, observations
(Fisher, 1980). Specific neurotoxicity tests are required only for drugs that are intended to be neuroactive; for
other drugs, a review for neurotoxicity is undertaken only if such effects are suspected (OTA, 1990). FDA
requires neurotoxicity testing of food additives only if neurotoxicity is suspected for a particular chemical on the
basis of evidence developed in traditional toxicity testing. However, FDA requires testing when a stated "level of
concern" is reached on the basis of anticipated exposure, potential toxicity, or structure-activity relationships. A
general histopathologic assessment of several sections of the brain, spinal cord, and peripheral nerve is required,
with notation of any observed behavioral abnormalities in the test animals. Positive evidence that is revealed by
the screening tests might, at the agency's option, lead to further assessments, whose nature is decided case by
case. In fact, only instances of neuropathy or clinical signs of neurotoxicity—such as paralysis, tremor, or
conclusions—have an impact on FDA decisions (Sobotka, 1986).
Rodent reproductive studies, which might permit the observation of developmental problems, are ordinarily
conducted for drugs that will be administered to women of child-bearing age (OTA, 1990). On the whole,
however, current tests for developmental toxicity (particularly at FDA) are designed primarily to detect structural
malformations or fetal death, which are appraised after sacrifice before delivery. Even in the short-term Chernoff-
Kavlock protocol (1982) and in the standard protocols for reproductive and developmental effects, the pups are
sacrificed before they acquire much behavior, so opportunities for observation of behavioral teratogenicity and
other developmental deficits are not provided.
Other countries have explicitly prescribed behavioral tests in animals to screen for developmental
neurotoxic effects of new drugs that possibly will be used by pregnant women. To allow a new drug on the
market, Japan requires a fertility study, a teratology study with female exposures, and a perinatal study with
exposures during the last third of gestation and through lactation. In the latter two studies, some offspring are
examined for "development (including behavioral development)." Great Britain also provides general
specifications, requiring tests for "auditory, visual, and behavioral impairment" for premarket screening of new
drugs. The European Economic Community guidelines require assessment of "auditory, visual and behavioral
impairment'' (Vorhees, 1986).
The testing protocol of the National Toxicology Program (NTP) provides for fairly extensive
neuropathologic assessment, but relatively little functional observation. NTP tests have detected the neurotoxic
effects of sodium azide, for instance, because it caused neuronal destruction in chronically treated

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rodents. However, it is generally accepted that finding mild neuronal degeneration, at the light microscopic level,
in the absence of functional or biochemical information, is unlikely to be a sensitive means of detecting
neurotoxicity.

STRATEGIES FOR IMPROVED NEUROTOXICITY TESTING

Screening
Several current proposals for generalized approaches to neurotoxicity testing of new and existing chemicals
recommend a three-tiered scheme (Moser, 1989; Tilson, 1990a; OTA, 1990). The first tier (the screen) consists
of tests for the existence of a neurotoxic hazard. If no evidence of such a hazard is found within the first tier,
toxicity testing ceases. Positive findings in the first tier would terminate the development of many chemicals
being investigated for commercial applications, raising the dilemma of false positives discussed later. For other
apparently neurotoxic chemicals (those already in use and those whose commercial development is nonetheless
deemed promising), neurotoxicity testing would continue to the second tier.
The experiments undertaken in the second tier would characterize the nature of a substance's neurotoxicity,
e.g., its specific target tissue and dose-response and time-response relationships. The selection of specific
experiments in the second tier would be directed by the results of earlier testing. Preliminary second-tier dose-
response assays would establish the potency of the substance for the adverse neurotoxic end points identified in
the first tier. Comparison with the chemical's potency for other types of toxicity and with estimated magnitudes
of human exposure is likely to determine whether extensive additional effort is put into characterizing its
neurotoxicity or whether it progresses to the third tier for investigation of its mechanisms of action in detail. If
other effects would predominate or no neurologic consequences were likely to ensue from plausible exposures,
research resources would be better directed toward other questions.
Testing in the third tier would aim to specify the mechanism by which the chemical produces its neurotoxic
effects. This type of detailed investigation would be appropriate for substances with broad exposure that produce
serious effects (e.g., lead), substances with low or rare exposure that produce a particular noteworthy effect (e.g.,
MPTP), or series of substances that produce lesions of concern (e.g., demyelination). The information obtained
in the investigations would be of use in attempting to control the most serious existing exposure problems and in
establishing SARs for the more efficient prediction of neurotoxicity of chemicals considered in the future.
Despite an apparent simplicity in the design of such a tiered strategy, implementation is not easy; many
questions remain. Determination of the component tests of the first tier is the most crucial aspect of the three-
tiered approach, because truly positive substances will not continue to later tiers unless detected at this point, and
the course of substances that are studied further will tend to be tailored to their performance in earlier tests. The
first tier is the heart of the screening aspect of neurotoxicity testing; the later tiers might produce data of great
value in advancing understanding of neurotoxic processes and how the nervous system operates, but the first tier
is the first line in preventing neurotoxic disease.
How are chemicals ranked for entry into the first tier? Does the first tier represent a single battery of tests to
which all entering chemicals will be subjected, or is the flow of testing in the first tier itself directed by the
profile of results? One way to proceed is to have an initial tier composed of a number of tests that are applied to
all candidate substances. Substances that do not show signs of neurotoxicity are not tested further. Confidence in
such a battery is a function of

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the number of tests in it, the quality of the tests, the size of the experiments (number of subjects, doses, durations
of exposure, etc.), and the degree to which the tests examine a wide range of potential neurotoxic outcomes
(learning, memory, developmental effects, etc.) or other biologic markers that may serve as surrogate measures
for them. The sensitivity of a neurotoxicologic screen might be increased by including additional tests. Many of
the tests were mentioned earlier. Inclusion of some of them might increase confidence in screening procedures,
but at the price of consuming more resources. That suggests a tradeoff between testing an individual substance
thoroughly and processing more substances. Tests that might be considered for addition to the screen in the
future would have to be validated and their incremental value demonstrated.
The neurotoxicologic literature has many references to two-tiered and multitiered testing. In many
instances, what is being discussed is the multistage nature of complete neurotoxic testing, i.e., the validation of
the screening decision and the progression from screening (hazard identification) to experimental neurotoxic
tests designed to establish quantitative dose-response relationships, mechanisms of action, etc. However, other
authors appear to be discussing "multistaged screening" in the strict sense of testing for the purpose of hazard
identification (tier one). If all steps of screening are required for every substance, then the plan is no different
from a proposal for a greatly expanded comprehensive screening battery. However, if multistaged testing is
required of only some substances, then it is important to specify the criteria for deciding which substances should
pass beyond the initial level. Requiring additional levels or types of testing for substances in some chemical
classes is, in effect, merely adding more tests to the first tier.
Some type of functional observational battery (FOB) has most often been proposed for the screening phase
of neurotoxicologic assessment. It appears to be the simplest, most comprehensive option available and it, at a
minimum, should be included in the first tier of neurotoxicity testing. Depending on the particular test,
behavioral end points are often used in the detection phase of testing, because they may be global indicators of
many of the sensory, motor, and integrative processes of the central and peripheral nervous systems.
Observational batteries have been used in rodents by many investigators to assess chemicals for neurotoxicity
and have been recommended by several expert panels (see Tilson, 1990b). The EPA Office of Toxic Substances
prepared guidelines (EPA, 1985) for testing potential neurotoxicants that included an FOB consisting of several
measures that are relatively simple, noninvasive, and quick to perform. A description of the FOB and a summary
of responses produced by representative neurotoxicants were published recently by Moser and colleagues (Moser
et al., 1988; Moser, 1989). Provision should be made for acute and repeated dosing and for observation of the
animals over an extended period. It would also be desirable to measure motor activity and perform limited
neuropathologic tests on the treated animals, as recommended by EPA (1985). Suggestive screening findings or
structural alerts derived from knowledge of SARs should invoke a second level of tests, in which impaired
acquisition of complex behaviors after exposure during development is used as a general indication of
neurodevelopmental toxicity. In addition to tests covering the various aspects of neurotoxic effects of concern for
every chemical, a screening battery might contain specific procedures used only for particular classes of
chemicals, as is the case for testing OP pesticides for possible delayed distal neuropathy.

Validation
The procedures just described are the only general neurotoxicity methods whose validation approaches what
would be necessary

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TESTING FOR NEUROTOXICITY 90

for a routinely applied screen. Before they could be adopted, additional or other tests would have to be
thoroughly appraised for reproducibility and for relevance to human health. Their success in distinguishing the
properties of an extensive battery of nonneurotoxicants and substances that act by a full spectrum of mechanisms
to produce a given end point would have to be demonstrated. Establishing a control battery of appropriate
chemicals is in itself a challenge, given the fairly limited extent to which the universe of chemicals has been
tested for neurotoxicity. Ever-increasing experience might ultimately permit the substitution of a battery of short-
term in vitro assays for longer procedures in whole animals. In addition to its screening capacity, such an in vitro
battery would be expected to yield considerably more information about mechanisms of action than the whole-
animal approach to screening with an FOB and thereby allow much more directed testing in the later tiers.

Priority-Setting and Implementation

The proposed implementation of large-scale screening for neurotoxicity has generated controversy about the
appropriate strategy for approaching the first tier of testing, hazard identification. It is generally agreed that
substances already present in the environment for which there is any indication of neurotoxicity in the form of
structural alerts or preliminary observations should be evaluated thoroughly. Similarly, in the case of chemicals
being considered for future use, such signals of possible neurotoxicity should trigger rigorous testing, if
commercial development is to be pursued. When there are few or no data on which to base a judgment as to
whether a substance (new or in use) presents a neurotoxic hazard, the estimated volume of production and
expected pattern of human exposure (concentrations, frequencies, and numbers of people) are the most
reasonable bases on which to set priorities and begin screening.
As reviewed by the Office of Technology Assessment (1990), federal regulations provide the regulatory
agencies with the authority to demand that toxicity testing, including tests for neurotoxicity, be performed on
chemicals as a requirement for registration or to establish standards for continued use. Federal agencies have not
exercised that authority often, nor followed through completely in analyzing the data that have been generated
(GAO, 1990a,b). Although it might seem obvious that the sponsors of an innovative product should be
responsible for testing it adequately, the question remains of who should be responsible for conducting and
financing the necessary testing on substances already widely present in the environment. It would be desirable if
the burden of neurotoxicity screening not only could be divided and coordinated among U.S. industry,
government, and academy, but also could involve an international effort, as is being encouraged by OECD
(Brydon et al., 1990) for general toxicity testing.

Research Needs
An important question is how in vitro systems can contribute importantly to the neurotoxicologic evaluation
of a broad range of agents. Research is needed to determine whether culture systems can reliably identify known
neurotoxic agents and active related compounds and distinguish them from related, but inactive (or less active)
compounds. Research is also needed to develop in vitro techniques that can identify neurotoxicants that require
metabolic activation to be effective and those which do not, that affect either adult or developing organisms, and
that are injurious after either acute or chronic exposures.
Empirical results of the many in vitro systems available need to be correlated with

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 TABLE 4-10 Proposed Components for Evaluating In Vitro Neurotoxicity Screening Tests
Neurotoxicant Test System
Site of Action Positive Chemical Controls Culture System Test Measures
Environmental Neurotoxicology

Excitable membranes Pyrethroids, ouabain Cell lines: Neuronal survival:

PC-12 cell counts
C-6 tetanus toxin binding
Transmitter systems Anticholinesterases, 6-hydroxydopamine Dissociated cell cultures: Glia:
cerebral cortex glial fibrillary acidic protein
spinal cord
neurons
glia
TESTING FOR NEUROTOXICITY

Axons Acrylamide, 2,5-hexanedione, vincristine Cell cultures: Myelination:

sympathetic ganglion structure
biochemical measures
Dendrites Excitatory amino acids Reaggregates: Transmitter-related:
basal ganglia choline acetyltransferase
glutamic acid decarboxylase
aminergic enzymes
transmitter uptake
agonist binding
Soma Trimethyltin, doxorubicin (Adriamycin), ricin Explants: Structural changes
dorsal root ganglia and spinal
cord, cerebellum, cerebral cortex
Astroglia Mercury Special sensory: Excitable membranes:
retina sodium uptake
organ of Corti calcium uptake
saxitoxin binding

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TESTING FOR NEUROTOXICITY 92

studies in whole animals (indeed, human populations) to establish the neurotoxicologic implications of
findings from in vitro preparations. Substantial efforts will be needed to provide funding and organizational
resources for that type of work; but the work is essential if reliable, efficient test systems are to be developed
reasonably soon. In the long term, mechanistic studies are needed to increase understanding of the fundamental
molecular targets of neurotoxic chemicals. Various schemes can be used to outline a more general approach
(Table 4-10). Accumulated knowledge should eventually allow relatively efficient evaluation of agents that are
likely to fall in one or another general category of neurotoxicant. A general model has emerged from
developmental neurobiology that might similarly allow focusing of neurotoxicologic research with in vitro
systems that capture one or another step in the progression from developing embryo to degenerating adult
nervous system (Figure 4-1).
Routinized screening and mechanistic studies will produce a growing body of detailed neurotoxicologic
data on diverse chemicals that should be intensively analyzed. Ultimately, study of the data should reveal SARs
that are more generally useful than the few that are now considered well established and that will be much better
substantiated and more reliable than most current conjectures.
Much of the controversy over proper testing procedures arises from two intrinsically conflicting objectives:
minimizing the incidence of false positives (substances are incorrectly identified as hazardous) and minimizing
the incidence of false negatives (substances are incorrectly identified as nonhazardous). Both goals are desirable,
but they cannot be maximized simultaneously. Too high an incidence of false positives

Figure 4-1 Biologic markers in the stages between formation and degener ation of neural circuits. Selected markers
are listed under the stages that are named in boxes.

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Environmental Neurotoxicology

TESTING FOR NEUROTOXICITY 93

will waste our resources, worsen the already severe logistic problem of testing thousands of chemicals, and cause
at least some potentially valuable chemicals to be discarded. In the screening situation, however, a high
incidence of false negatives is probably more undesirable. Failure to detect a truly neurotoxic substance will
expose our society to health hazards, with the potential for tragic consequences like those associated with one
false negative, thalidomide. Testing for characterization should expose false positives; the abandonment of an
occasional new chemical on the basis of what are actually false-positive screening results is the likely cost of this
process.
The expense and effort of neurotoxicologic screening would be very great if it were undertaken in isolation.
Considerable practical advantages would accrue if neurotoxicologic screening were integrated with a
multidisciplinary program of general toxicologic and teratologic screening.

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TESTING FOR NEUROTOXICITY 94

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SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS 95

5
Surveillance to Prevent Neurotoxicity in Humans

Need for Surveillance

Neurologic disease and dysfunction after exposure to toxic chemicals in the environment could be
prevented at least in part through premanufacturing and premarket screening (Chapter 4) of all newly
synthesized chemicals, coupled with retroactive screening of chemical substances already in commerce. Such
screening is intended as primary prevention; it is meant to prevent neurologic disease and dysfunction of
environmental origin by identifying new neurotoxicants before they enter the environment and before human
exposure has occurred and then handling them in such a way that human exposure will be as close to zero as
possible.
Current American programs for detecting neurotoxicants, including industrial chemicals, through premarket
testing have serious deficiencies (NRC, 1984). A major problem is that many potentially neurotoxic compounds
that came into use before passage of the Toxic Substances Control Act (TSCA) in 1976 remain untested and are
still not required to be tested. Current neurotoxicity testing procedures developed under the TSCA mandate have
not been standardized for new industrial chemicals (other than pesticides) and are not sensitive enough to detect
the effects of chronic low-dose exposures. Neurotoxins in natural products are most likely to escape testing and
control, in part because of confusion as to which federal agencies are responsible for this control. Primary
prevention is far from perfect; it has sometimes failed in the past, and it cannot be expected to be totally
successful in the future.
The early identification of disease or dysfunction and acting on the identification constitute secondary
prevention. Its goals are to prevent progression of disease in identified cases, to treat early enough to cure if that
is possible, and to prevent the occurrence of additional cases by providing a basis for primary prevention. The
methods of postmarketing secondary prevention of environmental neurotoxic disease include environmental
monitoring and surveillance of the human population to detect neurotoxic disorders as early as possible, which
are combined with efforts to keep potentially toxic exposures to a minimum. Epidemiologic studies and clinical
screening are complementary facets of maintaining alertness so that neurotoxic illness will not go unnoticed.
Secondary prevention constitutes a necessary and critical backup to primary prevention.

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SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS 96

The training of physicians, particularly industrial physicians, to recognize neurotoxic manifestations should
substantially improve secondary prevention efforts.
Secondary prevention is not a substitute for premarket testing of chemicals for potential neurotoxicity;
rather, the data from a secondary prevention program complement and supplement the data derived from
premarket laboratory testing. Although toxicologic information derived from future in vitro and in vivo
investigations might be expected to form an ever-increasing proportion of the neurotoxicity data base,
responsibility to conduct epidemiologic surveillance, to monitor toxic exposures, and to study incidents of
human exposure to neurotoxicants when they occur will remain.
Surveillance and monitoring play oversight or exploratory roles. Fundamentally, they should be viewed as
tools for identifying possible problems-hypothesis-generating exercises. In contrast, classical epidemiologic
studies of populations exposed to neurotoxicants (e.g., Needleman, 1986), clinical trials of pharmaceuticals, and
occasionally laboratory experiments on humans (e.g., Dick and Johnson, 1986; Johnson, 1987) are hypothesis-
testing and will not be considered here in detail.
Effective surveillance systems are characterized by systematic and rapid collection of data, efficient
analysis and evaluation of data, and prompt communication of the results obtained. In discussing general
principles of health surveillance, Foege et al. (1976) noted that dissemination of a program's information often
enhances cooperation in contributing more information to the system.
Surveillance of occupational and environmental health can be divided broadly into systems for monitoring
of exposure (to detect hazards) and monitoring of disease (to detect health responses). Hazard surveillance
systems monitor the use, storage, and environmental release of chemical toxicants and tabulate the numbers of
people exposed to them. Their purpose might be to detect instances of poor industrial hygiene, inappropriate or
excessive use of pharmaceuticals, or improper waste disposal or to generate exposure data to complement
epidemiologic and clinical data on toxic end points. Health surveillance systems record the occurrence of
designated diseases. They might have the general population as their target or focus on specific high-risk groups
(Baker, 1989; Landrigan, 1989). Both types of surveillance are necessary, and both are practiced by public-
health agencies in the United States and elsewhere; the data generated by the two types of systems are
complementary. Surveillance systems can be categorized according to their purpose: to monitor the handling of
known neurotoxicants, to follow the effect of control measures, or to detect previously unrecognized problems.
Recognizing a previously unrecognized problem is most difficult, because no system can be expected to
find reliably what is not specifically sought or to engender confidence that something not specifically looked for
is really not present. Because the measurement of environmental concentrations of chemicals requires discrete
decisions about what substances will be sampled and where and when, it is clear what information has and has
not been sought. Medical monitoring can be somewhat more open-ended, and that raises the possibility of
uncertainties about the definitiveness of negative findings; but the storage and retrieval of observations
necessitate specification of format and limitations on the type of information accepted. In addition, having a data
base truly useful for quantitative estimation of occurrence rates demands that the means by which the data were
ascertained be clearly defined, so that it is known what the sample represents.
A perennial problem in epidemiologic surveillance is that information on the baseline incidence and
prevalence of neurologic diseases is sparse. Thus, it is difficult to detect anything but large outbreaks or clusters

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of neurologic disease. The development of a substantial data set would require significant resources. Much
knowledge about neurotoxicants and neurotoxic disease has, however, come from observation of humans—from
case reports to large-scale epidemiologic studies. Most reports of outbreaks of neurologic diseases have come
from medical clinics, state health departments, industrial medical groups, or consultative programs, such as the
Health Hazard Evaluation Program of the National Institute for Occupational Safety and Health (NIOSH).
Outbreaks have all too often served as late-stage sentinel events identifying the neurotoxicity of chemical
substances. Many times, apparent excesses or clusters of disease have, on close examination, proved to be false
leads.

NEUROBEHAVIOURAL TEST BATTERIES

In Chapter 1 (Table 1-1), the great diversity of forms that neurotoxicity can take was demonstrated by the
extensive list of neurologic signs and symptoms attributed to exposure to various chemicals. Table 5-1 shows
that a temporal pattern of exposure and response can characterize particular chemicals. A complete neurologic
examination and occupational history (Table 5-2) are essential for identifying specific neurologic syndromes
(Johnson, 1987). The examination summarized in Table 5-2 is typically supplemented by tests of CNS function,
which can be as simple as asking the subject to name the current president of the United States or asking the
subject at the end of the examination for three words (e.g., hat, dog, and tree) that were presented at the
beginning of the examination. More sophisticated neuropsychologic tests can be used when there is reason to
suspect CNS problems.
Testing human subjects for neurotoxic injury combines clinicians' efforts to identify the cause of diseases or
disorders and neuropsychologists' or experimental psychologists' use of techniques to assess performance. Field
investigators might adopt a unique battery of tests for each chemical to be assessed or select from among the
5-10 standardized batteries now in use. If latitude is possible in selecting tests, selection is based on signs
reported in the exposed group and the known neurotoxic effects of the chemical under study (or of structurally
related chemicals). That general approach has led to the use of hundreds of tests in various worksite studies
(Johnson and Anger, 1983; Anger, 1990) and has typified NIOSH research (Anger, 1985). Several human
neurotoxicity test batteries have been developed and are being validated. Otto and Eckerman (1985), Anger
(1990), and Johnson et al. (1990) have described the major batteries, including several implemented on
computers.
Finland's Institute of Occupational Health pioneered neurotoxicity testing at the worksite in the 1950s.
Investigators at the institute developed a test battery well adapted to monitoring the main concerns in Finnish
industry (primarily solvents). Their tests (Table 5-3), which have been optimized by factor analysis over the
years, appraise various psychologic domains (Hänninen and Lindström, 1979). The battery is now used in
neurotoxicity evaluations of several worker groups in Finland, including prospective studies on new workers.
An apical test battery (Table 5-3) based on information-processing theory (Marteniuk, 1976) has been
developed and tested in the field in Australia (Williamson et al., 1982; Williamson, 1990). For example, battery
and lead-smelter workers with blood lead concentrations of 1.2–3.9/µmol/L (25–81 µg/dL) demonstrated poorer
performance on all tests than a set of matched controls (Williamson and Teo, 1986).
At a 1983 meeting sponsored by the World Health Organization (WHO), an international team of experts on
testing for

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 TABLE 5-1 Characteristics of Responses to Exposure to Some Neurotoxicants
Neurotoxicant Exposure Pattern Susceptible Development Stage Reversibility of Response Neurologic Category of Typical
Outcome Neurotoxicity
Environmental Neurotoxicology

Acrylamide Chronic Late Reversible, irrerversible Sensory, motor Perepheral neuropathy,

permanent visual loss,
reversible hypoesthesia
n-Hexane Chronic Late Reversible in early stages Sensory, motor Peripheral neuropathy
Carbon disulfide Chronic Late Reversible in early stages, Psychologic, cognitive, motor, Subjective complaints, visual
irreversible sensory loss, peripheral neuropathy
MPTP Acute, chronic Late Irreversible, delayed Motor Parkinsonian syndrome
Methanol Acute Late Irreversible Sensory Blindness from retinal
damage, basal ganglia damage
Ethanol Chronic Prenatal Irreversible Cognitive, motor Fetal alcohol syndrome
SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS

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98
 Organophosphates Chronic Late Reversible, irreversible Psychologic, motor, sensory Acute dysfunction, long-term neuropathy, spinal-cord damage
Lead Chronic Childhood Irreversible Cognitive, motor Reduced scores on developmental tests, peripheral nueropathy,
encephalopathy
Mercury Chronic Early Reversible Psychologic, motor Erethism, tremor
Environmental Neurotoxicology

Methylmercury Chronic Early Irreversible Cognitive, motor Visual, sensory, and motor dysfunction; retarded development
Manganese Chronic Late Irreversible Motor Dystonia, behavioral aberrationsk extrapyramidal dysfunction
Chlorpromazine Chronic Late Irreversible Motor Tardive dyskinesia
Vincristine Chronic Late Reversible Sensory, motor Paresthesia, weakness, peripheral and cranial neuropathy
SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS

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SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS 100

TABLE 5-2 Components of Clinical Neurologic Examination

General appearance
Tremor—arms outstretched and at rest
Ability to sit still
Speech—clear, slurred
Gums, nails, color of skin
Vital signs
Cranial nerves
I Sense of smell tested
II Funduscopic examination, disk margins, visual acuity, visual fields
III Pupil size and reactivity to light; extraocular movements—nystagmus
IV Extraocular movements
V Pin and touch over face, corneal reflex
VI Extraocular movements
VII Symmetry of facial movement
VIII Hearing acuity, vestibular function
IX–X Presence of gag reflex, ability to swallow
XI Symmetry of shoulder bulk and movement
XII Tongue: midline presence of atrophy or abnormal movements
Motor examination
Presence of atrophy, fasciculations, tone—resistance to passive movement
Ability to hold arms outstretched with eyes closed
Grip strength, scored on scale of 0 (absence of any movement) to 5 (normal strength)
Deep knee bend
Hopping on each foot, walking on heels and toes
Finger-tapping
Extensor plantar response
Tendon reflexes, scored as 0 (absent), 1 (decreased), 2 (normal), 3 (increased), 4 (grossly exaggerated)
Biceps
Triceps
Knee
Ankle jerk
Coordination
Tandem gait
Finger-to-nose pointing
Rapid alternating movement
Foot-tapping
Gait
Stance, presence of arm swing, rapid mining
Ability to walk on toes and heels
Walk quickly
Run
Sensory
Vibration and pin-testing in arms and legs
Position sensation

Source: Johnson (1987).

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SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS 101

neurotoxic effects in humans recommended the Neurobehavioral Core Test Battery (NCTB). The NCTB
(Table 5-3) can be used to identify a broad range of neurotoxic effects. The primary goal was to generate
uniform, more consistent data from a broad spectrum of occupations and neurotoxic exposure conditions. Tests
included in the set had been used successfully in worksite studies (they had identified group differences
associated with chemical exposures) and were believed to reflect a wide range of functional areas in humans. A
supplemental series of tests was specified for further characterization (Johnson, 1987).
The Neurobehavioral Evaluation System (NES), available on IBM PC and portable Compaq computers,
combines several neurobehavioral tests that have been used successfully in clinical settings or in field studies
(Table 5-3). Tests are occasionally added to the battery, which is following an evolutionary course dictated by
current interest in the field. The NES includes variants of five of the seven WHO NCTB tests, and the developers
of the NES recommend that the user select tasks to be used according to specific exposure situations (Baker et
al., 1985). That is, the battery provides a menu of tests without an explicit decision strategy for selecting among
them for screening or other purposes. The NES has been used in more laboratory and field studies than any other
battery in the last 5 years (Letz, 1991).
Anger (1989) matched the components of the WHO NCTB, Finnish, and NES batteries to the
neurobehavioral effects that he had found to be reported most commonly after chemical exposures (Table 1-1,
Chapter 1). He concluded that the NCTB would be the most comprehensive in detecting those effects. The
Finnish battery lacks the ability to detect affective changes, and the NES does not have an established test of
motor function. Although cognitive and, to a smaller degree, motor effects should be recognized fairly well by
all three batteries, they all have limited potential for detecting sensory deficits. All three batteries include tests to
assess the more subtle CNS deficits produced by lower exposures, which are the subject of increasing concern.
The tests noted above have been developed primarily for screening, but other tests have been used to
characterize the effects found in the screen. They are not fundamentally different, but add information about the
nature of the problem. They include laboratory-based behavioral tests, clinical neuropsychologic examinations,
and imaging evaluations. Some neurologic and behavioral tests also have been adapted to the monitoring for
early identification of disease or indicators of neurotoxicity. Screening and monitoring tests have been developed
to identify peripheral problems (such as toxic distal axonopathies) resulting from exposure to n-hexane, methyl n-
butyl-ketone, acrylamide, and related neurotoxicants. The neurologic examination often identifies neurotoxicity
at an early, but clinically observable, stage. Specific monitoring or screening devices (e.g., the Optacon and the
Vibratron II for assessment of vibratory sensitivity and NTE II to detect losses in temperature sensitivity) have
successfully identified neuropathy at the preclinical stage (Arezzo and Schaumburg, 1980, 1989). A more
specific test has been used to assess vibratory sensitivity in monkeys, and it is now used in the workplace to
identify peripheral neuropathy (Arezzo et al., 1983).
Tests of auditory threshold have been used to detect hearing loss after exposures to noise and to drugs that
affect the auditory system (e.g., quinine, arsenic, and glycoside antibiotics) (Stebbins et al., 1987). Similarly,
visual function has been found to be impaired in animals and people exposed to methylmercury (Evans et al.,
1975). Each of those tests has been used to identify early sensory loss so that exposures can be stopped before
the development of irreversible effects.
Although there is debate about some screening tests for field applications, the

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TABLE 5-3 Test Batteries

A. Finland Institute of Occupational Health Test Battery
Benton visual-retention test Reaction time
Bourdon-Wiersma Santa Ana
Symmetry drawing Wechsler Memory Scale (portions)
Mira test Wechsler Adult Intelligence Scale (portions)
Source: Hänninen and Lindstrom (1979).
B. Australian Battery Based on Information-Processing Theory
Critical flicker fusion Sensory store (iconic) memory
Vigilance Sternberg Memory Test
Simple reaction time Paired associates
Visual pursuit Short-term memory
Hand steadiness Long-term memory
Source: Williamson et al. (1982).

C. World Health Organization Neurobehavioral Core Test Battery (NCTB)

Santa Ana Benton visual-retention test
Aiming motor Digit span
Simple reaction time Profile of mood states
Digit symbol
Source: Johnson (1987).
D. Computer-Administered Neurobehavioral Evaluation System (NES)
Psychomotor performance Memory and learning
Digit symbola Digit spana
Hand-eye coordination Paired-associate learning
Simple reaction timea Paired-associate recall
Continuous-performance test Visual retentiona
Finger-tapping Pattern memory
Memory scanning
Perceptual ability Serial digit learning
Pattern comparison
Cognitive
Affect Vocabulary
Mood testa Horizontal addition
Switching attention
Source: Adapted from Letz and Baker (1986).

aVariant of WHO core test (Battery C)

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tests can identify some forms of peripheral neuropathy when they are still reversible. Some companies test
routinely to monitor for neuropathy alleging the sensory axons—a kind of secondary prevention system. Of
course, such strategies cannot be applied to the testing of newly developed chemicals, to chemicals with effects
that are not clearly defined, or to situations in which it is not known what chemical, if any, could cause any
health effect.
It can be difficult to interpret the results of test batteries. On the whole, they do not yet have established
norms based on extensive population testing, but they do provide objective measures suited to test-retest (before-
and-after) assessments. Baseline (pre-exposure) performance data have not been routinely collected from
workers before exposure, so unexposed people must be used for comparison. These performance effects are now
usually evaluated not by self-matching or against established standards, but by comparison to a referent or
control group.
The immense diversity of behavior makes it difficult for a simple test battery to screen adequately in a
comprehensive manner; a battery cannot detect an effect for which it lacks a test. Although it is not likely that an
entirely new type of health effect will be generated by a new chemical or class of chemical, it is likely that tests
for additional end points will be needed as more chemicals are tested. As understanding of the various
mechanisms of neurotoxicity increases, confidence that a comprehensive battery can be assembled should grow.
The established batteries of human tests are clearly limited in their screening capacity. No comprehensive
rationale for test selection has been posited. The Australians Williamson et al. (1982) developed their screening
battery around cognitive information theory, but this theory is now dated and is insufficiently related to medical
approaches to neurologic evaluation or to neuropsychologic approaches. Eckerman et al. (1985) proposed a
battery in which the tests are selected to assess eight cognitive functions identified by factor analysis; this battery
has not been used in worksite evaluations. The field needs to develop a comprehensive rationale for the
development of a neurotoxicity screening battery. Ideally, it would be based on the functions of the nervous
system and the range of potential neurotoxic effects, and that is not yet possible.
In the absence of a comprehensive rationale for the selection of tests to compose a neurotoxicity screening
battery, a good rationale would be to select tests that are sensitive to the range of neurotoxic effects that have
been identified. In a comprehensive review of the literature, Anger and Johnson (1985) identified more than 120
neurotoxic effects. The range of neurotoxic effects might be far too broad to test comprehensively. Anger (1986)
reviewed the list of 120 effects and identified the 35 reported as occurring after exposure to 25 or more
chemicals (Table 1-1). The result formed a basis for selecting the most important functions to assess, given
current knowledge. None of the batteries assesses sensory effects thoroughly, and the computer-based NES does
not use a proven test of motor performance. However, the NES and some other batteries have the advantage of
being administered by computer, which reduces the costs of administering the tests substantially. Some of the
most common neurotoxic effects—particularly some forms of peripheral neuropathy and affective symptoms,
weakness, ataxia, and many sensory effects—would not be detected by any of the batteries. (Of course, CNS
changes can be correlated with some of these effects, in which case these batteries would be performing their
function of detecting health effects. As noted earlier, a screening battery is developed for detection, not for
characterization.)
The WHO NCTB contributors first suggested human neurotoxicity testing based on

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the core-test idea, which uses a standard set of tests in all studies, supplemented by additional tests when
appropriate (this is a variant of the tier-testing idea) and is consistent with the idea of evaluating the most
common health effects. The NES can be used to test most of the same functions tested by the WHO NCTB, and
new tests are added periodically. That will allow adaptation to new problems as they arise. The tests of the NES
are largely cognitive and so will approach the complex functions that are not identifiable without sophisticated
tests, although tests of motor and sensory function are certainly required for truly comprehensive assessment.
Both the NES and the NCTB have been used in different countries and languages; that fosters the development
of an international data base that will allow exchange of test results.
Although those test batteries appear to be best suited to assessments of neurotoxic exposures in humans,
their usefulness needs constant examination in light of new types of neurotoxicity, possible changes in the most
frequently reported health effects (as chemical use shifts), and new cognitive or neurobiologic theories of
nervous system function. That is, NES and NCTB batteries are the best choice today, but this judgment is based
on identified health effects, rather than on understanding of the nervous system and how it functions.
Fundamental progress will be based on advances in basic and theoretical neurobiology; data from continued
testing of exposed population can be expected to lead only to minor improvements in test selection. An
intermediate goal is the collection of data with a small number of reliable and valid test batteries, to provide a
solid base of relatable data on various chemicals. Such a collection could lead to the development of specific
criteria for prediction of effects (most desirably on the basis of chemical structure) or the development of a better
screening battery.

CURRENT EXPOSURE SURVEILLANCE EFFORTS

Several sources of information provide data on potential for occupational exposure to neurotoxic substances
in the United States. However, none of them is comprehensive, represents a well-defined worker population or
industrial setting, or corresponds to any existing set of information on adverse health effects (NRC, 1987a).
The Integrated Management Information System (IMIS) of the Occupational Safety and Health
Administration (OSHA) contains information obtained as a result of inspections for accidents or complaints,
followups on violations, and general scheduled inspections. The only exposure data systematically entered into
the IMIS are data obtained through analysis of environmental samples taken at worksites where citations were
issued. Because IMIS samples are not random samples, generalizable information on the distribution of
neurotoxic occupational exposures does not exist.
Industry-generated data constitute a second source of information on neurotoxic hazards. For 11 toxic
substances (including lead) for which OSHA has set standards, industry is required to maintain records of
exposure. However, because the records are not part of a central data system, such as the system maintained by
OSHA, they are most useful for monitoring specific industries and employers and are far less useful for
surveillance and discovery.
NIOSH conducts health hazard evaluations (HHEs) at the request of employers, employees, or their
representatives. At times, both exposure monitoring and biologic sampling are done. The records provide insight
into particular occupations and industries, but, like the OSHA data, they are not readily generalizable. NIOSH
has conducted two national surveys of chemical exposure in nonagricultural businesses: the National
Occupational Hazard Survey (NOHS) from 1972 to 1974 and the National

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SURVEILLANCE TO PREVENT NEUROTOXICITY IN HUMANS 105

Occupational Exposure Survey (NOES) from 1981 to 1983. Each entailed walk-through inspections of
approximately 5,000 plants, and potential exposures were tabulated by trained observers. However, no industrial-
hygiene samples were collected. As a result, data on actual exposures are not available. The NOHS-NOES data
have been used to identify general industrial groups in which neurotoxic exposures may be anticipated, and they
have served as a valuable resource for epidemiologists and public-health officials (ASPH, 1988). Information on
exposure of workers and consumers to pesticides is available from the Environmental Protection Agency (EPA).
Evidence of exposures to toxic substances in the general environment might be detected by the National
Human Adipose Tissue Survey (NHATS), periodic sample surveys done by the National Center for Health
Statistics (the National Health and Nutrition Examination Surveys, or NHANES), and toxic inventories
maintained by states and cities under right-to-know legislation. Those data bases provide information on a range
of chemicals—some that are known to be neurotoxic (e.g., lead, surveyed under NHANES II [NCHS, 1984]),
some that accumulate in fat (such as pesticides, surveyed in the NHATS), and some that are stored and released
in substantial quantities in industrial and other sites. The National Research Council has published a review of
the NHATS program and made several recommendations for its modification and improvement (NRC, 1991b).
A particularly valuable new source of information is the Toxic Release Inventory (TRI), compiled annually
by EPA since 1988 to monitor more than 300 chemicals released by industry into the environment. The
inventory is mandated under the Superfund Amendments and Reauthorization Act (SARA). Seventeen of the 25
toxic substances with the highest volumes of release into the environment have neurotoxic potential (OTA, 1990).
Efforts are also needed to improve the availability and use of exposure-surveillance data. IMIS, the OSHA
inspection data base, would be more useful if it covered all states in a more uniform fashion and if it included a
larger, more representative sample of exposure data. Other types of exposure-reporting systems (such as those
under federal and state right-to-know laws) should be evaluated for their utility in surveillance. For the
preponderance of industrial settings, adequate exposure data are not available. Such information should be
collected according to explicit sampling plans with up-to-date measurement techniques and stored in accessible
data bases. It could then be used to target efforts to control known dangerous exposures or matched to health-
effects files in an effort to detect new problems, as discussed below. Exposure surveillance is a continuous effort
both to monitor changes in exposure and to monitor new industries or new uses of well-known chemicals
(Fowler and Silbergeld, 1989).
When the objective is to assess broad trends in population exposure to neurotoxicants and to gather
information on groups potentially at risk, selection of an appropriate surveillance strategy will depend on the
expected degree of toxicity of the chemicals under study, the size of the exposed population, and the intensity of
exposures. In essence, hazard or exposure surveillance consists of obtaining information on the distribution and
use of chemicals, on the distribution of exposures, and on the size and distribution of the exposed population.

CURRENT DISEASE-SURVEILLANCE EFFORTS

A disease-surveillance system directed toward detecting problems caused by unrecognized neurotoxicants
must be much more open and versatile than a system intended only to tabulate the incidence and prevalence of
and mortality from a known

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disease. Neurotoxic disorders are particularly difficult to monitor (NIOSH, 1986; Friedlander and Hearne, 1980),
and present surveillance systems are not very effective. Neurotoxic disorders are easy to overlook or
misdiagnose, because their signs and symptoms often develop slowly and subtly. And some can be reversible or
self-limiting. The patterns of effects produced by many neurotoxic chemicals are similar, consisting of common,
nonspecific complaints, often mimicking diseases with other etiologies. The association between an observed
health effect and the culpable environmental agent is rarely obvious. Clinical tests used in diagnosis are usually
too imprecise or insensitive to identify the full spectrum of adverse health effects. A latent period between
exposure and overt response will further complicate determination of causation.
Several existing data systems have been suggested as potentially useful for surveillance for neurotoxic
disease that might be attributable to environmental exposures (Gable, 1990). They include vital records, health
surveys, specifically designed surveillance systems, and the U.S. Census.
Since 1971, the Bureau of Labor Statistics (BLS) has conducted the Annual Survey of Illness and Injury
with a well-constructed (and well-defined) probability sample; the most serious problem with this data base for
the detection of neurotoxic illness is that the occurrence of illnesses, especially those involving latent periods, is
vastly under-reported. BLS also maintains a Supplementary Data System in which data from the states' workers'
compensation programs are compiled; the data gathered differ from state to state, and the lack of total reference
populations precludes the calculation of rates.
The National Center for Health Statistics (NCHS) compiles data (from death certificates) on every death in
the country through the National Death Index. The basic data on death certificates are fairly uniform across
states, and demographic cause-of-death information and some occupational data are almost always available.
Occupational data are of uneven quality, which usually depends on whether anyone uses them for research
purposes. The categories ''housewife'' and "retired" are often reported in the place of out-of-home employment or
one-time major employment. Data from the Bureau of the Census are appropriate as denominators in calculating
rates. NCHS makes the encoded mortality data available to researchers on computer tapes. The National Death
Index is valuable for determining the vital status of subjects in epidemiologic followup studies; for subjects who
have died, researchers are directed to the appropriate state for detailed information. NCHS also collects data
from the states on every birth and fetal death, including birthweight, Apgar scores, congenital anomalies, and
complications of pregnancy or birth. The birth records are not as uniform across states as are death certificates,
and only a few states record information on parental occupation. NCHS has conducted the National Health
Interview Survey, a large (120,000 persons), stratified probability sample of the civilian, noninstitutionalized
population, every year since 1957. In addition to standard demographic information, both occupational status and
data on illnesses, injuries, disabilities, and use of medical services are gathered. The fourth NHANES, a
probability sample of the civilian, noninstitutionalized population also conducted by NCHS, is currently being
carried out on a full-scale basis (the first was carried out in 1970). Physical examinations, laboratory tests, and
responses to a questionnaire yield a detailed medical picture, but occupational information was not gathered in
the previous surveys. In the current survey, job histories are being requested, and neurotoxicity is among five
work-related conditions about which information is sought.
Other data bases are of potential value for the detection of neurotoxic disease. Regional poison-control
centers, insurance

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companies, the Social Security Administration, and the Department of Veterans Affairs might have information
useful for a specific surveillance project, but most fall short by not having both health and occupational data
(data on actual exposures are almost never found), by being drawn from an inadequately defined population, by
having restricted availability, or by being limited to a period that is not of interest.
The National Research Council Panel on Occupational and Health Statistics (NRC, 1987a), after reviewing
the various sources of information on adverse health events, concluded that the information available on
occupational injuries is poor and that the information on occupational illnesses is considerably worse. Those data
sources were not designed to determine the causes of disease, nor do they emphasize occupational or
environmental exposures. Thus, the contribution of such exposures to the etiology of a given disease is not
usually recognized (ASPH, 1988). The end points selected for surveillance are usually restricted to overt clinical
disease or death.
Once cases are reported, officials can collect further information and provide appropriate followup and
suggestions for prevention in the workplace. The reporting systems must be interactive and provide a means for
followup of reported cases to ensure continued physician involvement in the surveillance program. State and
local health departments, in collaboration with occupational clinic groups, probably provide the best basis for
such reporting systems.
When an excess or cluster of a particular symptom associated with neurotoxicity (National Conference on
Clustering of Health Events [1990]) or a new syndrome with involvement of the nervous system is detected,
determination of the likely causes is the next stage of the epidemiologic process. It requires careful comparison
between observed physical and temporal patterns of exposures to possible toxicants and the occurrence of the
cases.
The NIOSH Health Hazard Evaluation Program and similar activities of academic or government groups
provide mechanisms for rapid evaluation of reported outbreaks and can therefore confirm initial reports of a
possible neurotoxic problem. California has pioneered the development of a reporting system for outbreaks of
pesticide poisoning in which collaborating health and agricultural departments can intervene. However, similar
systems for the reporting of such diseases by physicians are not well established elsewhere in the United States
(ASPH, 1988).
The committee has considered several ideas for improving disease surveillance systems:
• Training of medical professionals. Many medical professionals who are in direct contact with persons with
neurotoxic disorders of environmental origin lack the knowledge necessary to recognize these ailments or to
identify their etiology. The ability to diagnose the disorders and to associate them with workplace or
community exposures must be improved (U.S. House of Representatives, 1986). Physicians need to be made
more aware that environmental exposures can produce toxic effects and should be trained to obtain an
account of exposures received occupationally, avocationally, etc., as well as medically, when taking a
history (Goldman and Peters, 1981). Better initial training in occupational and environmental medicine and
the dissemination of more current information on neurotoxic illnesses would benefit both health
practitioners and their patients (NRC, 1987a). The development of improved diagnostic tests and criteria
would facilitate the recognition of an occurrence of neurotoxicity when it appears.
• Standardized disease definitions. Uniform clinical definitions of the disorders in question are needed to
provide a common reporting basis for physicians. Such a nomenclature would also make the assembled

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data more amenable to analysis and interpretation. NIOSH has a project to develop definitions for a series of
occupational disorders.
• Sentinel health events. A sentinel health event (SHE) is a preventable disease, disability, or untimely death
whose occurrence serves as a warning that the quality of preventive or therapeutic medical care might need
to be improved (Rutstein et al., 1976). The idea has been refined for application to occupational situations; a
SHE(O) is a disease, disability, or untimely death that is related to an occupation and whose occurrence can
provide the impetus for epidemiologic or industrial-hygiene studies or serve as a warning signal that
material substitution, engineering control, personal protection, or medical care might be required (Rutstein
et al., 1983). Wagener and Buffler (1989) showed how NCHS's Compressed Mortality File could be
screened with SHE(O) codes to derive mortality rates for as small an area as a county, which would be a
step toward identifying geographic areas with increased incidences of sentinel diseases. The industrial or
occupational information routinely entered on death certificates is encoded on higher-level, more easily
retrievable records in only a few states, however, and that diminishes the usefulness of the SHE(O) idea and
of other occupational-surveillance programs that use mortality data. Recently, the SHE(O) principle has
been adopted as the core of demonstration reporting projects between NIOSH and 10 state health
departments (Baker, 1989). Lead poisoning is one of the eight target occupational-health conditions focused
on by this project, the Sentinel Event Notification System for Occupational Risk (SENSOR). This trial
surveillance project was described in conjunction with the reporting of a case of adult lead poisoning (blood
lead was 170 µg/dl, and reporting is required for concentrations above 25 µg/dl). The index case led to
finding secondary cases in the subject's workplace and family (Johnson et al., 1989). The investigators at
NIOSH and Harvard Medical School (Rutstein et al., 1983), who developed the original list of SHE(O)s,
intended that new associations between toxic environmental exposures and disease conditions be added as
they were recognized. The extent to which neurotoxic health effects are indistinct entities will interfere with
their conversion into SHEs or SHE(O)s. Neurotoxic conditions that might be added to the list include
occupational neuropathy due to Lucel 7 (Horan et al., 1985), paralysis of the urinary bladder after exposure
to the NIAX catalyst dimethylaminopropionitrile (Gad et al., 1979; Pestronk et al., 1979), and the Kepone
(chlordecone) syndrome (Guzelian, 1982).
• Biologic markers. The complexity and inaccessibility of many parts of the nervous system make it difficult
to use biologic markers to study neurotoxicology. But events in physiologic systems that are substantially
controlled by neuronal processes, such as some aspects of endocrine and immune function, can be
monitored. And objective, computer-based systems for assessment of the function of the central and
peripheral nervous systems have been developed to yield biologic markers of neurologic function in exposed
populations (Letz and Baker, 1986); the tests are rapid, inexpensive, and noninvasive. Some indicators of
exposure to neurotoxicants are measurable in easily sampled media, such as blood, urine, and hair.
Surveillance for exposure to neurotoxicants is possible with such easily sampled surrogates, as in the
measurement of peripheral esterase in workers exposed to organophosphates to monitor CNS effects
indirectly (see also Chapter 3).
• Disease and exposure registries. Groups of people with known or suspected large exposures to
neurotoxicants, such as occupationally exposed populations or residents near hazardous-waste sites, can be
included in programs of targeted medical surveillance or followup, e.g., by the Agency for Toxic Substances
and Disease Registry (ATSDR).

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ATSDR is charged under the Comprehensive Environmental Response, Compensation, and Liability Act
(CERCLA, or Superfund) with maintaining national registries of people exposed to hazardous substances.
ATSDR's current registries are of people exposed to trichloroethylene, dioxin, or β-naphthylamine. The
latter two are not regarded as a neurotoxicants, but the program will be expanded and could provide some
unanticipated information. Available information from the NOHS (1972–1974) or the more recent NOES
(1981–1983), OSHA compliance monitoring, health-hazard evaluations, and similar programs can be
reviewed to identify occupational and industrial groups with the potential for substantial exposure. Another
group of persons for potential followup consists of patients (probably of HMOs) who are on long-term
pharmaceutical treatment regimens with potentially neurotoxic drugs. Depending on the nature of the
chemical, targeted medical surveillance might consist of periodic determination of blood concentrations,
measurement of cholinesterase, nerve-conduction testing, or other testing of neurologic or psychiatric
function.
Prospective surveillance of populations exposed to neurotoxicants in defined settings, such as the
workplace, is a promising approach to the early and sensitive detection of functional impairment of the
nervous system. The methodologic advantage of prospective surveillance over one-time or cross-sectional
surveillance is that each subject can serve as his or her own control. Initial information on a person can be
taken to represent baseline or pre-exposure measures of function, and later data can be compared with the
baseline data to detect possible toxic injury. In this study design, effects of modifiers, such as drug or
alcohol abuse, on the nervous system would be largely canceled out. Thus, compared with cross-sectional
evaluations, prospective surveillance should be able to detect neurologic damage at an early stage and at low
levels of exposure to such neurotoxic agents as lead in children and solvents in adults. Such surveillance is
ideally targeted to the identification of functional impairment while it is still potentially reversible; detection
of subclinical impairment can, in such instances, trigger a person's removal from exposure or steps to reduce
overall exposures so others will not be harmed.
In another epidemiologic approach to the detection of environmental causes of neurologic disease, time
trends or geographic patterns of occurrence of illness are measured. For example, in studies of multiple
sclerosis, a clear north-south gradient in incidence is noted, with higher rates at more northerly latitudes.
Moreover, people who spend their early years in northern latitudes and then move south seem to carry much
of their earlier risk of multiple sclerosis into later life. Those observations suggest some undefined
environmental factors in the etiology of this chronic neurologic disease. Likewise, strong time trends have
been seen in the incidence of Parkinson's disease and motor neuron disease. The explanation is not known,
but again an environmental factor is suggested (Lilienfeld et al., 1989).
Case-control studies of groups with chronic neurologic disease provide another approach to the detection
of environmental causation. People with and without a chronic neurologic disease are interviewed to
determine whether there are systematic and statistically significant differences in exposure between the two
groups (Hertzman et al., 1990). Such studies help to detect diseases of long latency caused by exposure
many years in the past.
• Imaging techniques. New methods are becoming available for studying the nervous system noninvasively.
Computed axial tomography (CAT) allows visualization of the structure of internal organs; CAT scanning
has been used, for instance, to reveal structural abnormalities in the brains of schizophrenics;

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(Zec and Weinberger, 1986). The recently developed positron- emission tomography (PET) and magnetic
resonance imaging (MRI) allow visualization of physiologic and biochemical processes as they occur in the
brain (Battistin and Gerstenbrand, 1986). PET scanning has been used in diagnosis of neurotoxicity in
people exposed to MPTP (Calne et al., 1985). At present, however, PET and MRI are not routine screening
tools.

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6
Risk Assessment

Risk assessment, as defined by Crouch and Wilson (1987), is "a way of examining risks, so that they might
be better avoided, reduced, or otherwise managed." It is a process based on science whose objective is to provide
criteria for policy-making. Risk assessment can be qualitative or quantitative. The development of quantitative
methods for risk assessment has been most extensive for carcinogenicity, that is, estimating the potential risks of
cancer associated with exposure to oncogenic chemicals and other agents. Risk Assessment in the Federal
Government (NRC, 1983) and Identifying and Regulating Carcinogens (OTA, 1987) discussed the development
of those methods. Compared with the work on risk assessment of neurotoxicants, the development of methods
for quantitative risk assessment of carcinogens has been built on a larger body of mechanistic information and
hypotheses that permits quantitative description of the cellular and molecular events involved in chemical- and
radiation-induced carcinogenesis. But uncertainties still attend almost all risk assessments that involve
extrapolation from acute to chronic responses, from response at high doses to response at low doses, from one
route of administration to another, and from species (laboratory animals) to species (human).
In neurotoxicology, critical mechanistic information is not available, except for a few substances. Thus, risk
assessment in neurotoxicology is still relatively undeveloped. Moreover, neurotoxicologic risk assessment is
likely to be highly complex, compared with carcinogenicity risk assessment. Carcinogenesis at the molecular
level has been proposed to involve a relatively limited set of cellular events common to the induction of all
tumors; although specific carcinogens can act at different stages of carcinogenesis, the process is assumed to be
fundamentally similar among tumor types and sites. In contrast, the diversity of cells and processes in the
nervous system that are potentially subject to toxic action is great. Moreover, the response of those targets can
depend on the developmental stage (including aging) of the organism at the time of exposure (Weiss, 1990). For
example, during the cytoarchitectural organization of the human CNS after birth, neurotransmitters can function
as trophic factors guiding the migration and localization of cells in brain regions; after synaptogenesis, they
function primarily as information transducers (Schwartz, 1985).

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An agent (such as α-methyl-p-tyrosine) that interferes with neurotransmitter synthesis during development can
cause permanent alterations in regional neuronal organization; the same agent acting on the same mechanisms
later in development can interrupt cell-cell communication without causing structural disarray.
No general mathematical model is likely to be appropriate for the quantitative risk assessment of
neurotoxicants as a class. Defining and validating risk-assessment methods for neurotoxicants will probably
require the development of a set of methods that are restricted in application to specific end points, rather than to
specific toxicants. The rest of this chapter discusses some approaches to that task.
The process of risk assessment has been divided into the following stages (NRC, 1983; Cohrssen and
Covello, 1989): hazard identification, dose-response analysis, exposure assessment, and risk characterization,
followed by risk management. Dividing the process into those conceptual stages can assist in allocating
resources and critical analyses to specific aspects of science-based policy analysis. In hazard identification, the
goal is to obtain sufficient information to determine the qualitative nature of any biologic activity associated with
a specific agent. Short-term tests are useful, including even those with a high rate of potentially false-positive
results, such as the Ames bacterial mutation assay used in carcinogen hazard identification. When a hazard has
been qualitatively identified, the next stage is to develop quantitative information on the relationship between
dose and response. The relevance of the test system to predicting outcomes in humans—such as route and timing
of exposure—becomes more important. Differences between conditions of the test and those anticipated to occur
in human populations might be unavoidable, so implicit and explicit assumptions as to extrapolation must then
be incorporated into experimental design and data analysis. Information on metabolic differences between
species can also be relevant. An important outcome of the analysis is the development of sufficient information
to evaluate the overall shape of the dose-response relationship (linear, sub-linear, hyperbolic, or sigmoidal),
which can assist in generating inferences on likely responses outside the range of measured doses and responses.
It should be noted, however, that the shape of the dose-response curve at the low end is often difficult to
determine. In the third stage, exposure-assessment data are accumulated on external doses expected to be
encountered by humans, including groups whose exposure or response might be expected to be greater than
average (for instance, children). In some cases, information on internal dose (and toxicokinetics) can also be
collected. Finally, all the preceding information—the nature of the expected outcome, the relationship between
dose and outcome (response), and the expected ranges and distribution of doses from individual to individual—
are incorporated into a risk characterization.
In the case of dichotomous events, such as the presence or absence of a diagnosable malignancy or death,
the process results in a probability estimate that can be expressed as an increase in individual risk or, with
appropriate adjustments, as an estimate of increased population risk. An approach based on a yes-no definition of
outcome does not provide an accurate quantitation of events that vary in severity. In neurotoxicity, we are almost
always concerned about outcomes other than death and effects that vary in severity (unless they are more or less
arbitrarily defined in dichotomous terms, such as defining mental retardation solely on the basis of a critical IQ
score). Because many neurobiologic functions are expressed on a continuum, it is likely that the effects of many
neurotoxicants similarly fall on a continuum of severity. In modeling terms, neurotoxicants act to change the
distribution of biologic properties that themselves assume a spectrum of values in a population. Gaylor

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and Slikker (1990) have provided an example of a procedure that uses data on neurochemical, neurohistologic,
and behavioral effects of exposure to methylenedioxymetham-phetamine (in rats or monkeys) to estimate risk as
a function of dose of a potentially neurotoxic substance.
One of the most fully examined instances of this type of neurotoxicity is related to the effects of lead on
cognitive function. Most measures of cognition are normally distributed around a population mean (e.g., the
Stanford-Binet IQ score has a mean of 100 and a standard deviation of 15). The IQ-lowering effect of lead in
children can be detected as a change in mean score (Needleman and Gatsonis, 1990). More important, however,
the change in mean implies that the entire distribution of IQ scores in the exposed population is shifted.
Although the change in mean IQ in lead-exposed children might seem relatively small (less than 10 IQ points in
most studies and so well within the population's normal variability), an examination of the effects of such a
decrease on an overall population distribution of IQ scores (Figure 6-1) reveals the potential for very important
consequences of low-level lead exposure for society as a whole. The displacement of the overall curve toward
lower IQ scores would reduce the proportion of children with superior IQs (i.e., 130 or more) and increase the
number of children with seriously compromised intellectual potential (i.e., IQs less than 70).
Current risk-assessment methods based on carcinogenicity risk-assessment models do not capture such
types of impacts, built as they are on an assumption that the end point of concern is an all-or-none (dichotomous)
variable, rather than a continuous one. The problems of risk management, although not among the specific
concerns of this volume, are compounded by the phenomenon of the continuum of both frequency and intensity
of response related to dose. At some doses, the "most sensitive" response might be some mild form of
neurotoxicity; at another, usually higher, dose, the response might be more serious, more life-threatening, or
more intense.

Figure 6-1 Effect of a shift in mean IQ score on the population distribution. The top figure represents a theoretical
population distribution of IQ scores with a mean of 100 and a standard deviation of 15. In a population of 100
million, 2.3 million (stippled area) will score above 130. The bottom figure represents distribution of intelligence
test scores with a shift of 5%, yielding a mean of 95. Here, the number of individuals scoring above 130 falls to 990
thousand, with a corresponding inflation of those scoring below 70. Source: Weiss (1990).

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APPROACHES TO RISK ASSESSMENT FOR NEUROTOXICITY

Risk-assessment models ideally are based on two sources of biologically based information: statistical
analyses of rich data sets and knowledge of mechanisms. Approaches involving curve-fitting require a great deal
of data to develop predictions of risk with any reliability. Although, in appropriate cases, statistical analysis can
be important in validating models based on hypothesized mechanisms of action, the available data are usually
insufficient to resolve critical issues, such as the predicted response at exposures below those on which the data
were collected. An essential part of developing risk-assessment methods is therefore the integration of
biologically based models that incorporate our understanding of mechanism of action. It is important to
emphasize that statistical models should conform to biologic hypotheses—that is, curve-fitting must be
consistent with what is known of the biology of the affected system.
The most commonly used approach for risk assessment of neurotoxicants, as of most noncarcinogens, is the
uncertainty- or safety-factor approach (NRC, 1986; Kimmel, 1990). It is based on identifying either the lowest
dose of a toxicant at which adverse effects are observed (lowest observed-adverse-effect level, LOAEL) or the
highest dose at which no adverse effects are observed (no-observed-adverse-effect level, NOAEL) and then
deriving a presumably safe dose by dividing by a safety or uncertainty factor. Its weakness is related to the
unreliability of observation as a means of determining doses at which biologic effects actually do or do not
occur. A small study might give negative results, and no study is large enough to exclude the possibility of any
effect. Furthermore, the importance of subtle alterations in behavior or small neurochemical or structural changes
is a matter of debate. Before undertaking tests to determine the LOAEL or NOAEL, it is important to define
what is meant by "adverse effect" and to develop appropriately sensitive measures of such effects. For example,
the persistence of the neurotoxic effects of small exposures to such solvents as trichloroethylene,
dichloromethane, and toluene is not known, so the importance of observed decrements in performance is still
under debate (Gade et al., 1988; Parkinson et al., 1990). This approach also provides no information on the slope
of the dose-response curve or intensity of effect above the "safe" dose calculated (Gaylor and Slikker, 1990;
Kimmel, 1990; IOM, 1991).
Thus, the determination of a LOAEL or NOAEL is strongly influenced by the sample size and design of the
experiment (Gaylor, 1983; Crump, 1984). In most cases, it is difficult to determine an effect smaller than a 20%
increase or decrease. As an alternative, Crump (1984) first suggested the use of a benchmark dose (BD), defined
as "a statistical lower confidence limit corresponding to a small increase in effect over the background level."
The increase in effect used to determine the BD would be near the lower limit of change, which can be
determined with reasonable accuracy in toxicologic studies. The BD is calculated with a mathematical model;
however, because BDs correspond to risks in the experimental range, they are less affected by the particular
shape of the dose-response model used in the calculations. The acceptable dose is extrapolated from the BD.
Using a BD, rather than the more usual LOAEL or NOAEL, avoids problems inherent in defining a negative (the
NOAEL) and in the lack of precision associated with defining a LOAEL (Kimmel, 1990).
Whether a conventional approach or a BD approach is used, the dose identified is usually modified by some
safety factor to yield a so-called safe dose. The incorporation of a safety factor (usually 10 raised to some power)
reflects a policy decision to incorporate in a quantitative manner some of the uncertainties associated with risk
assessment. In general, safety factors range

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between 10 and 1,000 (NRC, 1977; Kimmel, 1990), so the risk assessment is the final estimate of dividing the
BD, NOAEL, or LOAEL by 10, 100, or 1,000. Selection of the safety factor involves processes of judgment,
including considerations of weight of evidence, availability of information on human response, type of toxicity
observed, and probability of variations in response among susceptible groups. Some rules of thumb have been
adopted to accommodate intraspecies variability, cross-species extrapolation, experimental duration, etc.; they
correspond fairly well to observed variation (Dourson and Stara, 1983).
As noted above, statistical approaches to risk assessment should be consistent with what is known about
mechanisms of action or about the biology of the affected system. The NOAEL-LOAEL approach is based on an
assumption of a threshold, a dose below which an effect does not change in incidence or severity. However, the
evidence of the general applicability of that assumption for all neurotoxicants is relatively weak. Even though a
given neurotoxic response may require a threshold dose of a specific toxicant, other toxicants in the environment
that cause the same or similar response may in effect lower the threshold dose of the specific neurotoxicant of
interest. That is, a person may have a threshold of effect in a pure environment, but there may not be a threshold
for a heterogeneous population in a heterogeneous environment. It is likely that some neurotoxicants have
thresholds and others do not. (A lack of a threshold for a neurotoxicant makes the safety-factor approach
biologically indefensible.) On the one hand, the nervous system appears to have considerable reserve capacity
and plasticity, which would support the assumption of a threshold. On the other hand, it is composed of cells that
are nonreplaceable, which would argue that no damage can be considered innocuous. Moreover, both
susceptibility to injury and the nature of the injury can vary with developmental stage; early neurologic
development includes a selective pruning of apparently excess neurons and connections (Rakic and Riley, 1983),
whereas senescence includes a progressive loss of specific neurons and an apparently selective loss of function.
Because the CNS develops from a limited set of cells early in development, it is appropriate to assume that
exposure to neurotoxicants during critical periods of brain development can be without a threshold; data on x-
irradiation-induced brain injury support this assumption (Schull et al., 1990). As shown in Figure 6-2, when in
utero radiation exposure occurred during the period between 8 and 15 weeks gestation, the effects on intellectual
status

Figure 6-2 Estimated developmental scores at various ages for three blood-lead concentrations. Application of a
model developed by Hattis and Shapiro (1990) to data from Bellinger et al. (1987a,b) that assumes the group is
exposed to yield constant blood lead concentrations of 0, 5, and 10, µg/dL leads to estimates of developmental
scores for populations of exposed children. Source: Adapted from Wyzga (1990).

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were shifted to the left, possibly without a threshold. Whether the PNS and CNS differ with regard to thresholds
is not known. Studies of PNS neurotoxicants, such as acrylamide, suggest a capacity for recovery through axonal
regeneration, but it varies with the dose and duration of exposure; Hattis and Shapiro (1990) have investigated
quantitative dose-response models as alternatives to the NOAEL-LOAEL and uncertainty-factor approach for
this situation. The recent Institute of Medicine (IOM, 1991) publication on safety of seafood takes a similar
quantitative modeling approach for methylmercury. In any event, the application of the NOAEL-LOAEL (or
BD) approach to all neurotoxicants is unlikely to be biologically defensible.

CURVE-FITTING IN RISK ASSESSMENT FOR NEUROTOXICITY

When quantal information (the proportion of response at a given dose) is available, but little is understood
about underlying biologic mechanisms, a tolerance distribution, such as the probit (log-normal) or logistic
models, can be adopted to fit population data (Wyzga, 1990). Those sigmoid dose-response models assume that
the distribution of individual thresholds follows the specified model, but that the population as a whole might not
have a threshold.
If a substantial body of data is available, it might be possible to test goodness-of-fit of various statistical
models. For most neurotoxicants, that is not now possible. Attempts have been made to fit various models to data
on the PNS and CNS toxicity of lead. Data on nerve-conduction velocity in children exposed to environmental
lead (through living near a smelter) as a function of blood lead concentration fit a ''hockey-stick'' type of dose-
response curve (Schwartz et al., 1988). The data were sufficient to permit discrimination, in terms of goodness-
of-fit, from several other models, including a logistic model and a quadratic model. In another approach to
assessing lead neurotoxicity, Wyzga (1990) assumed that individual thresholds for a dichotomized measure,
muscle weakness, were log-normally distributed in the population. After producing maximal-likelihood
estimates of the mean and standard deviation of the threshold distribution, the distribution of blood lead
concentrations was estimated. Wyzga thereby determined that the probability that a randomly chosen male from
the general population would exceed the threshold and exhibit extensor muscle weakness would be 0.01. In a
second analysis, Wyzga applied the model developed by Hattis and Shapiro (1990) for acrylamide exposure that
compensates for past damage with the rate of repair proportional to the cumulative-damage data on children's
blood lead concentrations and mental development gathered by Bellinger et al. (1987a,b). The dose-response
estimation related group response to group exposure levels, and Wyzga concluded that this model, albeit only
one of many alternative models that could be fitted to the data, indicates some inhibition of development at
exposures that resulted in very low blood lead concentrations.
The hockey-stick model was also evaluated for its consonance with the available information on lead's
effects and on peripheral neurophysiology. As discussed by Schwartz et al. (1988), it is plausible to propose a
threshold for the peripheral neurotoxicity of lead for reasons of both mechanism of action and physiology: lead
inhibits synaptic release of acetylcholine through inhibition of ion-dependent neurotransmitter release, and lead
reduces velocity of peripheral-nerve conduction (as measured transdermally with surface electrodes). The
mechanism of action of lead thus involves events that are nonlinear: the changes in ion permeability that are
associated with nerve depolarization and transmitter release involve many discrete events (e.g., channel
openings, exocytotic events of electric propagation), and nerve conduction is a summation

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involving the function of many nerve fibers. Thus, both basic biology and mechanism of action are consistent
with the results of curve-fitting in this case.

MECHANISTIC MODELS FOR RISK ASSESSMENT FOR NEUROTOXICITY

Information on mechanisms of action provides the most powerful tools for developing models for risk
assessment (Silbergeld, 1990). For neurotoxicants, it is unlikely that a single mechanism or even a small number
of mechanisms will be elucidated for the neurotoxicants already identified, given the variety of potential cellular
and molecular targets of action. Nevertheless, in some cases, information is available on mechanisms of action of
some neurotoxicants and can be used for purposes of developing quantitative approaches to risk assessment. It
must be emphasized that the generalizability of such models is narrow, possibly even for similar molecules, so
the approach might be useful only for assessing, case by case, the risks of the substances for which it was
developed. Further developments in structure-activity analysis might permit extension of specific mechanistic
models to chemicals within appropriate structural classes.
As noted in Chapter 1, many pesticides are designed to be neurotoxic to a target organism. They should be
relatively nontoxic to humans in their intended uses, but will be neurotoxic to humans who are sufficiently
exposed. The reason is that their mechanisms of action in target species involve biologic substrates that are also
present in humans.
For example, the organochlorine pesticide dieldrin once was widely used in agriculture and in household
formulations, but now is restricted in application because of its persistence and potential carcinogenicity.
Dieldrin acts as a neurotoxicant by blocking the GABA channel in nerve cells (Narahashi and Frey, 1989). It is a
competitive antagonist for the recognition site (receptor) associated with the channel, and it can be shown to bind
to the site with a high affinity. Assuming that to be its fundamental mechanism of action, one can develop a
mathematical model based on the biology of receptor-ligand interactions. Receptor-ligand binding is well
described by classical mathematical models derived from Lineweaver-Burk equations of enzyme kinetics
(Silbergeld, 1990). (Enzyme-substrate interactions are a class of receptor-ligand interactions, as is carcinogen-
adduct binding to DNA.) As with all receptor-ligand interactions, when the concentration of the receptor is
greater than that of the ligand at low concentrations of dieldrin, receptor-ligand binding is linearly related to the
concentration of the ligand, dieldrin. If receptor binding is directly related to regulation of the ionophore and the
function of the ionophore is critical to neuronal function, a linear model based on receptor-binding kinetics can
be constructed for the neurotoxic risks associated with dieldrin. The model agrees well with electrophysiologic
data. This approach provides a prediction of risk that can be validated in terms of whole-organism response to
dieldrin.
It might be possible to model other neurotoxicants that act through receptor mechanisms with similar
approaches to quantitative risk assessment. (Table 6-1 lists such neurotoxicants.) Obviously, these approaches
describe the predicted behavior of a toxicant only at its cellular site of toxic action, e.g., relations between target-
organ dose and response. For risk assessment, knowledge of pharmacokinetics—absorption, distribution, and
metabolism—is relevant for estimating the overall relationship between external exposure and target-organ dose
(NRC, 1987b).
Another mechanism of neurotoxic action could be applicable to an understanding of the consequences of
cytotoxic agents that act on neural cells during development. On the basis of the principle of nonreplaceability, a

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TABLE 6-1 Some Neurotoxicants That Act on Receptors

Receptor or Channel Blocker (Antagonist) Modulator (Agonist)
Acetycholine receptor Lacticotoxin
Erabutoxin
α-Conotoxins
Anatoxin-a
Nereistoxin
Atropine
Scopolamine
Acetycholine-activated channel Histrionicotoxin
Amantidine
N-Alkylguanidines
Excitatory amino acid AP-5 (2-amino-5-phosponopentanoate) Oxotremorine
AP-7 (2-amino-5-phosponopentanoate)
Nephila orb web spider toxins
Argiope orb web spider toxins
γ-Philanthotoxin
MK 801
Ketamine
GABAA receptor or channel Bicuculline N-Methyl-D-aspartate
Lindane 1-Glutamate
Dieldrin Kainate
Picrotoxinin Quisqualate
GABAB receptor or channel Phaclofen Muscimol
Avermectin Bla
Barbituates
Benzodiazepines
Ethanol
Glycine receptor or channel Strychnine Baclofen
Presynaptic tunnel β-Bungarotoxin α-Latrotoxin
Botulinum toxin
Tetanus toxin
Taipoxin
linear no-threshold model might be appropriate to estimate he effects of cytotoxic agents encountered
during critical periods of development. Radiation is the classic example of such an exposure. The results of
animal studies are consistent with the hypothesis of a lack of threshold for prenatal irradiation during the critical
period of organogenesis of the cortex (Schull et al., 1990). The available human data are also consistent with the
hypothesis (Otake and Schull, 1984; NRC, 1990). As shown in Figure 6-3, there is no clear evidence of a
threshold for the effects of prenatal irradiation encountered during the critical period of fetal development, with
respect to

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RISK ASSESSMENT 119

mental retardation, in children irradiated in utero as a consequence of the atomic bombs in Hiroshima and
Nagasaki.
Of greatest interest and challenge in the development of appropriate models is the possibility, discussed in
Chapter 2 of this report, that early neurotoxic exposure can result in cumulative or progressive damage expressed
as a late-stage degenerative disease, such as dementia or a major motor disorder. Clinical data on the
parkinsonism-dementia complex on Guam and on MPTP intoxication in drug abusers in the United States
indicate that such damage does occur.
Although models for cumulative damage have not been extensively developed for neurotoxicity, Hattis and
Shapiro (1990) exploited the abundance of existing data on acrylamide-induced neurotoxicity in developing a
model that is relevant for reversible, cumulative neurotoxic effects. Alternatively, some of the time-to-tumor
models (Peto et al., 1980; Krewski et al., 1983) considered in cancer risk assessment might be relevant to such
mechanisms of neurotoxicity.
Design and reporting of both animal and epidemiologic experiments could be modified in several respects
to facilitate modeling of neurotoxic end points for risk assessment (Wyzga, 1990). It would be useful to conduct
studies at a number of carefully measured exposure concentrations, including ones of environmental relevance.
Recording of specific quantitative responses is more informative than dichotomizing or categorizing
observations, and making data available in nonaggregated form would enable thorough exploration and
interpretation of research data by other researchers. Summary statistics are sufficient only if the model assumed
to condense the data is appropriate, and that is seldom known with certainty. Comparable sets of animal and
human data should be analyzed in parallel, to establish the most effective methods of using animal data when
human data are not available.

Figure 6-3 Percentage of severe mental retardation among those exposed in utero by dose and gestational age in
Hiroshima and Nagasaki. Vertical lines indicate 90% confidence intervals.
Source: NRC (1990).

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SUMMARY
The critical focus for basic and applied research in neurotoxicology remains in the primary stage of risk
assessment, that is, hazard identification. Few chemicals have been adequately tested for potential neurotoxicity,
and data even on many of those are still insufficient for evaluating risk. In basic research, we still need
investigations to develop models for integrating molecular, biochemical, cellular, and organ-level events into
rational explanations of behavior, function, and learning. Applied research is needed in both clinical and
experimental disciplines, to develop appropriate methods for the detection of neurotoxicity, particularly in
developing and aging organisms. Efforts in the risk assessment of neurotoxicants must focus first on improving
hazard identification. Concerted efforts at improving and validating tests and increasing the vigilance and
sensitivity of epidemiologic surveillance must be undertaken. With improved hazard identification, more data
can be accumulated on dose-response relationships for particular neurotoxicants. The increased data base might
make it possible to undertake additional exercises in statistically based modeling and curve-fitting. At the same
time, further basic research in neuroscience will improve our understanding of the cellular and molecular biology
of the sites of action at which toxicants affect the nervous system.
The visual and visuomotor systems hold great promise for the integration of toxicity data and fundamental
neurobiology. Some chemicals affect vision, others perturb visuomotor control, and some affect both. The great
increase in knowledge about the mechanics of these systems has enabled researchers to develop highly
sophisticated models, including the neurobiology of the retina (e.g., Stryer, 1983), the organization of the visual
cortex (e.g., Hubel and Livingstone, 1987), and the neuromotor control of visual tracking (e.g., Fromm and
Evarts, 1981). At least two neurotoxicants—methylmercury (Friberg, 1977) and methanol (Politis et al., 1980)—
affect visual function. However, no attempt has yet been made to test the fit of existing toxicity data on those two
agents to any of the complex neurobiologic models to predict the results of small exposures or exposures at
different stages of development.
Mechanistic understanding is the most powerful tool in developing models for quantitative risk assessment,
for evaluating predictions of dose-response relationship outside the measurable range, and for extrapolating
across species reliably. Model-building in science is important in identifying critical data gaps. Neurotoxicants
have always served an important role as tools in basic neurobiology (Narahashi, 1989); and basic neurobiology is
essential in neurotoxicity risk assessment. Attempting risk assessment for neurotoxicants can assist in the
identification of critical data gaps to be addressed in developing and validating mechanistic models of
neurotoxicity. For instance, one important general issue in neurotoxicity risk assessment is the potential
irreversibility of damage. There are reasons to anticipate that neurotoxicity will be irreversible or will be
reversible, depending on which components of the nervous system are affected and at what stage of development
or senescence. Research is needed to identify which elements of the CNS are irreplaceable and how
irreplaceability might change to affect reversibility. The research will also increase our understanding of
neurologic development. Similarly, we know little about processes of aging in the brain. If some brain regions
inevitably lose neurons, are they more susceptible to neurotoxic damage, particularly of the delayed type? Would
such damage be appropriately estimated by a linear approach based on the assumption of additivity to
background?
The greatest need in developing quantitative approaches to risk assessment of neurotoxicants is for data on
neurotoxic agents. The intensive study of well-characterized

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RISK ASSESSMENT 121

neurotoxicants can be of great importance, as demonstrated by recent research on lead and n-hexane. In studies
of neurotoxicants, attention should be given to the concentration, duration, and timing of exposure. Especially
rich data sets on specific neurotoxicants should be examined for opportunities to test goodness-of-fit (statistical)
models, as has been done for lead.
Efforts should be directed to developing a broader range of biologically based models for risk assessment of
neurotoxicants, with emphasis on nondichotomous events. It might be useful to examine the research on model-
building that is already under way in neuroscience, particularly cognitive science. Caution should be exercised in
relying on simple uncertainty-factor and threshold models for the risk assessment of all neurotoxicants under all
exposure conditions in all populations.
Basic neurobiologic research (e.g., research on the neurobiology of cortical and cerebellar development and
on the neuronal changes in the aging brain) should include investigation of the effects of toxicants. Research
should be undertaken to further understanding of the role of pharmacokinetics in neurotoxicity so as to
encourage the development of physiologically based pharmacokinetic modes such as are being developed to aid
in carcinogenicity risk assessment, as well as of the differences and similarities between animals and humans
with regard to neurotoxicity. Research should also be undertaken to test the utility of cellular models, such as
receptor-binding models, for hazard identification and for predicting an organism's overall response.

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RISK ASSESSMENT 122

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CONCLUSIONS AND RECOMMENDATIONS 123

7
Conclusions and Recommendations

The Committee on Neurotoxicology and Models for Assessing Risk identified the following six major gaps
in information or conceptual development in environmental neurotoxicology (listed in a sequence to parallel the
structure of the report):
• The extent to which disease and dysfunction of the human nervous system are the result of exposure to toxic
environmental agents
• The neurotoxicity of most chemicals in commerce
• Biologic markers of neurotoxic exposures or effects that could be applied in epidemiologic and clinical studies
• Well-designed, hypothesis-driven neuroepidemiologic research to increase knowledge about the scope of the
problem of neurotoxicity
• Strategies for the rational, efficient testing of chemical substances for neurotoxicity
• Risk-assessment paradigms for evaluation of neurotoxic end points.
The committee's specific conclusions and recommendations to address these gaps are as follows.
The committee concludes that neurotoxic effects can be caused by exposure to chemical agents in the
environment. Environmental chemicals have been shown to cause neurotoxic effects in individual cases and in
epidemics. Neurotoxicity caused by environmental toxicants results in a range of neurologic and psychiatric
disorders; the complexity of the disorders reflects the enormous diversity of the nervous system's functions and
the presence in the nervous system of a large number of cellular and subcellular targets. Neurotoxic outcomes
range from devastating illnesses, such as parkinsonism and dementia, to subtle changes, such as alterations in
behavior and limitations on memory and cognition. In addition to immediate and progressively developing
effects, there is increasing evidence that neurotoxic effects can occur after long latent periods. It is postulated
that intervals as long as many decades can elapse between exposure to a chemical and the appearance of
neurologic illness. Concern over the potential neurotoxic effects of chemical substances is greatest for agents that
cause irreversible or progressive changes. Chemicals can permanently alter brain development and cause
subclinical dysfunction, or they can reduce reserve capacity of the nervous system, which

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CONCLUSIONS AND RECOMMENDATIONS 124

may become manifest as disease in the elderly. On the basis of the available evidence, the committee
hypothesizes that an as-yet-unspecified fraction of human neurologic and psychiatric disease is attributable to
chemical agents in the environment.
The committee concludes that a major obstacle to assessing the extent to which chemicals in the
environment cause nervous system diseases and dysfunction is that little qualitative or quantitative information is
available on possible adverse effects of most environmental chemicals on the nervous system. Some chemicals in
commerce are known to have neurotoxic potential, but most commercial chemicals have not been assessed for
neurotoxicity. Although resources are not readily available to undertake across-the-board testing of all chemicals
already in commerce, prudent public policy dictates that all chemicals, both old and new, be subject to at least
basic screening for neurotoxicity when use and exposure warrant. There is a particular lack of data on chronic
and long-latency neurotoxic effects. Structure-activity relationships, now the most widely used approach to
assessment of toxicity, provide a poor basis for predicting neurotoxic potential; however, greater understanding
of chemical structural correlations and the underlying mechanisms of toxicity can be expected to lead to the
discovery of more useful applications of structure-activity relationships.
The committee recommends that a more accurate estimate of the extent of the problem of neurologic
and psychiatric dysfunction attributable to chemical agents in the environment be made.
The estimate must be based on a combination of clinical, epidemiologic, and toxicologic studies coupled
with the techniques of quantitative risk assessment.
The committee concludes that additional biologic markers for the assessment of subclinical neurotoxic
effects are needed. Such markers can be biochemical, structural, or functional. They can be developed either
through in vitro analyses, through animal studies, or during observational studies in human populations exposed
to environmental neurotoxicants. Although associations between biologic markers and disease are usually
established initially in cross-sectional studies, a particular need exists to validate putative biologic markers in
prospective studies. Only in longitudinal prospective studies can the ability of biologic markers to predict the
occurrence of disease be accurately assessed.
The committee recommends that putative biologic markers in animal species be evaluated and
validated in in vivo and in vitro systems. The committee further recommends that biologic markers be
regularly incorporated into epidemiologic and clinical studies of neurologic disease, particularly
prospective studies.
The primary goal of the incorporation of biologic markers into such studies should be to validate their
predictive accuracy and to test hypothesized quantitative relationships between specific markers that are likely to
be on or close to causal pathways and neurotoxic outcomes.
The legislated authorities of the federal regulatory agencies (the Occupational Safety and Health
Administration, the Environmental Protection Agency, the Consumer Product Safety Commission, and the Food
and Drug Administration) to regulate or to require neurotoxicity testing might be too narrow to encourage
investigation of neurotoxicity. The potential authority of the existing mandates of those agencies to control toxic
substances, including neurotoxicants, has not been fully used.
The committee concludes that tests are available to construct a tiered approach to neurotoxicity testing. The
first tier, or screen, is intended for hazard identification. The results of the screen and a chemical's

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CONCLUSIONS AND RECOMMENDATIONS 125

exposure pattern would determine further characterization of dose-response relationship (second tier) and
mechanisms (third tier). No validated system satisfies all the necessary requirements for a screening program to
detect the neurotoxic potential of chemicals. The range of such a program should extend to the detection of
neurodevelopmental effects and effects on cognitive function and of neuroendocrine effects. No comprehensive
effort has yet been made to determine the predictive ability of individual screening tests by examining the
relationship between test results and data from long-term studies in animals or epidemiologic and clinical studies
in humans.
The committee recommends that a rational, cost-effective neurotoxicity testing strategy be developed
and adopted.
It should allow an accurate and efficient progression from the results of hazard-identification studies
(screening) to the selection and application of appropriate test methods for defining mechanisms of toxicity and
for quantitative characterization of neurotoxic hazards.
Benefits to be gained are several. First, it would permit development of tests that would confirm or disprove
each other at each of several levels of complexity; thus, it would incorporate a series of checks and balances.
Second, the results of the later phases of testing would provide the data necessary to evaluate and validate initial
screening batteries and thereby help to identify tests that should be excluded from or incorporated into an
efficient battery. Third, the information generated by such a strategy would reveal which types of data are most
useful for accurate, quantitative prediction of the risks to humans associated with exposure to similar chemical
compounds.
For reasons of efficiency, integrative studies combining a variety of end points need to be explored in the
development of the strategy. For example, tests of neurotoxicity on chronically exposed animals might be carried
out in conjunction with tests of other chronic effects. An objective testing strategy rationally applied would
enhance risk assessment by providing data rapidly for decision-making. Moreover, testing would identify toxic
effects of substances not previously known to be neurotoxic. To maximize detection of toxicity, some toxicity
studies encompassing the full life span of experimental animals should be encouraged.
The committee recommends that existing in vitro test methods be exploited more extensively than at
present to identify and analyze the mechanisms of neurotoxic action at cellular levels.
To that end, it is necessary to undertake a program to test the relationship between in vitro and in vivo
findings and between animal and human results for a set of welldefined substances. It should be understood that
some in vitro test methods will be most useful only for screening substances and will have little application in
assessing mechanisms. It is necessary to question how effective those methods will be in predicting chronic
effects in whole animals-a continuing objective of method validation. Additional validation might be
accomplished by determining the concordance between the results of tests and the findings of epidemiologic
studies.
The committee recommends that studies to define mechanisms of neurotoxicity in as much detail as
possible be encouraged, as well as studies to identify hazards.
Data are needed on the influence of dose, route of exposure, toxicokinetics, metabolism, and elimination on
the effects of a given neurotoxicant, and data are needed on interspecies differences. Moreover, a detailed
understanding of the pathogenesis of the neurotoxic injury caused by various agents is needed. What are the
toxic metabolites?

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CONCLUSIONS AND RECOMMENDATIONS 126

What are the molecular targets within the nervous system? How does the reaction of a toxicant or metabolite
with a target trigger the sequence of events that lead to functional changes or degenerative events? What is the
sequence of events? What biologic factors determine vulnerability, resistance, and capacity for reversibility and
repair? What is the basis for interspecies differences? Such understanding will make possible the better
prediction of relationships between exposure and neurotoxic response, the development of strategies to prevent
exposure, and eventually the treatment of the exposed.
Development of a mechanistic understanding of neurotoxicity might facilitate the discovery of biologic
markers of exposure to toxicants, as well as markers of early, subclinical neurotoxic effects. More complete
understanding of neurotoxic disease at a molecular level should also improve the ability to evaluate new
chemicals on the basis of structure-activity relationships, which as currently used can provide only minimal
guidance for hazard identification.
The committee concludes that attempts to quantify the exposure of populations to neurotoxic chemicals
have been limited. Clinical evaluation of neurotoxic illness and epidemiologic surveillance of populations at high
risk for neurotoxicity have been fragmentary and inadequate. Few attempts have been made to explore the
possible relationships between chemical exposures and chronic or progressive neurologic and behavioral
disorders. The disorders include developmental delays in the young and some forms of dementia and
parkinsonism in the elderly.
The committee recommends that exposure-surveillance systems cover a much broader range of
chemicals and use improved monitoring techniques for long-term assessment.
The committee also recommends that existing disease-surveillance systems, such as those of the Social
Security Administration, the Department of Veterans Affairs, and the National Center for Health
Statistics, be modified to provide more useful data on the incidence and prevalence of chronic neurologic
and psychologic disorders, some of which are likely to be of occupational and environmental origin.
A broader range of neurologic disease end points should be covered by surveillance programs.
The committee further recommends that anecdotal reports of neurotoxicity in humans be pursued
vigorously with clinical surveillance and followup.
The first step is to identify the substance or combination of agents apparently responsible for observed
effects. When animal models of the human disease have been established, laboratory studies can then determine
the mechanistic details that would assist in controlling the original situation and facilitate risk assessment.
The committee concludes that recognition of the possible environmental origin of neurologic and
psychiatric disease is hampered by the inadequate training of most physicians and other health providers in
occupational and environmental medicine. The committee concludes that uniform clinical definitions of
neurotoxic disorders are needed, to provide a common basis for reporting by physicians.
The committee recommends that improved disease reporting be supported by the dissemination of
information on neurotoxic illnesses to physicians and other health professionals, to increase their
awareness of environmental neurotoxicity as a possible explanation of specific illnesses or sets of symptoms.
The committee further recommends that

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CONCLUSIONS AND RECOMMENDATIONS 127

all physicians should be trained to take a thorough occupational-exposure history and to be aware of other
possible sources of toxic exposure, such as hobbies and selfmedication.
The committee recommends that standardized national reporting systems be established for
physicians to report outbreaks of suspected environmentally and occupationally caused neurologic and
psychiatric disorders.
The incorporation in surveillance systems of the concept of sentinel health events (SHEs) specifically for
neurotoxic illnesses should be encouraged.
The committee concludes that commonly used paradigms for risk assessment do not accurately or
adequately model the risks associated with exposure to neurotoxicants. Neurotoxicologic risk assessment has
been largely limited to the application of no-observed-effect levels and uncertainty factors, so it has not
generated specific risks for given magnitudes of exposure.
The committee recommends that further attention be paid to experimental designs for studies of
neurotoxic agents that provide information needed in the risk-assessment process.
Such variables as age, sex, duration of exposure, and route of exposure need to be more systematically
evaluated. Speciesspecific effects need to be recognized. Experiments should include a range of doses that spans
those relevant to expected human exposures. In addition to providing a firmer basis for estimating human risk,
such designs permit tests of the assumption that results obtained at high doses predict the pattern of effects at low
doses. The usefulness of that kind of information for quantitative risk assessment would be greatly amplified by
serial measures of neurotoxic end points and biologic markers.
The committee recommends that researchers make complete, original data sets available to other
investigators to facilitate full exploration of relationships and development of risk-assessment models.
The committee further recommends that to improve the assessment of the human risks associated
with exposure to possible neurotoxic agents, risk-assessment methods that capture the complexities of the
neurologic response, including dose-time-response relationships, multiple outcomes, and integrated organ
systems be developed.
A single model will not be adequate for all conditions of exposure, for all end points, or for all agents. It
might be necessary to build risk-assessment models to deal simultaneously with several end points produced by a
toxicant. Such models should incorporate biologic markers of neurologic dysfunction and be based on
fundamental information on mechanisms derived from experimental test systems and epidemiologic data.

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REFERENCES 129

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148

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INDEX 149

Index

A B
3-Acetyl-2,5-hexandedione (AcHD), 38 Behavioral assessment, 66, 69-70, 72-73, 87
Acetylcholinesterase, 47, 48, 79 Benchmark dose (BD), 114
Acrylamide, 76, 80, 98, 101, 116, 119 Biochemical assays, 59-62
Agency for Toxic Substances and Disease Registry Biologic markers,
(ATSDR), 1, 108-109 animal models, 49, 54
Alcohol, see Ethanol disease surveillance, 108,
Alzheiner's disease, 15, 40, 47, 48, 51, 83 concepts and definitions, 43-48, 51-52
American Conference of Governmental Industrial Hygien- of effect, 44, 45, 47-48, 51
ists (ACGIH), 17, 53 of exposure, 16, 36, 43, 44, 45, 46-47, 51
Ames test, 59, 112 for in vitro systems, list, 63
γ-Aminobutyric acid (GABA), 35, 63, 79, 117, 118 of neurotoxicity, list, 45
Amyotrophic lateral sclerosis (ALS), 15, 40, 51 quality assurance and quality control, 49-50
Animals recommendations, 5, 124
FOB end points, list, 71 selected, illustration, 92
models, 49, 54, 79 of susceptibility, 44, 45, 48, 51
neurotoxicity testing, 49-50, 53-93, 118, 119, 124, 125 use in risk assessment, 43, 50-51, 124
neurotoxic effects of representative agents, list, 67-68 validation, 44, 48-50, 56, 124
see also Testing Blood-brain barrier, 16, 28-29, 39, 55, 64
Annual Survey of Hiness and Injury, 106 Brain, imaging procedures, 81-83
Antidepressants, tricyclic, 16 Bureau of Labor Statistics (BLS), 106
Australian information-processing theory test battery, 97, C
102, 103
Carbamates, 58-59
Axons, see under Neurons
Carbon disulfide, 11

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INDEX 150

Cells Diisopropyl fluorophosphate (DFP), 75

culture techniques, 61-62, 63-64 γ-Diketones, 31, 32-33, 36-39, 40, 46, 48, 59, 64, 77, 79
ganglia, 27-28 3,4-Dimethyl-2,5-hexanedione (DMHD), 37, 38
glial, 21, 22, 24, 39, 61, 62, 63, 64, 79 Disease surveillance
loss, 51 biologic markers, 108
nuclei, 27, 28 clinical neurologic examination, list of components, 100
plasticity, 29, 30 disease and exposure registries, 108-109
Schwann, 22, 24 identification and prevention efforts, 95-97, 120
see also Nervous system; imaging techniques, 109-110
Neurons medical training, 107
Centers for Disease Control, 50 recommendations, 126-127
Central nervous system (CNS), 27-29, 30, 32, 34, 35, 40, 47, sentinel health events (SHEs), 108, 127
55, 64, 80, 81, 82, 84, 97, 103, 111, 115-116, 120 standardized disease definitions, 107-108
see also Nervous system surveillance efforts, 105-110
Cerebral cortex, 27-28 Dopamine, 29, 31, 33, 39, 40, 47, 78-79
Cerebral glucose, 83 Dose-response relationships, 2-3, 5, 6, 16, 43, 50, 51, 56, 72,
Chemicals 111 , 112, 113, 114-116, 120, 125
diagram of MPTP toxicity, 39 Dyskenesia, 15, 54
exposure response characteristics, list, 98-99 E
neurobehavioral effects, list, 11, 69-70
Electroencephalography (EEG), 78
neurotoxic effects in humans and animals, list, 67-68
Electrophysiology, 31
neurotoxicants, list, 10
Environmental Protection Agency (EPA)
receptors, list, 118
guidelines, 84, 87
regulation and testing, 18-19, 53-93, 95, 97, 123-127
premanufacturing notice (PMN) program, 86
see also Exposure;
regulation and testing of chemicals, 18, 19, 50, 53-54, 58,
Nervous system;
86-87, 89, 105, 124
Risk assessment;
review of substances, 53, 86-87
Testing;
Epinephrine, 48
and specific chemicals
Ethanol, 16, 17-18, 98
Comprehensive Environmental response, Compensation,
Explant cultures, 61, 62
and Liability Act (CERCLA), 109
Exposure
Computed axial tomography (CAT), 31, 81, 82, 83, 109-110
biologic markers, 16, 36, 43, 44, 45, 46-47, 51, 126
Consumer Product Safety Commission, 86, 124
characteristics of neurotoxicant exposure responses, list,
Cycad (Cycas circinalis), 15
98-99
D detection and control, 18-19, 43, 95-110
DDT, 25, 31, 34 environmental, 1-2, 16-19, 95, 96
Department of Veterans Affairs, 126 magnitude of problem, 17-18
Developmental neurotoxicology, 84 occupational, 2, 9, 16, 17, 96
Dieldrin, 117

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INDEX 151

during pregnancy, 15, 50-51 Imaging procedures, 81-83, 109-110

see also Risk assessment In vitro and in vivo testing, see Screening;
F Testing
Insecticides, 25, 34-36, 44, 59
Federal Insecticide, Fungicide, and Rodenticide Act
see also specific insecticides
(FIFRA), 86, 87
Integrated Management Information Systems (IMIS), 104,
Finland Institute of Occupational Health test battery, 97,
105
101, 102
Integrative functions
Food and Drug Administration (FDA), 50, 58, 84, 87, 124
cognitive processes, 66, 70
Functional observational batteries (FOBs), 66-72, 73, 74, 89,
motor performance, 65, 69, 72, 73
90
neurotoxic effects, 65-66, 69-70
G sensory acuity, 65-66, 69, 72, 73-74
Glial fibrillary acidic protein (GFAP), 79 memory, 66, 70, 74-76
Glucose, in nervous system, 32 Intelligence quotient (IQ), 9, 64, 112, 13
Good laboratory practices (GLPs), 50 L
H Lathyrism (spastic paraparesis), 11
Hazard identification, in neurotoxicity risk assessment, 4, Lead, 1, 2, 11, 15, 55, 58, 72, 78-79, 80-81, 98, 105, 108,
112, 120, 125-126 113, 116, 121
Health Care Financing Administration (U.S. Department of Learning/memory function, 66, 70, 74-76
Health and Human Services), 18 Lowest-observed-adverse-effect level (LOAEL), 56, 114-116
Health Hazard Evaluation Program, 97, 104, 107 Lymphocyte neurotoxic esterase (NTE), 79
Hemoglobin adducts, 79 Lysergic acid diethylamide (LSD), 33
n-Hexane, 33-34, 36, 37, 38, 59, 77, 98, 101, 121 M
2,5-Hexanedione (HD), 36, 37, 38, 47, 80
Magnetic resonance imaging (MRI), 31, 81-82, 83, 110
Homovanillic acid (HVA), 78
Malathions, 31
Hormone production, 80
Manganese, 15
Humans
Mental retardation, 15
effects of representative neurotoxic agents, 67-68
Mercury, 1, 2, 15, 51, 58, 79, 98
neurotoxicity testing, 95-110, 118-119, 124, 125
Metabolism, in central nervous system, 28
see also Risk Assessment;
Methanol, 1, 120
Screening;
Methyl n-butylketone, 36, 37, 48, 59, 77, 101
Surveillance;
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, see MPTP
Testing
Methylmercury, 49, 51, 55, 64, 72, 80, 85, 98, 101, 120
Hypothalmus, 27-28
Microencephaly, 15
I Modifying variables, in neurotoxicity testing

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INDEX 152

age at exposure, 83-84 see also Central Nervous System;

age at testing, 84-85 Neurons;
genetic differences, 85 Neurotransmitters;
sex, 85 Peripheral Nervous System
Motor activity and function, 65, 69, 72, 73, 77 Neurobehavioral Core Test Battery (NCTB), 97-101, 102,
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine), 15, 103-104
31, 34, 39-40, 49, 51, 58, 82-83, 98, 110, 119 Neurobehavioral Evaluation System (NES), 101, 102, 104
Myelin, 24, 30, 31, 38, 64, 80-81 Neurobehavioral test batteries, 97-104
N see also specific test batteries
Neurochemical procedures, 78-79
National Center for Health Statistics (NCHS), 106, 108, 126
Neuroendocrine interactions, 79-80, 125
National Center for Toxicological Research (NCTR)
Neurons
behavioral-teratology tests, list, 83
axons, 22-24, 25, 30, 31, 32-33, 38, 55, 64, 77, 80
Collaborative Study, 84
communication, 24-27, 33, 55
National Death Index, 106
development and functions, 21-22, 29-30 , 51
National Health and Nutrition Examination Surveys
glucose, 32
(NHANES), 105, 106
illustrations, 23, 26
National Health Interview Survey, 106
integrative functions, 65-66
National Human Adipose Tissue Survey (NHATS), 105
ions, 24-25, 29, 33, 116
National Institute for Occupational Safety and Health
nerve impulse, 24-25, 26
(NIOSH), 17, 97, 104-105, 107, 108
structure, 22-24
National Institute of Standards and Technology, 50
synaptic messengers, 26-27, 33, 55
National Occupational Exposure Survey (NOES), 104-105,
synaptic transmission, 25-26, 27, 33, 77
109
trophic interactions, 30
National Occupational Hazard Survey (NOHS), 104-105, 109
see also Nervous system;
National Toxicology Program (NTP), 87-88
Neurotransmitter
Nerve-conduction studies, 76-77
Neuropathology, 80-81
Nervous system
Neurophysiologic procedures, 76
biologic markers, 43-52
Neurotoxicity events, list of major, 12-14
cellular anatomy and physiology, 15-16, 21-27, 61, 62,
Neurotransmitters, 25-27, 31, 32, 33, 40, 47, 55, 61, 78,
63-64
111-112, 116
complexity, 29-30, 31
see also Nervous system;
effects of toxicants on, 2, 9-17, 21, 25, 30-40
Neurons
examples of neurotoxic mechanisms, 33-40
No-observed-adverse-effect level (NOAEL), 56, 114-116
lipid-soluble toxicants, 31-32
Nuclear magnetic resonance spectroscopy (MRS), 81, 82
neuropathic evaluation areas, list, 81
receptor sites, 46, 47 O
repair limitations, 31, 40, 51, 55 Occupational Safety and Health Administration (OSHA),
structure and function, 15-16, 21, 27-30, 40-41 104, 105, 109 , 124
tissues used in neurophatologic evaluation, list, 82 Office of Technology Assessment (OTA), 90

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INDEX 153

Office of Toxic Substances (EPA), 87 statistical models, 114-116, 121

Organization for Economic Cooperation and Development strategy, 2-3
(OECD), 50, 86, 90 see also Screening;
Organ cultures, 61-62 Surveillance;
Organophosphates, 47, 58-59, 77, 98 Testing
Organophosphorus (OP) esters, 59-60, 79, 81, 86 S
P Schedule-controlled operant behavior (SCOB), 72-73
Parkinsonism, 15, 31, 34, 39-40, 49, 51, 85, 119 Screening
Peripheral nervous system, 27, 31, 35, 81, 116 applications, 57-58
see also Nervous system evaluation of in vitro tests, 91-92
Pesticides, 1, 2, 17, 31-32, 33, 34, 47, 72, 79, 86, 95, 105, 117 in vitro batteries, 62-65, 90-92
see also specific pesticides in vivo batteries, 66-71
Pharmaceuticals, 2, 9, 16, 18, 33, 54 multitiered testing, 2-3, 88-89, 124-125
Phenylalanine, 85 priority-setting and implementation, 90
Poisoning, pyrethroid, 34-36 protocol for in vitro system, 65
Positron-emission tomography (PET), 31, 40, 81, 82, 83, 110 research needs, 2-3, 90-93
Potassium ions, 24, 25, 33 sensitivity, 56-57
Pregnancy, 15, 50-51 specificity, 56-57
Prevention (primary and secondary), 18, 53, 95-96 strategies for improved testing, 88-89, 124-125
Psychosis, toxic, 15 validation, 3, 57, 89-90, 125
Pyrethroids, 25, 31, 33, 34-36, 44, 59 see also Testing
Pyrrole, 37-38, 59 Sensory-evoked potentials, 77-78
R Sensory function, 65-66, 69, 72, 73-74, 77-78
Sentinel Event Notification System (SENSOR), 108
Radiation, 9, 15
Social Security Administration, 126
Reaggregate cultures, 61, 62
Sodium ions, 24-25, 33, 34, 35-36, 44, 59, 63
Risk assessment
Stimulus-response techniques, 74, 75-76
approaches to, 114-116
Structure-activity relationships (SARs), 4, 19, 54, 56, 58-59,
biologic markers, 43, 50-51, 124, 124, 125
117 , 124, 125
cognitive model, 113
Superfund Amendments and Reauthorization Act (SARA),
curve-fitting, 114, 116-117, 120
105
carcinogenicity models, 111-113, 119
Surveillance
hockey-stick model, 116-117
of disease, 105-110
neurotoxicity models, 111-113, 117-119, 120
of exposure, 104-105
neurotoxicity receptors, 117-118
need, 95-97, 126
no-threshold model, 117-118
neurobehavioral test batteries, 97-104
NRC paradigm, 56
recommendations, 4-5, 126-127
problems, 17-18, 19, 50-51, 111-112
see also Disease surveillance;
recommendations, 5-6, 123-127
Risk Assessment
safety-factor approach, 114-117

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Environmental Neurotoxicology

INDEX 154

T
Testing
behavioral assessment, 66
diagnostic techniques, 31, 47-48
difficulties, 54-55
effects of age, 83-85
effects of genetic differences, 85
effects of sex, 85
electroencephalography, 78
functional observational batteries (FOBs), 66-72, 73, 74,
89, 90
hazard characterization, 56
imaging procedures, 81-83
in vitro, 4, 5, 18-19, 54, 59-65, 90-92, 124, 125
in vivo, 5, 18-19, 54, 65-85, 124, 125
laboratory, 49-50;
see also Animals
modifying variables, 83
motor-activity, 72
nerve-conduction studies, 76-77
neurochemical procedures, 78-79
neuroendocrine interactions, 79-80, 125
neuropathology, 80-81
neurophysiologic procedures, 76
recommendations, 2-5
regulatory approaches, 86-93
schedule-controlled operant behavior, 72-73
screening characteristics and applications, 56-58
sensory evoked potentials, 77-78
specialized function tests, 73-76
standards and guidelines, 18, 19, 49-50, 53-54
strategy, 2-4, 56, 125
structure-activity relationships, 19, 54, 56, 58-59
see also Animals;
Environmental Protection Agency;
Humans;
Screening;
Surveillance
Toxic Release Inventory (TRI), 105
Toxic Substances Control Act (TSCA), 19, 86, 95
Tri-o-cresylphosphate (TOCP), 15
W
World Health Organization (WHO)
recommendations for neuropathologic evaluation, 81
Neurobehavioral Core Test Battery, 97-101, 102, 103-104

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