Wednesday
August 17, 1994
600294001
Part III
Environmental
Protection Agency
Final Report: Principles of Neurotoxicity
Risk Assessment; Notice
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Federal Register / Vol. 59. No. 158 / Wednesday, August 17, 1994 / Notices
ENVIRONMENTAL PROTECTION
AGENCY
[FRL-5050-8]
Final Report: Principles of
Neurotoxlcity Risk Assessment
AGENCY: U.S. Environmental Protection
Agency.
ACTION: Final Document.
SUMMARY: The U.S. Environmental
Protection Agency is publishing a
document entitled Final Report:
Principles of Neurotoxicity Bisk
Assessment, which was prepared by the
Working Party on Neurotoxicology
under the auspices of the Subcommittee
on Risk Assessment of the Federal
Coordinating Council for Science,
Engineering, and Technology (FCCSET).
The purpose of this report is to
articulate a view of neurotoxicology that
scientists generally hold in common
today and to draw on this
understanding to generate a series of
general principles that can be used to
establish guidelines for assessing
neurotoxicity risk. It is not the intent of
this report to provide specific directives
for how neurotoxicity risk assessment
should be performed. The intent of this
document is to provide the scientific
basis for the development of a cogent
strategy for neurotoxicity risk
assessment.
SUPPLEMENTARY INFORMATION: This
document is the result of the combined
efforts of senior scientists of 13 Federal
agencies comprising the ad hoc
Interagency Committee on
Neurotoxicology, including the Agency
for Toxic Substances and Disease
Registry, Center for Food Safety and
Applied Nutrition, Center for Biologies
Evaluation and Research, Center for
Drug Evaluation and Research,
Consumer Product Safety Commission,
Department of Agriculture, Department
of Defense, Environmental Protection
Agency, National Center for
Toxicological Research, National
Institutes of Health, National Institute
for Occupational Safety and Health, and
National Toxicology Program.
Discussions were held under the
auspices of the Working Party on
Neurotoxicology of the Subcommittee
on Risk Assessment of the Federal
Coordinating Council for Science,
Engineering, and Technology. The draft
report, a product of the Working Party
on Neurotoxicology, contains six
chapters: an introduction, an overview
of the discipline of neurotoxicology, a
review of methods for assessing human
neurotoxicity, a review of methods for
assessing animal neurotoxicity, an
overview of principles of neurotoxicity
risk assessment, and a general summary.
The draft report was prepared in view
of the decision-making processes
currently used by many regulatory
agencies relating to neurotoxicity risk
assessment. It is intended that the
principles reviewed in this document
will serve as the basis for consistent
regulatory neurotoxicity guidelines to be
used by Federal agencies to meet their
respective legislative mandates. This
document is not meant to be used to
perform risk assessment nor does it
recommend one approach or strategy.
The document reviews the science of
neurotoxicology and attempts to
formulate general assumptions and
principles that could lead to such
approaches or strategies.
The draft report has undergone
interagency review under the auspices
of the Subcommittee on Risk
Assessment of FCCSET. Public
comments received were used in the
preparation of the final report by the
Working Party on Neurotoxicology.
Dated: August 9, 1994.
Ken Sexton,
Director, Office of Health Research.
Final Report: Principles of
Neurotoxicology Risk Assessment
Contents
1. Introduction
1.1. Background
1.2. Purpose of This Report
1.3. Context of This Report
1.4. Content of This Report
2. Overview of Neurotoxicology
2.1. Scope of the Problem
2.1.1. Introduction
2.1.2. Examples of Neurotoxicity and
Incidents of Exposure
2.1.3. Federal Response
2.1.3.1. Food and Drug Administration
2.1.3.2. Occupational Safety and Health
Administration
2.1.3.3. National Institute for Occupational
Safety and Health
2.1.3.4. Environmental Protection Agency
2.1.3.5. Consumer Product Safety
Commission
2.1.3.6. Agency for Toxic Substances and
Disease Registry
2.2. Basic Toxicological Considerations for
Neurotoxicity
2.2.1. Basic Toxicological Principles
2.2.2. Basic Neurotoxicological Principles
2.3. Basic Neurobiological Principles
2.3.1. Structure of the Nervous System
2.3.2. Transport Processes
2.3.3. Ionic Balance
2.3.4. Neurotransmission
2.4. Types of Effects on the Nervous
System
2.5. Special Considerations
2.5.1. Susceptible Populations
2.5.2. Blood-Brain and Blood-Nerve
Barriers
2.5.3. Metabolism
2.5.4. Limited Regenerative Ability
3. Methods for Assessing Human
Neurotoxicity
3.1. Introduction
3.2. Clinical Evaluation
3.2.1. Neurologic Evaluation
3.2.2. Neuropsychological Testing
3.2.3 Applicability of Clinical Methods to
Neurotoxicology Risk Assessment
3.3. Current Neurotoxicity Testing Methods
3.3.1. Neurobehavioral Methods
3.3.1.1. Test Batteries
3.3.1.2. Investigator-Administered Test
Batteries
3.3.1.3. Computerized Test Batteries
3.3.2. Neurophysiologic Methods
3.3.3. Neurochemical Methods
3.3.4. Imaging Techniques
3.3.5.'Neuropathologic Methods
3.3.6. Self-Report Assessment Methods
3.3.6.1. Mood Scales
3.3.6.2. Personality Scales
3.4. Approaches to Neurotoxicity
Assessment
3.4.1. Epidemiologic Studies
3.4.1.1. Case Reports
3.4.1.2. Cross-Sectional Studies
3.4.1.3. Case-Control (Retrospective)
Studies
3.4.1.4. Prospective (Cohort, Follovvup)
Studies
3.4.2. Human Laboratory Exposure Studies
3.4.2.1. Methodologic Aspects
3.4.2.2. Human Subject Selection Factors
3.4.2.3. Exposure Conditions and Chemical
Classes
3.4.2.4. Test Methods
3.4.2.5. Controls
3.4.2.6. Ethical Issues
3.5. Assessment of Developmental
Neurotoxicity
3.5.1. Developmental Deficits
3.5.2. Methodologic Considerations
3.6. Issues in Human Neurotoxicology Test
Methods
3.6.1. Risk Assessment Criteria for
Neurobehavioral Test Methods
3.6.1.1. Sensitivity
3.6.1.2. Specificity
3.6.1.3. Reliability and Validity
3.6.1.4. Dose Response
3.6.1.5. Structure-Activity
3.6.2. Other Considerations in Risk
Assessment
3.6.2.1. Mechanisms of Action
3.6.2.2. Exposure Duration
3.6.2.3. Time-Dependent Effects
3.6.2.4. Multiple Exposures
3.6.2.5. Generalizability and Individual
Differences
3.6.2.6. Veracity of Neurobehavioral Test
Results
3.6.3. Cross-Species Extrapolation
4. Methods to Assess Animal Neurotoxicity
4.1. Introduction
4.1.1. Role of Animal Models
4.1.2. Validity of Animal Models
4.1.3. Special Considerations in Animal
Models
4.1.3.1. Susceptible Populations
4.1.3.2. Dosing Scenario
4.1.3.3. Other Factors
4.1.3.4. Statistical Considerations
4.2. Tiered Testing in Neurotoxicology
4.2.1. Type of Test
4.2.2. Dosing Regimen
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Federal Register / Vol. 59, No. 158 / Wednesday, August 17. 1994 / Notices 42361
4.3. Endpoints of Neurotoxicity
4.3.1. Introduction
4.3.2. Behavioral Endpoints
4.3.2.1. Functional Observational Batteries
4.3.2.2. Motor Activity
4.3.2.3. Neuromotor Function
4.3.2.4. Sensory Function
4.3.2.5. Learning and Memory
4.3.2.6. Schedule-Controlled Behavior
4.3.3. Neurophysiological Endpoints of
Neurotoxicity
4.3.3.1. Nerve Conduction Studies
4.3.3.2. Sensory Evoked Potentials
4.3.3.3. Convulsions
4.3.3.4. Electroencephalography
4.3.3.5. Electromyography
4.3.3.6. Spinal Reflex Excitability
4.3.4. Neurochemical Endpoints of
Neurotoxicity
4.3.5. Structural Endpoints of
Neurotoxicity
4.3.6. Developmental Neurotoxicity
4.3.7. Physiological and Neuroendocrine
Endpoints
4.3.8. Other Considerations
4.3.8.1. Structure-Activity Relationship
4.3.8.2. In Vitro Methods
5. Neurotoxicology Risk Assessment
5.1. Introduction
5.2. The Risk Assessment Process
5.2.1. Hazard Identification
5.2.1.1. Human Studies
5.2.1.2. Animal Studies
5.2.1.3. Special Issues
5.2.2. Dose-Response Assessment
5.2.3. Exposure Assessment
5.2.4. Risk Characterization
5.3. Generic Assumptions and Uncertainty
Reduction
6. General Summary
7. References
Tables
1-1. Major Regulatory Agencies
1-2. Authorities for Toxicity Testing
2-1. Human Neurotoxic Exposures
3-1. Neurobehavioral Methods
4-1. Examples of Potential Endpoints of
Neurotoxicity
4-2. Examples of Specialized Tests to
Measure Neurotoxicily
4-3. Summary of Measures in the Functional
Observational Battery and the Type of
Data Produced by Each
4—4. Neurotoxicants With Known
Neurochemical Mechanisms
4-5. Examples of Known Neuropathic Agents
4-6. Partial List of Agents Believed to Have
Developmental Neurotoxicity
5-1. General Assumptions That Underlie
Traditional Risk Assessments
1. Introduction
1.1. Background
Over the years, agencies and programs
have been established to deal with
hazardous substances, with a focus on
deleterious long-term effects, including
noncancer endpoints such as
neurotoxicity (Reiter, 1987). Recent
evidence indicates that exposure to
neurotoxic agents may constitute a
significant health problem (WHO, 1986;
OTA, 1990; chapter 2). Table 1-1 lists
the four Federal regulatory agencies
with authority to regulate either
exposure to or use of chemicals and that
require data reporting on assessment of
hazards. Regulatory bodies vary greatly
in their mandate to require approval of
chemicals prior to entering the
marketplace and to regulate subsequent
exposure (Fisher, 1980) (Table 1-2). The
Occupational Safety and Health
Administration (OSHA) cannot require
chemical testing by the manufacturer
whereas all other agencies can. Only the
Food and Drug Administration (FDA)
and the Environmental Protection
Agency (EPA) have authority for
premarketing testing of chemicals (i.e.,
FDA for drugs and food additives and
EPA for pesticides). EPA can, under
some circumstances, require premarket
testing of industrial and agricultural
chemicals. The Consumer Product
Safety Commission (CPSC) regulates a
number of consumer products including
household chemicals and fabric
treatments. Laws administered by CPSC
require cautionary labeling on all
hazardous household products whether
the hazard is based on acute or chronic
effects. These laws also provide the
authority to ban hazardous products and
to ask for data in support of product
labeling.
TABLE 1-1.—MAJOR REGULATORY AGENCIES
Agency
Statute and sources covered
Food and Drug Administration (FDA)
A unit of the Department of Health and Human Services with authority
over the regulation of medical and veterinary drugs; foods and food
additives; cosmetics.
Occupational Safety and Health Administration (OSHA)
A unit of the Department of Labor that regulates workplace conditions ..
Environmental Protection Agency (EPA).
Independent agency (i.e., not part of a Cabinet department); admin-
isters a number of diverse laws concerned with human health and
the environment.
Consumer Product Safety Commission (CPSC).
Regulates a variety of consumer products including household chemi-
cals and fabric treatments.
Food, Drug, and Cosmetics Act for food additives; color in cosmetics;
medical devices; animal drugs of medical and feed additives.
Occupational Safety and Health Act covers toxic chemicals in the
workplace.
Toxic Substances Control Act requires premanufacture evaluation of
all new chemicals (other than foods, food additives, drugs, pes-
ticides, alcohol, tobacco); allows EPA to regulate existing chemical
hazards not sufficiently controlled under other laws.
Clean Air Act requires regulation of hazardous air pollutants.
Federal Water Pollution Control Act governs toxic water pollutants.
Safe Drinking Water Act covers drinking water contaminants.
Federal Insecticide, Fungicide, and Rodenticide Act covers pesticides.
Resource Conservation and Recovery Act covers hazardous wastes.
Marine Protection Research and Sanctuaries Act covers ocean dump-
ing.
Federal Hazardous Substances Act covers "toxic" household products.
Consumer Product Safety Act covers dangerous consumer products.
Poison Prevention Packaging Act covers packaging of dangerous chil-
dren's products.
Lead-Based Paint Poison Prevention Act covers use of lead paint in
federally assisted housing.
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Federal Register / Vol. 59, No. 158 / Wednesday. August 17. 1994 / Notices
TABLE 1-2.—AUTHORITIES FOR TOXICITY TESTING
Agency
FDA
EPA
OSHA . .. .
CPSC
Law
Food, Drug, and Cosmetics Act
Federal Insecticide, Fungicide, and
Rodenticide Act.
Toxic Substances Control Act
Clean Air Act
Resource Conservation and Recov-
ery Act.
Occupational Safety and Health Act .
Federal Hazardous Substances Act .
Consumer Product Safety Act
Coverage
Food additives and
cosmetics.
Industrial chemicals ...
Air pollutants
Industrial waste
Occupational expo-
sure.
Consumer products ...
Consumer products ...
Authorities
Premarketing
approval
X
X
X
'X
Testing by man-
ufacturer
X
X
X
X
X
X
Reporting of
data
X
X
X
X
X
X
1 Can require testing based on available data.
1.2. Purpose of This Report
The purpose of this document is to:
(1) articulate a view of neurotoxicity
that scientists generally hold in
common today and (2) draw upon this
understanding to compose, as was done
here by senior scientists from a number
of Federal agencies, a series of general
principles that can be used to establish
general guidelines for assessing
neurotoxicity risk. It is not the intent of
this report to provide specific directives
to agencies with respect to their own
approach for neurotoxicity risk
assessment. This document is intended
to provide the scientific basis for the
development of a cogent strategy for
neurotoxicology risk assessment as
needed by each agency.
Because of present gaps in
understanding, the principles contained
in this document are based on the best
judgment of those involved in writing
this document, as well as statements of
what is generally accepted as fact. There
has been, however, an attempt to
distinguish where possible between the
different types of information presented.
The principles contained in this
document can serve as the basis for
consistent regulatory neurotoxicology
guidelines that the Federal agencies can
tailor to meet the requirements of the
legislative acts they are charged to
implement. This document should be
viewed broadly as part of an ongoing
process within the Federal Government
to periodically update and review the
current scientific understanding and
regulatory utility of neurotoxicity risk
assessment.
This document is the result of the
combined efforts of senior scientists
from the following Federal health-
related units, operating under the
direction of the Office of Science and
Technology Policy (OSTP):
Agency for Toxic Substances and
Disease Registry (ATSDR)
Center for Biologies Evaluation and
Research (CBER), FDA
Center for Drug Evaluation and Research
(CDER), FDA
Center for Food Safety and Applied
Nutrition (CFSAN), FDA
Consumer Product Safety Commission
Department of Agriculture (USDA)
Department of Defense (DoD)
Environmental Protection Agency
National Center for Toxicological
Research (NCTR), FDA
National Institutes of Health (NIH)
National Institute for Occupational
Safety and Health
National Toxicology Program (NTP)
1.3. Context of This Report
This document was prepared in light
of a decision-making process used by
many regulatory agencies pertaining to
the assessment of neurotoxicity risks
posed by chemical agents. The scientific
basis for such assessment can be best
understood by examining the decision-
making process in some detail.
Risk can be thought of as being
composed of two aspects, each of which
can be addressed by science, i.e., hazard
and exposure assessment. Although
other definitions have been used
historically, this document conforms to
present usage. Hazard generally refers to
the toxicity of a substance and is
deduced from a wide array of data,
including those from epidemiological
studies or controlled clinical trials in
humans, short- and long-term
toxicological studies in animals, and
studies of mechanistic information and
structure-activity relationships.
Exposure generally refers to the amount
of a substance with which people come
in contact. The risk in a quantitative risk
assessment is estimated by considering
the results of the exposure and hazard
assessments. As either the hazard or
exposure approaches zero, the risk also
approaches zero.
As a first step in assessing the
neurotoxic risk associated with the use
of a particular chemical substance, the
qualitative evidence that a given
chemical substance is likely to be a
human neurotoxicant must be
evaluated. In this step, as in the whole
process, a number of assumptions and
approximations must be made in order
to deal with inherent limitations found
in the existing data bases. Then,
estimates of human exposure and
distribution of exposures likely to be
encountered in the population are
made. In the absence of dose-response
relationships in humans, one or more
methods for estimating the dose-
response relationship including doses
below those generally used
experimentally must also be evaluated.
Finally, the exposure assessment is
combined with the dose-response
relationship to generate an estimate of
risk. The various ways in which these
steps are conducted and combined and
their attendant uncertainties constitute
what is generally referred to as
"neurotoxicity risk assessment."
Some legislation calls for action in the
presence of any risk. Other forms of
legislation use the concept of
unreasonable risk, defined in some acts
as a condition in which the risks
outweigh the benefits. A spectrum of
regulatory responses, from simply
informing the public of a risk through
restricted use to a complete ban, may be
available to bring the risks and benefits
into appropriate balance.
This document does not perform a
risk assessment nor does it suggest that
one method of neurotoxicology risk
assessment is better than another.
Rather, it attempts to review the science
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42363
of chemical neurotoxicology and
develops from this review a set of
general principles. It is not a
comprehensive review nor a document
written for the lay public; this document
is a semitechnical review that evaluates
the impact of scientific findings of the
last decade on general assumptions or
principles important to risk assessment.
This is based on the belief that
elucidation of the basic mechanisms
underlying neurotoxicity and the
identification of neurotoxic agents and
conditions, when coupled to research
aimed at identifying and characterizing
the problems caused by such agents,
should provide the best scientific bases
for making sound and reasonable
judgments. These overlapping
approaches to evaluating the problems
of neurotoxicology should form a strong
foundation for decision-making.
1.4. Content of This Report
Including the Introduction (chapter
1), this document contains six chapters.
Chapter 2 provides an overview of the
discipline of neurotoxicology. It is
important to understand the scope of
the problem as it relates to
neurotoxicology, including: (1)
Definitions of neurotoxicity and adverse
effect, (2) examples of neurotoxicity and
incidents of exposure, and (3) Federal
response to neurotoxicology. Chapter 2
also discusses the basic principles of
toxicology that apply generally to the
evaluation of neurotoxicity. Issues such
as dose, exposure, target site, and the
intended use of the chemical are
discussed, as are principles of
pharmacodynamics, chemical
interactions, and the concept of
threshold. Chapter 2 also lays the
neurobiological basis for understanding
how and where chemicals can affect the
nervous system and provides examples
of such chemical types. Finally, chapter
2 discusses special considerations for
neurotoxicology including the issue of
susceptible populations, the blood brain
barrier, and the limited capability of the
nervous system to repair following
chemical insult.
Chapter 3 examines methods for
assessing human neurotoxicity.
Neurologic evaluations,
neuropsychological testing, and
applicability of methods used in clinical
evaluations and case studies are
discussed in this chapter. Epidemiologic
study designs, endpoints, and methods
are also discussed, as well as problems
of causal inference and applications and
limitations of epidemiologic and field
study methods for risk assessment.
Chapter 3 also describes human
laboratory exposure studies, including
methods for assessing neurobehavioral
function, self-report methods for
assessing subjective states, and a
number of other methodological issues.
This chapter also discusses the
comparability of human and animal
laboratory methods and special
considerations in human neurotoxicity
assessments.
Chapter 4 assesses methods for
evaluating animal neurotoxicity.
Discussed in this chapter is the role that
animal models play in the assessment of
chemicals for neurotoxicity, the validity
of animal models, and experimental
design considerations in animal
neurotoxicological studies. Also
included in this chapter is a discussion
of tier-testing approaches in chemical
evaluations. Specific endpoints used in
animal neurotoxicological studies are
also discussed, including methods for
neurobehavioral, neurophysiological,
neuroanatomical, and neurochemical
assessments. Developmental
neurotoxicology and in vitro
neurotoxicology are also described in
this chapter.
Chapter 5 of this document discusses
principles of neurotoxicity risk
assessment. This chapter evaluates the
generic assumptions in neurotoxicity
risk assessment, ending with a
discussion of uncertainty reduction and
identification of knowledge gaps.
Chapter 6 is a general summary of the
material presented in the first five
chapters.
2. Overview of Neurotoxicology
2.1. Scope of the Problem
2.1.1. Introduction
Chemicals are an integral part of our
lives, with the capacity to both improve
as well as endanger our health. The
general population is exposed to
chemicals with neurotoxic properties in
air, water, foods, cosmetics, household
products, and drugs used
therapeutically or illicitly. Naturally
occurring neurotoxins, such as fish and
plant toxins, present other hazards.
During the daily life of an ordinary
person, there is a multitude of
exposures, both voluntary and
unintentional, to neuroactive
substances. Under conditions of
multiple exposures, identifying the
substance responsible for an adverse
response may be difficult. The EPA's
inventory of toxic chemicals is greater
than 65,000 and increasing yearly.
Concerns have been raised about the
toxicological data available for many
compounds used commercially (NRC,
1984).
It is not known how many chemicals
are neurotoxic to humans. However,
estimates have been made for subsets of
substances. A large percentage of the
more than 500 registered active
pesticide ingredients are neurotoxic to
varying degrees. Of 588 chemicals listed
by the American Conference of
Government and Industrial Hygienists
(ACGIH), 167 affected the nervous
system or behavior (Anger, 1984; CDC,
1986). Using a generally broad
definition of neurotoxicity, Anger
(1990a) estimated that of the
approximately 200 chemicals to which
1 million or more American workers are
exposed, more than one-third may have
adverse effects on the nervous system at
some level of exposure. Anger (1984)
also recognized neurotoxic effects as
one of the ten leading workplace
disorders. In addition, a number of
therapeutic substances, including some
anticancer and antiviral agents and
abused drugs, can cause adverse or
neurotoxicological side effects (OTA,
1990). It has been estimated that there
is inadequate toxicological information
available for more than three-fourths of
the 12,860 chemicals with a production
volume of 1 million pounds or more
(NRC, 1984). It should be noted,
however, that estimates concerning the
number of neurotoxicants vary widely.
O'Donoghue (1989), for example,
reported that of 488 compounds
assessed in his chemical evaluation
process, only 2.7% had effects on the
nervous sytem.
2.1.2. Examples of Neurotoxicity and
Incidents of Exposure
There is a long-standing history
associating certain neurological and
psychiatric disorders to exposure to a
toxin or chemical of an environmental
origin (OTA, 1990) (Table 2-1). Lead is
one of the earliest examples of a
neurotoxic chemical with widespread
exposure. This metal is widely
distributed with major sources of
inorganic lead including industrial
emissions, lead-based paints, food,
beverages, and the burning of leaded
gasolines. Organic lead compounds
such as tetraethyl lead have been
reported to produce a toxic psychosis
(Cassells and Dodds, 1946). If exposure
occurs at relatively low levels during
development, lead can cause a variety of
neurobehavioral problems, learning
disorders, and altered mental
development (Bellinger et al., 1987;
Needleman, 1990). Over the years,
Federal Government regulations have
been developed to decrease human
exposure to lead, and as a goal an
intervention level of 10 ug/dcl whole
blood has been recommended (CDC,
1991). Lead exposure in the United
States has decreased significantly
during the last several years.
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TABLE 2-1 .—HUMAN NEUROTOXIC EXPOSURES
Year(s)
370 B.C
1 st century A.D .
1837
1924
1930
1930's
1932
1937
1946
1950's
1950's
1950's
1950's
1956
1956
1959
1960
1964
1968
1969
1969
1971
1971
1972
1973
1974-1975
1976
1977
1979-1980
1980's
1981
1983-84
1985
1987
1988
1989
1991
Location
Greece
Rome
Scotland
United States (New Jer-
sey).
United States (South-
east).
Europe
United States (Califor-
nia).
South Africa
England
Japan (Mina-
mata)
France
Morocco
Guam
Turkey
Japan
Morocco
Iraq
Japan
Japan
Japan
United States (New
Mexico).
United States
Iraq
France
United States (Ohio)
United States (Virginia) .
United States (Texas) ....
United States (Califor-
nia).
United States (Texas) ....
United States
Spain
United States
United States and Can-
ada.
Canada
India
United States
Nigeria
Substance
Lead
Lead
Manganese
Tetraethyl lead
Tri-c-cresylphosphate
(TOCP).
Apiol
Thallium
TOCP
Tetraethyl lead
Methylmercury
Organotin
Manganese
Cycad
Clioouinol
TOCP
PCBs
n-Hexane
Hexachlorophene
Methyl n-butylketone
Chlordecone (Keptone) .
Leptophos (Phosvel)
Dichloropropene (Telone
II).
2-t-Butylazo-2-hydroxy-
5-methylhexane
(BHMH) (Lucel-7).
Methylphenyltetrahydro-
pyridine (MPTP).
Toxic oil
Vitamin 85
Aldicarb
Domoic acid
TOCP
L-tryptophan-containing
products.
Scopoletin
Comments
Lead toxicity recognized in mining industry.
Vapors recognized as toxic.
Chronic manganese poisoning described.
Workers suffer neurologic symptoms.
Chemical contaminant added to Ginger Jake, an alcoholic beverage
substitute; more than 5,000 paralyzed, 20,000 to 100,000 affected.
Drug containing TOCP causes 60 cases of neuropathy.
Contaminated barley laced with thallium sulfate poisons family, caus-
ing neurologic symptoms.
Paralysis develops after use of contaminated cooking oil.
Neurologic effects observed in people cleaning gasoline tanks.
Fish and shellfish contaminated with mercury are ingested, causing
neurotoxicity.
Medication (Stalinon) containing diethyltin diiodide results in poisoning.
Miners suffer chronic manganese intoxication.
Ingestion of plants associate^ with amyortrophic lateral sclerosis and
Parkinson-like syndrome.
Hexachlorobenzene causes poisoning.
Drug causes neuropathy.
Cooking oil contaminated with lubricating oil causes poisoning.
Mercury-treated seed grain causes neurotoxicity.
Methylmercury neurotoxicity.
Polychlorinated biphenyls are leaked into rice oil, causing
neurotoxicity.
Neuropathy due to n-hexane exposure.
Fungicide-treated grain results in alkyl mercury poisoning.
Hexachlorophene-containing disinfectant is found to be toxic to nerv-
ous system.
Methylmercury used as fungicide to treat seed grain causes poisoning.
Hexachlorophene poisoning of children.
Fabric production plant employees exposed to MnBK solvent suffer
polyneuropathy.
Chemical plant employees exposed to insecticide suffer severe
neurologic problems.
At least nine employees suffer serious neurologic problems after expo-
sure to insecticide.
People hospitalized after exposure to pesticide.
Employees of manufacturing plant experience serious neurologic prob-
lems.
Impurity in synthesis of illicit drug causes Parkinson's disease-like ef-
fects.
People ingesting toxic substance in oil suffer severe neuropathy.
Excessive intake, causes sensory neuropathy, numbness, parathesia,
and motor dysfunction.
People experience neuromuscular deficits after ingestion of contami-
nated melons.
Ingestion of mussels contaminated with domoic acid causes illnesses.
Ingestion of adulterated rapeseed oil cause polyneuritis.
Ingestion of a chemical contaminant associated with the manufacture
of L-tryptophan results in eosinophilia-myalgia syndrome.
Natural component of gari caused neuropathy associated with optic at-
rophy and ataxia.
Mercury compounds are potent
neurotoxic substances and have caused
a number of human poisonings, with
symptoms of vision, speech, and
coordination impairments (Chang,
1980). Erethism, a syndrome with such
neurologic features as tremor and
behavioral symptoms as anxiety,
irritability, and pathologic shyness, is
seen in people exposed to elemental
mercury (Bidstrup, 1964). One major
incidence of human exposure occurred
in the mid-1950's when a chemical
plant near Minamata Bay, Japan,
discharged mercury as part of waste
sludge. An epidemic of mercury
poisoning developed when the local
inhabitants consumed contaminated
fish and shellfish. Congenitally affected
children displayed a progressive
neurological disturbance resembling
cerebral palsy and manifested other
neurological problems as well. In 1971,
an epidemic occurred in Iraq from
methylmercury used as a fungicide to
treat grain (OTA, 1990).
Manganese is used in metal alloys and
has been proposed to replace lead in
gasoline. It is an essential dietary
substance for normal body functioning
yet parenteral exposure to manganese
can be neurotoxic, producing a
dyskinetic motor syndrome similar to
Parkinson's disease (Cook et al., 1974).
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Exposed miners in several countries
have suffered from "manganese
madness" characterized by
hallucinations, emotional instability,
and numerous neurological problems.
Long-term manganese toxicity produces
muscle rigidity and staggering gait
similar to that seen in patients with
Parkinson's disease (Politis et al., 1980).
A Parkinsonian-like syndrome was
also observed in people who
accidentally ingested l-methyl-4-
pheny 1-1,2,3,6-tetrahy dropyridine
(MPTP) (Langston et al., 1983). MPTP
was a byproduct of a meperidine
derivative sold illicitly as "synthetic
heroin."
Organic solvents are encountered
frequently in occupational settings.
Most solvents are volatile, i.e., they can
be converted from a liquid to a gaseous
state and readily inhaled by the worker.
They are also lipid soluble and readily
accumulate in the fat deposits of the
exposed organism. An example of a
solvent exposure in humans is carbon
disulfide. Workers exposed to high
levels of this solvent were found to have
an increased frequency of depression
and suicide (Seppalainen and Haltia,
1980). Furthermore, repeated exposure
to organic solvents is suspected of
producing chronic encephalopathy.
Workers exposed to methyl-n-butyl
ketone, a dye solvent and cleaning
agent, displayed peripheral nervous
system neuropathy involving
degeneration of nerve fibers (Spencer
and Schaumburg, 1980). Solvents
including ether, ketones, alcohols, and
various combinations are commonly
used in glues, cements, and paints and
when inhaled can be neurotoxic.
Repeated abuse of such solvents can
lead to permanent neurological effects
due to severe and permanent loss of
nerve cells (OTA, 1990).
Pesticides are one of the most
commonly encountered classes of
neurotoxic substances. These can
include insecticides (used to control
insects), fungicides (for blight and
mildew), rodenticides (for rodents such
as rats, mice, and gophers), and
herbicides (to control weeds). Active
ingredients are combined with so-called
inert substances to make thousands of
different pesticide formulations.
Workers who are overexposed to
pesticides may display obvious signs of
poisoning, including tremors, weakness,
ataxia, visual disturbances, and short-
term memory loss (Ecobichon and Joy,
1982). Chlordecone exposure results in
nervousness and tremors (Cannon et al.,
1978). The organophosphorous
insecticides have neurotoxic properties
and account for approximately 40
percent of registered pesticides. A
delayed neurotoxicity can be seen as a
result of exposure to certain
organophosphate pesticides, producing
irreversible loss of motor function and
an associated neuropathology
(Ecobichon and Joy, 1982).
Organophosphate and carbamate
insecticides are known to interfere with
a specific enzyme, acetylcholinesterase
(AChE) (Davis and Richardson, 1980).
Paralysis has also been reported
following consumption of nonpesticide
organophosphate products such as tri-o-
cresylphosphate (TOCP).
Neurotoxicities in humans, domestic
livestock, and poultry associated with
fungal toxins (mycotoxins) have been
well documented (Kurata, 1990; Aibara,
1986; Wyllie and Morehouse, 1978).
Mycotoxins not only have a negative
economic effect on animal production,
but they also represent a definite threat
to human health. Mycotoxins occur in
forages, field crops, and grains used for
livestock; they also are incorporated
into cereals, grains, and grain-based
products used for human consumption.
Therefore, human exposure may occur
either through direct consumption of
these products or secondarily through
consumption of meat, milk, or eggs. An
example of human exposure to fungal
toxins is Claviceps purpurea- or C.
paspay/-infected wheat, barley, and oats
used for bread and as a dietary
supplement for livestock. These fungal
toxins are notorious for producing the
gangrenous and convulsive forms of the
disease known as "ergotism" (Bove,
1970). These fungi are in the family
Clavicipitaceae and produce a group of
compounds known as ergot alkaloids,
which have neurotropic, uterotonic, and
vasoconstrictive activities. They may act
as dopamine agonists or serotonin
antagonists, and also block alpha-
adrenergic receptors. Since there are
numerous naturally occurring ergot
alkaloids, this represents only part of
their pharmacopoeia (Berde and
Schield, 1978). These alkaloids are
highly toxic and cause both acute and
chronic poisonings. Although
guidelines now limit the amount of
C/avi'ceps-contaminated, or "ergot"-
contaminated, grains, these compounds
may enter human food sources through
secondary mechanisms. Other fungi
associated with ergot-like syndromes in
livestock include Acremonium lolii
(Gallagher et al., 1984) and A.
coenophialum (Thompson and Porter,
1990).
Cyclopiazonic acid (CPA) is an indole
tetramic acid produced by Aspergillus
flavus, A. oryzae, Penicillium
cyclopium, and P. camemberti. This
mycotoxin is suspected of causing
"kodua poisoning" in humans who
consumed kodo millet seed in India
(Rao and Husain, 1985). Fusarium
moniliforme is a common fungal
infection in corn (Bacon et al., 1992)
and directly related to neurotoxic
syndrome in horses known as equine
leukoencephalomalaisia (ELEM).
Natural plant toxins also represent a
health risk to both livestock and
humans. Movement toward limited uses
of herbicides, fungicides, and no-till
agricultural practices increases the
possibility of noxious weeds and weed
seeds being incorporated into food
products. Ergot alkaloids also are
produced by morning glories (Ipomea
violacea) and may be incorporated into
soybeans, corn, peas, etc., during
harvest. Export regulations limit
morning glory-contaminated soybeans
because of the hallucinogenic and other
effects produced by ergot alkaloids.
Jimson weed (Datura stramonium),
another weed incorporated into
agricultural commodities, produced
scopolamine, hyocyamine, and stropine,
all of which have parasympatholytic
(anticholinergic) activities.
Recently, an outbreak of toxic
encephalopathy caused by eating
mussels contaminated with domoic
acid, an excitotoxin, was reported (Perl
et al., 1990).
2.1.3. Federal Response
In the United States, several agencies,
including EPA, FDA, OSHA, CPSC,
N1OSH, and ATSDR, have been given
the mandate to regulate or evaluate
public exposure to toxic chemicals
(Tilson, 1989).
2.1.3.1. Food and Drug Administration.
The FDA has the authority to regulate
the use of food and color additives as
well as to determine whether or not
various foods are unsafe for human
consumption because of adulteration by
environmental contaminants. The
manufacturer must supply adequate
data to establish the safety of the food
additives. Before marketing approval,
the potential toxicity of proposed food
and color additives is established in a
battery of animal toxicity studies.
During all of these studies, clinical signs
of toxicity, including abnormal
behavior, are monitored and
abnormalities recorded. At the
termination of these studies, tissues
from all organs, including the brain, are
sectioned and evaluated for both gross
and histopathological changes, in
addition to being evaluated for their
clinical chemistry and hematology.
None of the routinely required tests is
specifically designed to assess
neurotoxicity. If neurotoxic effects are
detected during any of the standard
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toxicity tests, however, they must be
reported. Specific neurotoxicity testing
may then be required. The FDA is
currently revising its guidelines for the
safety assessment of direct food and
color additives to include neurotoxicity
as a routine element in toxicological
testing.
The FDA is also authorized to regulate
substances in food considered to be
poisonous or deleterious. Unavoidable
environmental contaminants in food fall
into this category. The FDA determines
a level at which the risks from
realistically possible intakes are
negligible or acceptable. Based on this
risk assessment, an action level or
tolerance is established. Once the action
level or tolerance is formally
established, the FDA may take
appropriate action to restrict adulterated
food from the market if these standards
are exceeded.
The FDA is responsible for assessing
the toxicity of human therapeutic
products. Many products have been
shown to produce adverse effects on the
nervous system at standard therapeutic
doses as well as at higher doses. Before
marketing approval is given, the toxicity
of potential new products is assessed. A
battery of animal toxicity study
parameters relevant to the nervous
system, including gross behavioral
observation and gross and
histopathological examination of the
nervous tissue, are evaluated. This
information is used to help guide the
surveillance of human subjects for
adverse effects that are assessed during
clinical trials.
2.1.3.2. Occupational Safety and Health
Administration.
OSHA has been given the
responsibility to ensure that the working
environment is a safe and healthy place
of employment. In the early 1970's,
OSHA adopted the existing Federal
standards, most of which were
developed under the Walsh-Healy Act
(including the 1968 ACGIH Threshold
Limit Values), and approximately 20
consensus standards of the American
National Standards Institute (ANSI) as
Permissible Exposure Limits (PELs). Of
the 393 remaining original PELs, 145
were set in part to protect the individual
from neurotoxic effects.
Since the adoption of the initial
standards, OSHA has issued new or
revised health standards or work
practices for 23 substances. Of these, the
one concerning lead was based in part
on nervous system effects. Four other
compounds, inorganic arsenic,
acrylonitrile, ethylene oxide, and 1,2-
dibromo-3-chloropropane, were cited as
causing various disturbances in the
nervous system, but the standards for
these were based primarily on
carcinogenic effects.
In 1989, OSHA updated 428 exposure
limits for air contaminants. Of these, 25
substances were categorized by OSHA
as "substances for which limits are
based on avoidance of neuropathic
effects." In addition, 24 substances were
included in the category "substances for
which limits are based on avoidance of
narcosis." However, OSHA stated that
the categorization was intended as a tool
to manage the large number of
substances being regulated and not to
imply that the category selected
identified the most sensitive or the
exclusive adverse health effects of that
substance.
2.1.3.3. National Institute for
Occupational Safety and Health.
The Occupational Safety and Health
Act established NIOSH as a Public
Health Service (PHS) agency to develop
and recommend criteria for prevention
of disease and hazardous conditions in
the workplace. NIOSH also performs
research on occupational health issues
and conducts worksite evaluations of
suspected hazards. OSHA and the Mine
Safety and Health Administration
(MSHA) use NIOSH recommendations
in the promulgation of new or revised
health and safety standards.
In establishing recommended
exposure limits (RELs) for chemicals,
NIOSH examines all relevant scientific
information about a given compound
and attempts to identify exposure limits
that will protect nil workers from
adverse effects. NIOSH has
recommended standards for
approximately 644 chemicals or classes
of chemicals. For 214 (33 percent) of
these, neurotoxicity was cited as a
health effect considered when
formulating the REL (NIOSH, 1992).
2.1.3.4. Environmental Protection
Agency.
The Toxic Substances Control Act
(TSCA) and the Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA)
provide the legislative authority for EPA
to require data collection for premarket
approval of chemicals. Under section 5
of TSCA, after a manufacturer has
notified EPA of its plans to produce a
"new',' chemical that has not yet been
listed on the inventory, EPA has the
responsibility to assess possible health
hazards. Potential neurotoxicity is
included in the health hazards
assessment. If there are reasons to
suspect neurotoxicologic effects (e.g.,
from structure-activity analysis,
information in the literature, or data
submitted by the manufacturer), EPA
can issue a test rule requiring the
manufacturer to develop data directed
toward these effects. At the same time,
EPA can restrict the chemical or
prohibit it entirely from entering
commerce until the required data are
submitted and reviewed. In addition, for
"old" chemicals (under section 4 of
TSCA), if EPA suspects neurotoxicity, a
test rule would be the mechanism used
for obtaining the data. Many other
statutes provide authority to regulate
chemicals through the setting of
standards, including the Clean Air Act,
Clean Water Act, and Safe Drinking
Water Act.
Neurotoxicity is recognized as a
health effect of concern under FIFRA,
and there are neurotoxicity testing
requirements for premarketing
submission of data to EPA for
registration of a pesticide under FIFRA.
2.1.3.5. Consumer Product Safety
Commission.
The CPSC is an independent Federal
regulatory agency with jurisdiction over
most consumer products. Most chemical
hazards are regulated under the Federal
Hazardous Substances Act (FHSA)
administered by CPSC. The FHSA
requires appropriate cautionary labeling
on all hazardous household products
(hazards include chronic toxicity such
as neurotoxicity). While the FHSA does
not require premarket registration, a
manufacturer is required to assess the
hazards of a product prior to marketing
and assure that it is labeled with all
necessary cautionary information. Tho
FHSA also bans children's products that
are hazardous and provides the CPSC
with the authority to ban other
hazardous products.
2.1.3.6. Agency for Toxic Substances
and Disease Registry.
ATSDR has a mission to prevent or
mitigate adverse effects to both human
health and the quality of life resulting
from exposure to hazardous substances
in the environment. The ATSDR
publishes a National Priority List (NPL)
of hazardous substances that are found
at National Priority Waste Sites. The
order of priority is based on an
algorithm, taking into consideration
frequency with which substances are
found at NPL sites, toxicity, and
potential for human exposure; this list
is reranked on a yearly basis. So far, 129
toxicological profiles have been
developed for the priority hazardous
substances, and 92 substances have a
profile with a neurological health effect
endpoint (HAZDAT, 1992).
Neurotoxicity has been selected by the
ATSDR to be one of the seven high-
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priority health conditions resulting from
exposure to environmental toxicants.
2.2. Basic Toxicological Considerations
for Neurotoxicity
2.2.1. Basic Toxicological Principles
A chemical must enter the body,
reach the tissue target site(s), and be
maintained at a sufficient concentration
for a period of time in order for an
adverse effect to occur. Not all
chemicals have the same level of
toxicity; some may be very toxic in
small amounts while others may have
little effect even at extremely high
amounts. Thus, the dose-response
relationship is a major concept in
determining the toxicity of a specific
substance. Other factors in determining
toxicity include the physical and
chemical properties of the substance,
the route and level of exposure, the
susceptibility of the target tissue, and
the health, gender, and age of the
exposed individual.
Once the toxic substance has entered
the body, usually through the lungs
(inhalation), the skin (absorption), or the
gastrointestinal tract (ingestion), it is
partitioned into various body tissues
where it can act on its target sites. The
substance is eliminated from the
bloodstream by the process of
accumulation into the various sites in
the body, with the liver and kidney
being major sites of accumulation of
toxic substances. This is thought to be
associated with these organs' large
blood capacity and major role in
elimination of substances from the
body. Lipophilic chemicals accumulate
in lipid-rich areas of the body and
present a significant potential problem
for the nervous system. The nervous
system is unique in its high percentage
content of lipid (50 percent of dry
weight) and may be particularly
vulnerable to such chemicals. The site
or sites of accumulation for a specific
toxic substance may or may not be the
primary sites of action. Examples
include two known neurotoxicants,
carbon monoxide in the red blood cells
and lead in the bone. It must be noted
that some substances are not distributed
throughout the body, partially as a
function of their insolubility, polarity,
or molecular weight.
The effect that a substance has will
generally depend on the body burden or
level in the tissue and duration of
exposure. The time course of the levels
is determined by several factors,
including the amount at time of
exposure, duration of exposure, and
metabolic fate of the chemical. The
study of such metabolic processes,
pharmacokinetics, has demonstrated
complex patterns in the absorption,
distribution, possible biotransformation,
and elimination of various substances
(Klaassen, 1980).
Many substances are removed by the
kidney and excreted through the urine.
The liver can detoxify substances like
organic lead, which are excreted from
the liver into the bile and then the small
intestines, bypassing the blood and
kidney. Lipophilic toxic substances are
primarily removed from the body
through feces and bile, and water-
soluble metabolites are removed in the
urine, through the skin, and through
expiration into the air.
Biotransformation is a biochemical
process that converts a substance into a
different chemical compound, allowing
it to be excreted more easily. Substances
are more easily removed if they are
biotransformed into a more hydrophilic
compound. Biotransformation can either
aid in the detoxification of a substance
or produce a more toxic metabolite.
Therefore, the original substance may
not be the substance that is producing
the toxicity on the nervous system or
any other system. Thus, several factors
must be taken into consideration when
evaluating the potential neurotoxicity of
a chemical. They include the
pharmacokinetics of the parent
compound, the target tissue
concentrations of the parent chemical or
its bioactivated proximate toxicant, the
uptake kinetics of the parent chemical
or metabolite into the cell and/or
membrane interactions, and the
interaction of the chemical or metabolite
with presumed receptor sites.
2.2.2. Basic Neurotoxicological
Principles
Neurotoxicity can be manifest as a
structural or functional adverse
response of the nervous system to a
chemical, biological, or physical agent
(Tilson, 1990b). It is a function of both
the property of the agent and a property
of the nervous system itself.
Neurotoxicity refers broadly to the
adverse neural responses following
exposure to chemical or physical agents
(e.g., radiation) (Tilson, 1990b). Adverse
effects include any change that
diminishes the ability to survive,
reproduce, or adapt to the environment.
Neuroactive substances may also impair
health indirectly by altering behavior in
such a way that safety is decreased in
the performance of numerous activities.
Toxicity can occur at any time in the life
cycle, from conception through
senescence, and its manifestations can
change with age. The range of responses
can vary from temporary responses
following acute exposures to delayed
responses following acute or chronic
exposure to persistent responses.
Neurotoxicity may or may not be
reversible following cessation of
exposure. The responses may be graded
from transient to fatal and there may be
different responses to the same
neurotoxicant at different dose levels
but similar responses to exposure to
different agents. Displays of a
neurotoxic response may be progressive
in nature, with small deficits occurring
early in exposure and developing to
become more severe over time.
Expression of neurotoxicity can
encompass multiple levels of
organization and complexity including
structural, biochemical, physiological,
and behavioral measurements.
Caution must be exercised in labeling
a substance neurotoxic. The intended
use and effect of the chemical, the dose,
exposure scenario and whether or not
the chemical acts directly or indirectly
on the nervous system, must be taken
into consideration. A substance that
may be neurotoxic at a high
concentration may be safe and
beneficial at a lower concentration. For
example, vitamin A, vitamin B6, are
required in the diet in trace amounts,
yet all result in neurotoxicity when
consumed in large quantities.
Pharmaceutical agents may also have
adverse effects at high dose levels or
where the beneficial effects outweigh
the adverse side effects. For example,
antipsychotic drugs have allowed many
people suffering from schizophrenia to
lead relatively normal lives; however,
chronic prescribed use of some of these
drugs may result in severe tardive
dyskinesia characterized by involuntary
movements of the face, tongue, and
limbs. Other examples include toxic
neuropathies induced by
chemotherapeutic agents like cis-
platinum, toxic anticholinergic effects of
high doses of tricyclic antidepressants,
disabling movement disorders in
patients treated with anti-Parkinsonian
agents and major tranquilizers, and
hearing loss and balance disruption
triggered by certain antibacterials
(Sterman and Schaumburg, 1980). Drugs
of abuse such as ethanol also have
neurotoxic potential. Opiates such as
heroin may lead to dependence, which
is considered to be a long-term adverse
alteration of nervous system
functioning. Simultaneous exposure to
drugs or toxic agents may produce toxic
interactions either in the environment
or occupational settings. For example,
exposure to noise and certain antibiotics
can exacerbate the loss of hearing
function (Boottcher et al., 1987; Lim,
1986; Bhattacharyya and Dayal, 1984).
The nervous system is a highly
complex and integrated organ. It is
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possible that nonlinear dose-response
relationships or a threshold effect could
exist for some agents. It has been
hypothesized that the nervous system
has a reserve capacity that masks subtle
damage and any exposure that does not
overcome this reserve capacity may not
reach the threshold and no observable
impairment will be evident (Tilson and
Mitchell, 1983). However, the functional
reserve may be depleted over time and
the manifestations of toxicity may be
delayed in relationship to the exposure.
The reserve may be depleted by a
number of factors including aging,
stress, or chronic exposure to an
environmental insult, in which case
functioning will eventually be impaired
and toxicity will become apparent. If a
number of events occur simultaneously,
the response is progressive in nature, or
there is a long latency between exposure
and manifestation of toxicity, the
identification of a single cause of the
functional impairment may not be
possible.
2.3. Basic Neurobiological Principles
2.3.1. Structure of the Nervous System
The nervous system is composed of
two parts: the central nervous system
(CNS) and the peripheral nervous
system (PNS) (Spencer and
Schaumburg, 1980). Within the nervous
system, there exist predominantly two
general types of cells—nerve cells
(neurons) and glial cells. Neurons have
many of the same structures found in
every cell of the body; they are unique,
however, in that they have axons and
dendrites, extensions of the neuron
along which nerve impulses travel. The
structure of the neuron consists of a cell
body, 10 to 100 um in diameter,
containing a nucleus and organelles for
the synthesis of various components
necessary for the cell's functioning, e.g.,
proteins and lipids. There are numerous
branch patterns of elongated processes,
the dendrites, that emanate from the cell
body and increase the neuronal surface
area available to receive inputs from
other sources. Neurons communicate
with each other by releasing chemical
signals onto specific surface regions,
receptors, of the other neuron. The axon
is a process specialized for the
conduction of nerve impulses away
from the cell toward the terminal
synapses and eventually toward other
cells (neurons, muscle cells, or gland
cells).
Neurons are responsible for the
reception, integration, transmission, and
storage of information (Raine, 1989).
Certain nerve cells are specialized to
respond to particular stimuli. For
example, chemoreceptors in the mouth
and nose send information about taste
and smell to the brain. Cutaneous
receptors in the skin are involved in the
sensation of pressure, pain, heat, cold,
and touch. In the retina, the rods and
cones sense light. In general, the length
of the axon is tens to thousands of times
greater than the cell body diameter. For
example, the cell body whose processes
innervate the muscles in the human foot
is found in the spinal cord at the level
of the middle back. The axons of these
cells are more than a meter in length.
Many, but not all, axons are surrounded
by the layers of membrane from the
cytoplasmic process of glial cells. These
layers are called myelin sheaths and are
composed mostly of lipid. In the PNS,
the myelin sheaths are formed by
Schwann cells, while in the CNS the
sheaths are formed by the
oligodendroglia. The myelin sheath
formed by one glial cell covers only a
short length of the axon. The entire
length of the axon is ensheathed in
myelin by numerous glial cells. Between
adjacent glial sheaths, a very short
length of bare axon exists called the
node of Ranvier. In unmyelinated axons,
a nerve impulse must travel in a
continuous fashion down the entire
length of the nerve. The presence of
myelin accelerates the nerve impulse by
up'to 100 times by allowing the impulse
to jump from one node to the next in a
process called "saltatory conduction."
The nerve cells of the PNS are
generally found in aggregates called
ganglia. The brain and spinal cord make
up the CNS and the neurons are
segregated into functionally related
aggregates called nuclei. They
synthesize and secrete
neurotransmitters, which are
specialized chemical messengers that
interact with receptors of other neurons
in the communication process. Various
nuclei together with the interconnecting
bundles of axonal fibers are functionally
related to one another to form higher
levels of organization called systems.
For example, there is the motor system,
the visual system, and the limbic
system. At the base of the brain, several
small nuclei in the hypothalamus form
the neuroendocrine system, which plays
a critical role in the control of the
body's endocrine (hormone-secreting)
glands. Nerve cells in the hypothalamus
secrete chemical messengers into a short
loop of blood vessels that carries the
messengers to the pituitary gland which,
in turn, releases chemical messengers
into the general circulation. These
pituitary messengers regulate other
glands (e.g., the thymus and the
gonads). The entire system maintains a
state of optimal physiological function
for all of the body's organ systems.
2.3.2. Transport Processes
All types of cells must transport
proteins and other molecular
components from their site of
production near the nucleus to the other
sites in the cell (Hammerschlag and
Brady, 1989). Neurons are unique in
that the neuronal cell body must
maintain not only the functions
normally associated with its own
support, but it must also provide
support to its various processes. This
support may require transport of
material over relatively vast distances.
Delivery of necessary substances by
intracellular transport down the axon
(axonal transport) represents a supply
line that is highly vulnerable to
interruption by toxic chemicals. In
addition, the integrity of the function of
the neuronal cell body is often
dependent on a supply of trophic factors
from the cells that it innervates. These
factors are continually supplied to the
neural cells by the process of retrograde
axonal transport, often as a process of
normal exchange between two or more
cells. They play a significant factor in
the normal growth and maintenance of
the neural cells, and a continual supply
of certain trophic factors is necessary for
cell functioning.
The majority of axonal transport
occurs along longitudinally arranged
fiber tracks called neurofilaments. This
movement along neurofilaments
requires energy in the form of oxidative
metabolism. Toxicants that interfere
with this metabolism or that disrupt the
spatial arrangement or production of
neurofilaments may block axonal
transport and can produce neuropathy
(Lowndes and Baker. 1980). This can be
seen following exposure to many
substances, such as n-hexane and
methyl n-butyl ketone as well as the
drugs vincristine, vinblastine, and taxol.
Acrylamide produces a dying-back
axonopathy but by an alternative
mechanism involving altered axonal
transport.
2.3.3. Ionic Balance
The axonal membrane is
semipermeable to positively and
negatively charged ions (mostly
potassium, sodium, and chloride)
within and outside of the axon. There
are several enzyme systems that
maintain an ionic balance that changes
following depolarization of the
membrane (Davies, 1968). This is
maintained only by the continual active
transport of ions across the membrane,
which requires an expenditure of
energy. The nerve impulse is a traveling
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42369
wave of depolarization normally
originating from the cell body; however,
in sensory neurons it originates at the
terminal receptive end of specialized
axons (Davies, 1968). The wave is
continued by openings in the membrane
that allow ions to rush into the axon.
This sudden change in the charge across
the axon's membrane is the nerve
impulse. It is an amplified
depolarization that reaches the
threshold value and spreads down the
axon from one length to another until
the next length of membrane reaches the
threshold value. It continues in this
fashion until it reaches the synaptic
terminal regions. There are a number of
varieties of membrane channels (e.g.,
calcium) that rapidly open and close
during impulse generation; the common
ones are the sodium and potassium
channels. They are very small and allow
only ions of a certain size to pass.
Several classes of drugs (e.g., local
anesthetics) and natural toxins (e.g.,
tetrodotoxin) inhibit nerve impulse
conduction by blocking these channels.
2.3.4. Neurotransmission
The terminal branches of the axon
end in small enlargements called
synaptic "boutons." It is from these
boutons that chemical messengers will
be released in order to communicate
with the target cell at the point of
interaction, the synapse (Hammerschlag
and Brady, 1989). When the nerve
impulse reaches the terminal branches
of the axon, it depolarizes the synaptic
boutons. This depolarization causes the
release of the chemical messengers
(neurotransmitters and
neuromodulators) stored in vesicles in
the axon terminal (Willis and Grossman,
1973). Classical neurotransmitters
include serotonin, dopamine,
acetylcholine, and norepinephrine and
are typically secreted by one neuron
into the synaptic cleft where they are on
the postsynaptic membrane.
Neuropeptides, however, may travel
long distances through the bloodstream
to receptors on distant nerve cells or in
other tissues. Following depolarization,
the amount of secretion is dependent on
the number of nerve impulses that reach
the synaptic bouton, i.e., the degree of
depolarization. The chemical
messengers diffuse across the synaptic
cleft or into the intraneuronal space and
bind to receptors on adjacent nerve cells
or effector organs, thus triggering
biochemical events that lead to
electrical excitation or inhibition.
When information is transmitted from
nerves to muscle fibers, the point of
interaction is called the neuromuscular
junction and the interaction leads to
contraction or relaxation of the muscle.
When the target is a gland cell, the
interaction leads to secretion. Synaptic
transmission between neurons is
slightly more complicated, but still
dependent on the opening and closing
of ion channels in the membrane. The
binding of the messenger to the receptor
of the receiving cell can lead to either
the excitation or inhibition of the target
cell. At an excitatory synapse, the
neurotransmitter-receptor interaction
leads to an opening in certain ion-
specific channels. The charged ions that
move through these opened chambers
carry a current that serves to depolarize
the cell membranes. At inhibitory
synapses, the interaction leads to an
opening in a different type of ion-
specific channel that produces an
increase in the level of polarization
(hyperpolarization). The sum of all the
depolarizing and hyperpolarizing
currents determines the transmembrane
potential and when a threshold level of
depolarization is reached at the axon's
initial segment, a nerve impulse is
generated and begins to travel down the
axon.
The duration of neurotransmitter
action is primarily a function of the
length of time it remains in the synaptic
cleft. This duration is very short due to
specialized enzymes that quickly
remove the transmitter either by
degrading it or by reuptake systems that
transport it back into the synaptic
bouton. A toxic substance may disrupt
this process in several different ways. It
is important that the duration of the
effect of synaptically released chemical
messengers be limited. Some
neurotoxicants, e.g., cholinesterase-
inhibiting organophosphorous
pesticides, inhibit the enzyme (AChE),
which serves to terminate the effect of
the neurotransmitter (acetylcholine) on
its target. The result is an
overstimulation of the target cell. Other
substances, particularly biological
toxins, are able to interact with the
receptor molecule and mimic the action
of the neurotransmitter. Some toxic
substances, like neuroactive
Pharmaceuticals, may interfere with the
synthesis of a particular
neurotransmitter, while others may
block the neurotransmitter's access to its
receptor molecule.
2.4. Types of Effects on the Nervous
System
The normal activity of the nervous
system can be altered by many toxic
substances. A variety of adverse health
effects can be seen ranging from
impairment of muscular movement to
disruption of vision and hearing to
memory loss and hallucinations (WHO,
1986; Anger, 1984,1990). Toxic
substances can alter both the structure
and the function of cells in the nervous
system. Structural alterations include
changes in the morphology of the cell
and its subcellular structures. In some
cases, agents produce neuropathic
conditions that resemble naturally
occurring neurodegenerative disorders
in humans (Calne et al., 1986). Cellular
alterations can include the
accumulation, proliferation, or
rearrangement of structural elements
(e.g., intermediate filaments,
microtubules) or organelles
(mitochondria) as well as the
breakdown of cells. By affecting the
biochemistry and/or physiology of a
cell, a toxic substance can alter the
internal environment of any neural cell.
Intracellular changes can result from
oxygen deprivation (anoxia) because
neurons require relatively large
quantities of oxygen due to their high
metabolic rate.
Many times the response of the
nervous system to a toxic substance can
be a slow degeneration of the nerve cell
body or axon that may result in
permanent neuronal damage.
Substances can act as a cytotoxicant
after having been transported into the
nerve terminal. A complete loss of nerve
cells can occur following exposure to a
number of toxic substances. Sensory
nerve cells may be lost following
treatment with megavitamin doses of
vitamin B6; hippocampal neurons
undergo degeneration with trimethyltin
and trimethyl lead poisoning; motor
nerve cells are affected in cycad
toxicity, which has been loosely linked
to Guam-ALS-Parkinsonism dementia.
Acute carbon monoxide poisoning can
produce a delayed, progressive
deterioration over a period of weeks of
portions of the nervous system that may
lead to psychosis and death. Substances
such as mercury and lead can cause
central nervous system dysfunction. In
children, mercury intoxication can
cause degeneration of neurons in the
cerebellum and can lead to tremors,
difficulty in walking, visual
impairment, and even blindness. Lead
affects the cortex of the immature brain,
resulting in mental retardation.
At the cellular level, a substance
might interfere with cellular processes
like protein synthesis, leading to a
reduced production of
neurotransmitters and brain dysfunction
(Bondy, 1985). Nicotine and some
insecticides mimic the effects of the
neurotransmitter acetylcholine.
Organophosphorous compounds,
carbamate insecticides, and nerve gases
act by inhibiting AChE, the enzyme that
inactivates the neurotransmitter
acetylcholine. This results in a buildup
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of acetylcholine and can lead to loss of
appetite, anxiety, muscle twitching, and
paralysis. Amphetamines stimulate the
nervous system by releasing and
blocking reuptake of the
neurotransmitters norepinephrine and
dopamine from nerve cells. Cocaine
affects the release and reuptake of
norepinephrine, dopamine, and
serotonin. Both drugs can cause
paranoia, hyperactivity, aggression, high
blood pressure, and abnormal heart
rhythms. Opium-related drugs such as
morphine and heroin act at specific
opioid receptors in the brain, producing
sedation, euphoria, and analgesia. They
also tend to slow the heart rate and
cause nausea, convulsions, and slow
breathing patterns. Other substances can
alter the synthesis and release of
specific neurotransmitters and activate
their receptors in specific neuronal
pathways. They may perturb the system
by overstimulating receptors, blocking
transmitter release and/or inhibiting
transmitter degradation, or blocking
reuptake of neurotransmitter precursors.
Also at the cellular level, the flow of
ions such as calcium, sodium, and
potassium across the cell membrane
may be changed and the transmission of
information between nerve cells altered.
A substance may interfere with the ionic
balance of a neuron. Organophosphate
and carbamate insecticides produce
autonomic dysfunction and
organochlorine insecticides increase
sensorimotor sensitivity, produce
tremors and in some cases cause
seizures and convulsions (Ecobichon
and Joy. 1982). Lindane, DDT,
pyrethroids, and trimethyltin also
produce convulsions. Conversely,
solvents act to raise the threshold for
eliciting seizures or act to reduce the
severity or duration of the elicited
convulsions.
The role of excitatory amino acid
(EAA)-mediated synaptic activation is
critical for normal function of the CNS.
Because endogenous EAA-mediated
synaptic transmission is a widespread
excitatory system in the brain and is
involved in the process of learning and
memory, the issue of the effects of
endogenous and exogenous EAA-related
toxicity has broad implications for both
CNS morbidity and mortality in
humans. Much of the injury and
neuronal death associated with toxicity
is mediated by receptors for excitatory
amino acids, especially glutamic acid.
When applied in sufficient excess from
either endogenous or exogenous
sources, EAAs have profound
neurotoxic effects that can result in the
destruction of neurons and, as a
consequence, lead to acute phase
confusion, seizures, and generalized
weakness or to persistent impairments
such as memory loss (Choi, 1988).
A final common path in the activation
of these receptor classes is an increase
in free cytosolic Ca~ that can result in
the release and activation of
intracellular enzymes (which break
down the cytoskeleton) and in further
release of glutamate, both of which can
be cytotoxic (Choi, 1988). Critical to an
understanding of the etiopathology
associated with at least some of the
neurotoxic degeneration may be the link
that impaired energy metabolism could
have with excitotoxic neuronal death. It
is likely that reduced oxidative
metabolism results in the partial
depolarization of resting membrane
potential, the activation of ionotropic
membrane receptor/channels, and the
influx of Ca~ or its release from
intracellular stores.
The nervous system is dependent on
an extensive system of blood vessels
and capillaries to deliver large
quantities of oxygen and nutrients as
well as to remove toxic waste products.
Damage to the capillaries in the brain
can lead to the swelling characteristic of
encephalopathy. This can be seen
following exposure to higher
concentrations of lead. Other metals
(e.g., cadmium, thallium, and mercury)
and organotin (e.g., trimethyltin) cause
rupturing of vessels that can also result
in encephalopathy.
One large aspect of function that may
be affected by neurotoxicants is
behavior, which is the product of
various sensory, motor, and associative
functions of the nervous system.
Neurotoxic substances can adversely
affect sensory or motor functions,
disrupt learning and memory processes,
or cause detrimental behavioral effects;
however, the underlying mechanisms
for these effects have yet to be
determined. Although changes may be
subtle, the assessment of behavior may
serve as a robust means of monitoring
the well-being of the organism (Tilson
and Cabe, 1978).
2.5. Special Considerations
2.5.1. Susceptible Populations
Everyone is at a certain level of risk
of being adversely affected by
neurotoxic substances. Individuals of
certain age groups, health states, and
occupations, however, may be at a
greater level of risk. Fetuses, children,
the elderly, workers in occupations
involving exposure to relatively high
levels of toxic chemicals, and persons
who abuse drugs are among those in
high-risk groups. Neurotoxic substances
may exacerbate existing neurological or
psychiatric disorders in a population.
Although controversial (Waddell, 1993),
recent evidence suggests that there may
be a subpopulation of people who have
become sensitive to chemicals and
experience adverse reactions to low-
level exposures to environmental
chemicals (Bell, et al., 1992).
Confounded in all of these groups is the
role that nutrition plays in the response
of the organism to exposure. Both
general nutritional status and specific
nutritional deficiencies (for example,
protein, iron, and calcium) can
significantly influence the response to a
toxic substance.
It is widely accepted that during
development adverse effects can result
from exposure to some chemicals at
lower levels than would be necessary
for the average adult (Suzuki, 1980). The
developing nervous system appears to
be differentially sensitive to some kinds
of damage (Cushner, 1981; Pearson and
Dietrich, 1985; Annau and Eccles, 1986;
Hill and Tennyson, 1986; Silbergeld,
1986). During the developmental period,
the nervous system is actively growing
and establishing intricate cellular
networks. Both the blood-brain and
blood-nerve barriers that will eventually
protect much of the adult brain, spinal
cord, and peripheral nerves are
incomplete. The protective mechanisms
by which the organism deals with toxic
substances, such as the detoxification
systems, are not fully developed.
Exposure to chemicals during
development can result in a range of
effects. At the highest exposure, effects
include death, gross structural
abnormalities, or altered growth. Larger
populations are generally exposed to
more moderate levels resulting in more
subtle functional impairments. The
qualitative nature of some injuries
during development may differ from
those seen in the adult, such as changes
in tissue volume, misplaced or
misoriented neurons, or delays or
acceleration of the appearance of
functional or structural endpoints
(Rodier, 1986). In many cases, the
results of early injuries may become
evident only as the nervous system
matures and ages (Rodier, 1990). There
are several instances in which
functional alterations have resulted
from exposure during the period
between conception and sexual maturity
(Riley and Vorhees, 1986; Vorhees,
1987).
Early exposure to relatively low levels
of lead can result in reduced scores on
tests of mental development (Bellinger
et al., 1987; Needleman, 1990). Early
gestational exposure to neurotoxicants
such as cocaine can produce long-term
neurobehavioral abnormalities
(Anderson-Brown et al., 1990;
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Hutchings et al.. 1989); heavy alcohol
exposure produces craniofacial
abnormalities and mental retardation
(Jones and Smith, 1973), while moderate
levels of alcohol consumption during
gestation can delay motor development
(Little et al., 1989).
With aging, the level of risk for a
number of health-related factors
increases; it has been hypothesized that
the risk for toxic perturbations to the
nervous system also increases with age
(Weiss, 1990). It is generally believed
that with increasing age comes a
decreased ability of the nervous system
to respond to adverse events or to
compensate for either biological,
physical, or toxic effects. At the tissue
and cellular level, the aging process can
result in nerve cell loss, formation of
neurofibrillary tangles (abnormal
accumulation of certain filamentous
proteins) and neuritic plaques
(abnormal clusters of proteins and other
substances near synapses). As cells die,
the complex neuronal circuitry of the
brain becomes impaired.
Neurotransmitter concentrations and the
enzymes involved in their synthesis
may be altered. Some axons can
gradually lose their myelin sheath,
resulting in a slowed conduction of
nerve impulses along the axon. It has
been postulated that with age, not only
might the nervous system become more
susceptible to new insults, but the
effects of previous exposures also may
become evident, with a diminished
capacity for compensation (Weiss,
1990). The increased incidence of
multiple drug-taking in the elderly
population might also lead to
interactions, either drug/drug or drug/
chemical, which can adversely affect the
nervous system. Nutritionally, the aged
experience increased incidences of both
general undernutrition and deficits of
specific nutrients such as iron or
calcium, which might influence the
response to toxic substances.
In the geriatric population, the
clinical manifestation of
neurodegenerative disorders may have a
contributing component of past
exposures to environmental chemical
agents. Calne et al. (1986) hypothesized
that various agents contribute to
Alzheimer's disease, Parkinson's
disease, or amyotrophic lateral sclerosis
(ALS, motoneurone disease, or Lou
Gehrig's disease) by depleting neuronal
reserves to an extent that perturbations
become observable in the context of the
natural aging process. B-N-
methylamino-L-alanine, from the seed
of the false sago palm (Cycas circinalis
L.), has been reported to induce a form
of amyotrophic lateral sclerosis
(Spencer et al., 1987). Alzheimer-type
syndromes have been reported in
individuals occupationally exposed to
organic solvents or metal vapors (Freed
and Kandel, 1988). Severe cognitive
dysfunction has been noted in
Alzheimer's disease and aluminum
intoxication (Yokel et al., 1988).
At any age, preexisting physical as
well as mental disorders of the
individual may play a significant role in
the manifestation of a toxic response
following exposure to a potentially toxic
substance. Both types of disorders
compromise the system in some way so
that either the defense mechanisms of
the organism are not able to deal with
the toxic substance or are not able to
repair themselves quickly. In addition to
the basic altered biology, for individuals
with a physical or mental disorder who
are under some form of medical
intervention, the combination of
therapeutic drugs and toxic substances
may have an interactive effect on the
nervous system. For example, due to the
delicate electrochemical balance of the
nervous system, mental disorders may
be exacerbated by exposure to a toxic
substance.
2.5.2. Blood-Brain and Blood-Nerve
Barriers
The bioavailability of a specific
chemical to the nervous system is a
function of both the target tissue and the
chemical. The brain, spinal cord, and
peripheral nerves are surrounded by a
series of semipermeable tissues referred
to as the blood-brain and blood-nerve
barriers (Katzman, 1976; Peters et al.,
1991). In the central nervous system, the
blood-brain barrier is composed of tight
junctions formed by endothelial cells
and astrocytes. These tight junctions
and cellular interactions forming the
barrier restrict the free passage of most
bloodborne substances. By doing this,
they create a finely controlled
extracellular environment for the nerve
cells. Certain regions of the brain and
nerves are directly exposed to chemicals
in the blood because the barrier is not
present in some areas of the nervous
system. For example, it is absent in the
circumventricular area, around the
dorsal root ganglion in the peripheral
nervous system, and around the
olfactory nerve, which may allow
chemicals to penetrate directly from the
nasal region to the frontal cortex.
The existence of these blood-brain
and blood-nerve barriers suggests that
proper functioning of the nervous
system is dependent on control of the
substances to which nerve cells are
exposed. The term "barrier," however,
is somewhat of a misnomer. Although
water-soluble and polar compounds
enter the brain poorly, lipophilic
substances readily cross the barrier. In
addition, a series of specific transport
mechanisms exist through which
required nutrients (hormones, amino
acids, peptides, proteins, fatty acids,
etc.) reach the brain (Pardridge, 1988). If
toxicants are lipid soluble or if they are
structurally similar to substances that
are normally transported into the brain,
they can achieve high concentrations in
brain tissue. It has been proposed that
one reason why the developing nervous
system may be differentially sensitive to
some toxicants is that the blood-brain
barrier is less effective than in an adult.
The effectiveness of the blood-brain
barrier may also be changed by
chemical-induced physiological events
such as metabolic acidosis and
nutritional deprivation.
2.5.3. Metabolism
The central nervous system has a very
high metabolic rate and, unlike other
organs, the brain depends almost
entirely on glucose as a source of energy
and raw material for the synthesis of
other molecules (Damstra and Bondy,
1980). The absence of an alternative
energy source makes the CNS critically
dependent on an uninterrupted supply
of oxygen as well as the proper
functioning of enzymes that metabolize
glucose. Substances can be toxic to the
nervous system if they perturb neuronal
metabolism. Without glucose, nerve
cells usually begin to die within
minutes. Despite its relatively small
size, the energy demands of the brain
require 14 percent of the heart's output
and consumes about 18 percent of the
oxygen absorbed by the lungs.
2.5.4. Limited Regenerative Ability
The nervous system has a
combination of special features not
found in other organ systems. It is
composed of a variety of metabolically
active neurons and supporting cell types
that interact through a multitude of
complex chemical mechanisms. Each
cell type has its own functions and
vulnerabilities. At the time of puberty,
the system is fully developed and
neurogenesis (the birth of new neurons
from cell division of precursor cells
called neuroblasts) ceases. This is in
marked and significant contrast to
almost all other tissues, where cell
replacement is continual.
It is this loss of neurogenesis that
limits the nervous system's ability to
recover from damage and influences the
plasticity of the system. Neurons are
unable to regenerate following damage;
therefore, they are no longer able to
perform their normal functions. Toxic
damage to the brain or spinal cord that
results in cell loss is usually permanent.
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If nerve cell loss is concentrated in one
of the CNS's functional subsystems, the
outcome could be debilitating; for
example, a relatively small loss of
neurons that use acetylcholine as their
neurotransmitter may produce a
profound disturbance of memory. A
relatively minor insult concentrated in a
subsystem that relies on dopamine as its
neurotransmitter may drastically impair
motor coordination. However, in
response to injury, neurons are able to
show considerable plasticity both
during development and after
maturation. Damage to the nervous
system alters connectivity between the
surviving neurons, permitting
functional adjustments to occur to
compensate for the damage. Such
responsiveness may, in and of itself,
have profound consequences for
neurological, behavioral, and related
body functions.
After damage to axons in the
peripheral nerves, if the neurons are not
damaged, the axons have the ability to
regenerate and to attempt to reach their
original target site. This is the basis, for
example, of the eventual return of
sensation and muscle control in a
surgically reattached limb. Neurons in
the CNS also have the ability to
regenerate interrupted axons; however,
they have a much more difficult task in
reaching their original targets due to
both the presence of scar tissue formed
by proliferating glia and to the increased
complexity of the connectivity in the
CNS.
3. Methods for Assessing Human
Neurotoxicity
3.1. Introduction
This chapter outlines and discusses
current methods for detecting
neurotoxicity in humans. In contrast to
studies of neurotoxicity in animals
where functional changes readily can be
correlated with neuroanatornic and
neurochemical alterations, there are
ethical and technical barriers to the
direct observation of neuronal damage
in humans. Neurotoxicity in humans is
most commonly measured by relatively
noninvasive neurophysiologic and
neurobehavioral methods that assess
cognitive, affective, sensory, and motor
function. The evaluation of human
neurotoxicity and the relevance to risk
assessment will be discussed within the
context of clinical evaluation,
epidemiologic/worksite studies, and
human laboratory exposure studies.
3,2. Clinical Evaluation
Neurobehavioral assessment methods
are used extensively in clinical
neurology and neuropsychology to
evaluate patients suspected of having
neurologic disease. An extensive array
of examiner-administered and paper
and pencil tasks are used to assess
sensory, motor, cognitive, and affective
functions and personality states/traits.
Neurobehavioral data are synthesized
with information from neurophysiologic
studies, imaging techniques, medical
history, etc., to derive a working
diagnosis. Clinical diagnostic
approaches have provided a rich
conceptual framework for
understanding the functions (and
malfunctions) of the central and
peripheral nervous systems and have
formed the basis for the development of
methods for measuring the behavioral
expression of nervous system disorders.
Human neurobehavioral toxicology has
borrowed heavily from neurology and
neuropsychology for concepts of
nervous system impairment and
functional assessment methods.
Neurobehavioral toxicology has adopted
the neurologic/neuropsychologic model,
using adverse changes in behavioral
function to assist in identifying
chemically or drug-induced changes in
nervous system processes.
3.2.1. Neurologic Evaluation
Assessment of neurobehavioral
function by the clinical examination of
a patient has long been used as a
primary tool in neurologic diagnosis.
The domains of cognitive function,
motor function, sensation, reflexes, and
cranial nerve function are a standard
part of the clinical neurologic exam.
Movement and gait, speech fluency and
content, verbal memory, deep tendon
reflexes, muscle strength, symmetry of
movement and strength, ocular
movements, sensory function (pressure,
vibration, visual, auditory), motor
coordination, and logical reasoning are
only a few of the functions assessed by
neurologists (Denny-Brown et al., 1982).
Trained and experienced clinicians
gather these data by observation, verbal
exchange, and direct examination.
Neurologic exams arc sensitive
indicators of neurologic disease; the
data have predictive value for the
diagnosis of underlying nervous system
disease, and the methods have been
extensively validated against other
diagnostic procedures (e.g., imaging,
neurophysiologic testing), the course of
the illness, and autopsy findings.
Examination of the patient in a
semistructured procedure can yield a
wealth of information and insights
about functional impairment and the
underlying neuropathology.
3.2.2. Neuropsychological Testing
Neuropsychologists have developed
quantitative methods to supplement
clinical neurologic exam and laboratory
data for the diagnosis of neurologic
disease. Currently, two assessment
batteries, the Luria-Nebraska and the
Halstead-Reitan, and shorter versions
are used in clinical practice. The
batteries consist of subtests that quantify
a wide spectrum of cognitive, motor,
sensory, intellectual, affective, and
personality functions. The pattern of
relative performance on the subtests can
be interpreted along with historical and
medical data to suggest the presence or
absence of neurologic disease and the
possible anatomic location of any focal
lesions or degeneration. Clinical
interpretation of the data is enhanced by
data on age-related population norms
for many subtests and by the systematic
observation of the patient during testing.
Several neurotoxicity assessment
batteries use components of
neuropsychological tests and have
adapted and shortened analogs of some
subtests. Tests derived from the
Wechsler Adult Intelligence Scale—
Revised (WAIS-R) have been used
frequently to assess neurobehavioral
impairment from chemical agents, and
other abbreviated variations of
neuropsychological battery subtests
have been incorporated into
neurobehavioral toxicity batteries and
used in field and laboratory studies.
3.2.3. Applicability of Clinical Methods
to Neurotoxicology Risk Assessment
Neurologic and neuropsychologic
methods have long been employed to
identify the adverse health effects of
environmental workplace exposures.
Peripheral neuropathies (with sensory
and motor disturbances),
encephalopathies, organic brain
syndromes, extrapyramidal syndromes,
demyelination, autonomic changes, and
dementia are well-characterized
consequences of acute and chronic
exposure to chemical agents. The range
of exposure conditions that produce
clinical signs of neurotoxicity also has
been defined by using these clinical
methods. It is very important to make
external/internal dose measurements in
humans in order to determine the actual
dose(s) which can cause unwanted
effects.
Aspects of the clinical neurologic
examination approach limit its
usefulness for neurotoxicologic risk
assessment. Information obtained from
the neurologic exam is mostly
qualitative and descriptive rather than
quantitative. Estimates of the severity of
functional impairment can be reliably
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42373
placed into only three or four categories
(for example, mild, moderate, severe).
Much of the assessment depends on the
subjective judgment of the examiner; the
magnitude and symmetry of muscle
strength are often judged by having the
patient push against the resistance of
the examiner's hands. The datum is
therefore the absolute and relative
amount of muscle load sensed by the
examiner in his or her arms.
Compared with other methods, the
clinical neurologic exam may be less
sensitive in detecting early
neurotoxicity in peripheral sensory and
motor nerves. While clinicians'
judgments are equal in sensitivity to
quantitative methods in assessing the
amplitude of tremor, tremor frequency
is poorly quantified by clinicians. Thus,
important aspects of the clinical
neurologic exam may be insufficiently
quantified and lack sufficient sensitivity
for detecting early neurobehavioral
toxicity produced by environmental or
workplace exposure conditions.
However, a neurologic evaluation of
persons with documented
neurobehavioral impairment would be
helpful for identifying nonchemical
causes, such as diabetes and
cardiovascular insufficiency.
Administration of a
neuropsychological battery also requires
a trained technician, and interpretation
requires a trained and experienced
neuropsychologist. Depending on the
capabilities of the patient, 2 to 4 hours
may be needed to administer a full
battery; 1 hour may be needed for the
shorter screening versions. These
practical considerations may limit the
usefulness of neuropsychological
assessment in large field studies of
suspected neurotoxicity.
In addition to logistical problems in
administration and interpretation,
neuropsychological batteries and
neurologic exams share two
disadvantages with respect to
neurotoxicity risk assessment. First,
neurologic exams and
neuropsychological test batteries are
designed to confirm and classify
functional problems in individuals
selected on the basis of signs and
symptoms identified by the patient,
family, or other health professionals.
Their usefulness in detecting low-base
rate impairment in workers or the
general population maybe generally
thought to be limited, decreasing the
usefulness of clinical assessment
approaches for epidemiologic risk
assessment.
Second, neurologic exams and
neuropsychologic test batteries were
largely developed to assess the
functional correlates of the most
common forms of nervous system
dysfunction: brain trauma, focal lesions,
and degenerative conditions. The
clinical tests were primarily validated
against these neurologic disease states.
There has been insufficient research to
demonstrate which tests designed to
assess functional expression of
neurologic disease are most useful in
characterizing the modes of CNS
impairment produced by chemical
agents and drugs. More research is
needed to validate the usefulness of
neuropsychologic test methods in
neurotoxicology.
3.3. Current Neurotoxicity Testing
Methods
3.3.1. Neurobehavioral Methods
Chemical agents directly or indirectly
affect a wide range of nervous system
activities. Many of these chemical
actions are expressed as alterations of
behavior; Anger (1990a) lists 35
neurobehavioral effects of chemical
exposure that illustrate alterations in
sensory, motor, cognitive, affective, and
personality function. Professional
judgment is important in the
interpretation of data from studies using
neurobehavioral methods since some
endpoints can be subjective.
Dozens of tests of neurobehavioral
function have been proposed or used in
field or laboratory studies to assess the
neurotoxicity of chemical agents. Table
3-1 lists some frequently used tests of
motor, sensory, cognitive, and affective
neurobehavioral function.
TABLE 3-1 .—NEUROBEHAVIORAL
METHODS
TABLE 3-1 .—NEUROBEHAVIORAL
METHODS—Continued
Neurobehavioral
function
Sensation
Motor/Dexterity
Cognition
Test
Neurobehavioral
function
Affect
Test
Wechsler Memory
Scale.©
Profile of Mood
States© (POMS).
Flicker Fusion.
Lanthony (color
vision).
Pursuit Aiming.
Finger Tapping.
Postural Stability.
Reaction Time.
Santa Ana Peg
Board.
Benton Visual Re-
tention.
Continuous Per-
formance Task.
Digit-Symbol.
Digit Span.
Dual Tasks.
Paired-Associate.
Symbol-Digit Task.
Wechsler Adult In-
telligence Scale—
Revised© (Com-
ponents).
In contrast to the individual focus in
clinical evaluation, neurobehavioral
tests primarily have been used to
evaluate differences between groups,
comparing unexposed groups with
persons environmentally or
occupationally exposed to a suspected
neurotoxic agent. An ideal evaluation of
groups for quantitative evidence of
chemically induced neurobehavioral
impairment would involve the
assessment of a wide variety of
functions, but testing all possible
neurobehavioral functions that might be
affected in a group of exposed workers,
for example, would be impossible.
Therefore, a testing strategy has been to
use limited number tests that sample
representative neurobehavioral
functional domains such as dexterity,
visual memory, and reaction time.
3.3.1.1. Test batteries.
Many field and laboratory studies
have selected neurobehavioral methods
according to available information about
the spectrum of effects of the suspected
neurotoxic agent(s). This focused
strategy is useful for answering specific
questions about known neurotoxins. To
identify unspecified neurotoxic effects
in groups of workers or to characterize
the effects of less well-studied
chemicals or mixtures of chemicals,
several tests that sample a
representative range of functional
domains have been grouped into test
batteries. The advantage of a
standardized battery is that data from
different study populations and
chemical classes can be compared, and
similarities in effects observed (Johnson,
1987). Standardized batteries can be
categorized into investigator-
administered and computer-
administered types.
3.3.1.2. Investigator-administered test
batteries.
The WHO-recommended
Neurobehavioral Core Test Battery
(NCTB) (Johnson, 1987), the Finnish
Institute of Occupational Health (FIOH)
(Hanninen, 1990), and the Pittsburgh
Occupational Exposures Test Battery
(POET) (Ryan et al., 1987) are three
commonly used batteries. The NCTB is
frequently used in field studies
worldwide and can be fit inside a
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medium-sized suitcase for transport.
The NCTB consists of the following
tests: simple reaction time task, digit-
symbol coding task, timed motor
coordination test (Santa Ana pegboard),
digit span memory test. Benton Visual
Retention test, pursuit aiming test, and
the Profile of Mood States (POMS).
Based on factor-analytic studies
(Hooisma et al., 1990). these tests are
believed to measure the functional
domains of immediate memory,
attention, dexterity/hand-eye
coordination, reaction time, and mood.
Long-term memory, verbal and language
functions, auditory sensation, judgment,
and so forth are not assessed.
3.3.1.3. Computerized test batteries.
Computerized tests and batteries have
been developed for field and laboratory
use. The Neurobehavioral Evaluation
System (NES) (Baker et al., 1985),
MicroTox (Eckerman et al., 1985), the
SPES (Iregren et al., 1985), and the
NCTR Operant Battery (Paule et al.,
1990) are computerized systems
developed for neurotoxicity assessment.
Current versions of the NES, for
example, consist of about 15 different
neurobehavioral tests, and the battery
has been used in epidemiologic studies
of groups exposed to solvent, pesticide,
and mercury, and in laboratory studies
of NCb, ethanol, and toluene (Letz,
1990).
Although many computerized tests
appear to tap similar neurobehavioral
domains as noncomputerized batteries,
the visual mode of presentation, the
manual mode of response, and the
emphasis on speed of responding are
believed to have led to significant
differences in results obtained from
computerized versus noncomputerized
forms of similar tests. Attempts to
clarify the differences between
computerized and noncomputerized test
batteries have met with difficulty.
Although some tests are similar in each
type of battery, size and duration of
stimuli, presentation and response
modality, number of trials, and scoring
vary arbitrarily, preventing direct
comparison. An example is the digit-
symbol test on the NCTB and the
symbol-digit test on the NES. Although
almost identical in task requirements,
procedural and scoring differences
prevent direct comparison of the results
from these two tests.
Postural stability is an aspect of
integrated sensory and motor function
that increasingly is being evaluated in
clinical, epidemiologic, and laboratory
investigations of effects of pesticides
and solvents, and would be useful for
assessing therapeutic drug-induced
movement disorders such as
neuroleptics. Measurement of postural
stability requires a computer, special
software, monitor, and a force
transduction platform on which the
subjects must stand (Dick et al., 1990).
Mechanical and capacitive field
methods for assessing the amplitude
and frequency of tremor also are seeing
more frequent use.
An advantage of computerized testing
is the standardization of test
presentation, but a disadvantage is the
need for delicate, expensive computers
and measurement devices that require
transport for field studies.
Noncomputerized test batteries may be
less costly to purchase and easier to
transport, enhancing their desirability in
field studies, but test administrators
require training and small differences in
test administration may affect the data.
3.3.2. Neurophysiologic Methods
With improvements in the capabilities
and size of equipment, quantitative
neurophysiologic measurement of
sensory and motor function will be
increasingly useful in human
neurotoxicity evaluations. A major
advantage of these methods for risk
assessment is that they can be assessed
in both human and animal subjects and
the data can be interpreted in an
homologous manner.
Electromyographic responses (EMG)
and nerve conduction velocity (NCV)
have been used in the assessment of
peripheral nerve neurotoxicity. Some
techniques require that needle
electrodes be placed beneath the skin
for stimulation and recording and are
therefore somewhat uncomfortable for
the subject. However, the methods are
quantitative, provide multiple
endpoints of 1'NS function, and have
clinical relevance.
The adverse effects of solvents,
pesticides, and metals have been
identified with EMG/NCV
neurophysiologic measures. Although
not reduced as a function of duration of
employment, maximum nerve
conduction velocity (MCV) has been
reported to vary systematically with
cumulative exposure to carbon disulfide
(Johnson et al.. 1983), suggesting that
this measure may be particularly
valuable for quantitative risk assessment
of some types of peripheral motor nerve
toxicity.
Noninvasive neurophysiologic test
methods used in neurotoxicity
evaluations include the
electroencephalogram (EEC), visually
evoked response (VER), somatosensory
evoked potential (SEP), and the
brainstem auditory evoked response
(BAER). The EEC is the summed
electrical activity of neurons measured
with scalp electrodes; voltage and
frequency are primary measures. Evoked
methods employ specific eliciting
stimuli applied to the sense organs to
measure nervous system electrical
response. Visual patterns, sounds, and
cutaneous stimuli are presented to the
subject, and "evoked" voltage changes
in the nervous system are measured
with skin electrodes.
While EEGs were developed as a tool
in the neurologic diagnosis of seizure
disorders and other brain diseases, dose-
related EEC changes in chemically
exposed (especially solvents and
styrene) individuals have been noted
(Seppalainen and Harkonen, 1976). EEC
measurement requires large recording
devices that can be used in the
laboratory or clinic, but are difficult to
use in field studies. However, compact
computerized recording equipment has
been developed, and automated spectral
analyses of EEGs have recently been
applied to neurotoxicity evaluation
(Piikivi and Tolonen, 1989).
In contrast to EEGs, evoked response
technology is improving, and
equipment, while expensive, is
becoming more portable. VERs have
been used to detect the sensory toxicity
of solvents and carbon monoxide in
human subjects, and a relationship has
been suggested between BAER and
blood lead levels in children exposed to
lead-containing dust in the environment
(Otto and Hudnell, 1990). Evoked
potentials also may be conditioned,
allowing the use of sensory methods to
investigate associative processes.
Dose-response functions have been
found with evoked methods. A
curvilinear relationship was found
between BAER and blood lead
concentrations in children (Otto and
Hudnell, 1990), and a biphasic function
described visual evoked potential (VEP)
latency and visual contrast sensitivity
and perchloroethylene exposure
concentration in a laboratory study
(Altmann et al., 1991). In the latter
study, the direction of the response was
jointly dependent on dose and stimulus
parameters. In addition, changes over
time in the effect of the solvent on VEP
were dose and stimulus parameter
dependent.
Two important methodologic
considerations are illustrated by BAER
and VEP data. One is that low
concentrations of some chemical agents
may produce effects (shorter latencies in
these examples) that could be
inaccurately interpreted as facilitation
rather than impairment. Changes in
neuronal latencies in either direction
could be a result of a neurotoxic
process. The second is that the detection
of neurotoxic effects is dependent on
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42375
dose-time-testing parameter
interactions. A thorough understanding
of the effects of testing parameters on
the dose-response relationship and the
time course of chemical effect will be
necessary for interpreting neurotoxicity
studies.
The development of neurophysiologic
methods, such as evoked and
conditioned potentials, for neurotoxicity
risk assessment should be encouraged.
These methods provide relatively
unambiguous quantitative data on
sensory function that may have clear
implications for health, are influenced
by fewer extraneous variables than are
self-report and neurobehavioral
performance tests, and allow relatively
direct extrapolation of effects between
animals and humans.
3.3.3. Neurochemical Methods
One of the major difficulties in risk
assessment is estimating exposure
parameters and the dose or body burden
actually absorbed by the individual. In
epidemiologic studies, the actual
absorption and bioavailability of a
chemical from an exposure are
frequently unknown.
Measurement of chemical
concentrations in biologic fluids or
tissues is one way to measure more
precisely the concentration at the site(s)
of toxic effect. In epidemiologic studies,
this has been possible only for chronic
exposure and for acute exposure to
chemicals with long biologic half-lives
in the body, such as lead, other metals,
and bromides. Blood lead levels show
correlations with neurobehavioral
impairment, but blood lead levels are
representative correlates of toxicity only
for relatively acute doses. In children,
for example, the majority of lead-related
impairment is the result of chronic,
rather than acute, absorption. The
cumulative amount of lead sequestered
in tissues (such as deciduous teeth) may
be a more representative indicator of the
area under the time-concentration
curve.
For chemicals with half-lives in the
body too short for estimating absorbed
dose, the biochemical products from the
chemical or from the physiologic effects
of the chemical may serve as an index
of exposure. Serum enzyme
concentrations (cholinesterase) and
esterases in other tissues (lymphocyte
target esterase) have been employed in
field studies to detect pesticide
exposure, while vanillylmandelic acid
(product of catecholamine
neurotransmitter biotransformation) and
erythrocyte protoporphyrin
concentrations have been used with
varying success in differentiating
between lead-exposed and control
workers. The addition of similar
"exposure biomarker" measures to
laboratory studies may allow the
development of quantitative estimates of
absorbed dose under various exposure
conditions.
The measurement of metabolic
products of neurotoxic agents may be
extremely useful in risk assessment; an
example comes from cancer risk
assessment. Human data from the early
1970s on saturation of microsomal
methylene chloride biotransformation to
carbon monoxide (Stewart et al., 1972),
along with subsequent animal
carcinogenesis data garnered in the
1980s, provided a quantitative basis for
a physiologically based
pharmacokmetic model of methylene
chloride cancer risk assessment
(Andersen et al., 1991). The information
on human CO pathway kinetics
provided the homologous key that
allowed extrapolation of risk from
animals to humans on a comparative
physiologic basis rather than using
default assumptions.
3.3.4. Imaging Techniques
A number of recently developed
computerized imaging techniques for
evaluating brain activity and cerebral/
peripheral blood flow have added
valuable information to the neurologic
diagnostic process. These imaging
methods include thermography,
positron emission tomography, passive
neuromagnetic imaging
(magnetoencephalography), magnetic
resonance imaging, magnetic resonance
spectroscopy, computerized
tomography, doppler ultrasonography,
and computerized EEC recording/
analysis (brain electrical activity
mapping). The research application of
these invasive and noninvasive
quantitative methods has primarily been
in neurology, schizophrenia research,
drug abuse, AIDS research and toxic
encephalopathy (Hagstadius et al.,
1989). Although the equipment for brain
imaging is expensive and not portable,
neuroimaging techniques promise to be
valuable clinical and laboratory research
tools in human neurotoxicology.
3.3.5. Neuropathologic Methods
Neuropathologic examination of
nervous system tissue has been used to
confirm data from clinical testing and to
contribute to the understanding of
mechanisms of action of neurotoxicity.
Peripheral nerve biopsies have
confirmed chemically induced
peripheral neuropathies and evaluated
rates of recovery (Fullerton, 1969).
Postmortem examination of nervous
tissue also has elucidated the
neuropathological effects of carbon
disulfide, clioquinol, and doxorubicin
(Spencer and Schaumburg, 1980).
3.3.6. Self-Report Assessment Methods
Self-report measures relevant to
neurotoxicity risk assessment consist of
histories of symptoms, events,
behaviors, and environmental
conditions. Information is obtained by
face-to-face interviews, structured
interviews (often conducted for
diagnostic purposes), medical histories,
questionnaires, and survey instruments.
Self-report instruments are the only
means for measuring some symptoms
and all interoceptive states, such as pain
and nausea. Self-reports also are used to
obtain information on behaviors and
events (e.g., exposure conditions)
especially when practical, legal, or
ethical limitations prevent direct
observation.
Subjective symptoms elucidated from
self-report instruments are responsive to
dose. Hanninen et al. (1979) found that
subjective symptoms were positively
correlated with blood lead levels in
exposed workers. Subjective pain
estimations are correlated with dose and
type of centrally and peripherally acting
analgesics, and anxiety scores on a
variety of scales are responsive to the
size of the anxiolytic dose.
Symptom checklists are used in
epidemiologic research to identify the
pattern of subjective complaints, which
can be used to guide the selection of
objective assessment methods. The
distribution of symptoms can be
correlated with indices of exposure to
determine if particular symptoms are
more prevalent in exposed persons
(Sjogren et al., 1990).
Self-report data are notable for biases
that may influence them; these biases
are well known in epidemiology,
clinical practice, and social science.
Even in the most superficial of
questions, respondents may consciously
or unknowingly bias the answer to fit
what they believe to be the examiner's
expectations. Details of objective events
or subjective states are subject to
alteration; recall and reporting of
remembered occurrences may be biased
to fit interpretations and expectations.
The socioeconomic status, gender, and
affiliation of the tester also have been
identified as biasing variables. Bias
occurs when information is requested
about behaviors, beliefs, or feelings
believed by the respondent to be
socially undesirable or when
reinforcement contingencies (e.g.,
litigation) strongly favor selective
reporting. •
Biases in self-report data can be
reduced by making the questionnaire
anonymous or highly confidential;
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objective data can be used to validate
self-reports. Ethnographic observations,
objective measurement of behavior,
biologic samples, and the observations
of significant others are employed to
validate self-report data. Consistent
descriptions of events by several
persons lend credence to the reliability
of the report. Many clinical interviews
and self-report assessment instruments
include some mechanisms for detecting
self-report bias, either by looking for
endorsement of improbable behaviors,
or by examining the consistency of
information gathered in several ways or
from several sources. Concordance
among biologic indices, observations,
and physical examinations increases the
judged validity of self-reports.
3.3.6.1. Mood scales.
Changes in mood and emotionality
can be consequences of neurotoxicity.
For example, case reports have
identified mood changes from exposure
to mercury, lead, solvents, and
organophosphate insecticides. The
Taylor Manifest Anxiety Scale and the
Profile of Mood States (POMS) are
standardized self-report assessment
instruments for which there is some
evidence of sensitivity to chemical
insult.
The POMS, a component of the
Neurobehavioral Core Test Battery, is a
self-report measure that asks
respondents to use a 5-point scale to
rate the magnitude of 65 subjective
states, such as "tense," "relaxed,"
"hopeless," "guilty," etc., that they have
experienced within the past week. The
responses are scored according to six
mood factors, and a Total Mood
Disturbance Score also may be
calculated. Liang et al. (1990) used the
POMS to evaluate lead-exposed workers
(mean blood lead concentration of 41
ug/dL) from a battery plant and a control
group from a fabric-weaving
manufacturer. Exposed workers were
significantly higher on tension,
depression, anger, fatigue, and
confusion scales.
Mood scales were developed to aid in
assessment of psychological disorders,
such as depression, and to track
treatment response. In addition, mood is
modulated by metabolic and endocrine
variables in health and disease and can
change rapidly in response to
interpersonal, workplace, and
environmental events. The large number
of nonchemical variables and the
lability of mood make inclusion of
carefully selected controls essential in
using affect as an endpoint in
neurotoxicity research.
The validity of mood scales may be
limited to the specific populations in
which the validity studies were
performed. As characterizations of
internal states, the meaning of the
descriptors in the POMS established for
one culture may not be the same as the
meaning of that concept or term in other
cultures or in other language systems.
There may be variations in
interpretation of the terms by
respondents across English-speaking
subcultures, perhaps as a function of
education or the size of the verbal
community. While these differences
may not impede a global clinical
interpretation, the reduction in
generalizability across study
populations may be sufficient to
decrease the usefulness of subjective
scales in quantitative neurotoxicity risk
assessment.
3.3.6.2. Personality scales.
The Minnesota Multiphasic
Personality Inventory (MMPI), the
Cattell 16 PF, and the Eysenck
Personality Inventory have occasionally
been used in neurotoxicity research.
Exposed and nonexposed groups have
differed on several scales derived from
these standardized questionnaires. The
diagnostic power of the MMPI, for
example, is not in the individual scales
but in the pattern of scores on the 10
clinical and 3 validity scales. Because
interpretation of the MMPI requires a
trained diagnostician with experience in
the population of interest, it is less
likely to be useful in quantitative
neurotoxicity assessment.
3.4. Approaches to Neurotoxicity
Assessment
3.4.1. Epidemiologic Studies
Epidemiology has been defined as
"the study of the distributions and
determinants of disease and injuries in
human populations" (Mausner and
Kramer, 1985). Knowing the frequency
of illness in groups and the factors that
influence the distribution is the tool of
epidemiology that allows the evaluation
of causal inference with the goal of
prevention and cure of disease.
Epidemiologic studies are a means of
evaluating the effects of neurotoxic
substances in human populations, but
such studies are limited because they
must be performed shortly after
exposure if the effect is acute. Most
often these effects are suspected to be a
result of occupational exposures due to
the increased opportunity for exposure
to industrial and other chemicals.
3.4.1.1. Case reports.
The first type of human study
undertaken is the case report or case
series, which can identify cases of a
disease and are reported by clinicians or
discerned through active or passive
surveillance, usually in the workplace.
For example, the neurological hazards
of exposure to Kepone,
dimethylaminopropionitrile, and
methyl-n-butyl ketone were first
reported as case studies by physicians
who noted an unusual cluster of
diseases in persons later found to have
been exposed to these chemicals (Cone
et al., 1987). However, case histories
where exposure involved a single
neurotoxic agent, though informative,
are rare in the literature; for example,
farmers are exposed to a wide variety of
potentially neurotoxic pesticides.
Careful case histories assist in
identifying common risk factors,
especially when the association between
the exposure and disease is strong, the
mode of action of the agent is
biologically plausible, and clusters
occur in a limited period of time.
Case reports are inexpensive
compared with other types of
epidemiologic studies and can be
obtained more quickly than more
complex studies. They provide little
information about disease frequency or
population at risk, but their importance
has been clearly demonstrated,
particularly in accidental poisoning or
acute exposure to high levels of
toxicant. They remain an important
source of index cases of new diseases
and for surveillance in occupational
settings. These studies require
confirmation by additional
epidemiologic research employing other
study design.
3.4.1.2. Cross-sectional studies.
In cross-sectional studies or surveys,
both the disease and suspected risk
factors are ascertained at the same time
and the findings are useful in generating
hypotheses. A group of people is
interviewed, examined, and tested at a
single point in time to ascertain a
relationship between a disease and a
neurotoxic exposure. This study design
does not allow the investigator to
determine whether the disease or the
exposure came first, rendering it less
useful in estimating risk. These studies
are intermediate in cost and time
required to complete compared with
case reports and more complex
analytical studies.
3.4.1.3. Case-control (retrospective)
studies.
Last (1986) defines a case-control
study as one that "starts with the
identification of persons with the
disease (or other outcome variable) of
interest, and a suitable control
population (comparison, reference)
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42377
group of persons without the disease."
He states that the relationship of an
"attribute" to the disease is measured by
comparing the diseased with the
nondiseased with regard to how
frequently the attribute is present in
each of the groups. The cases are
assembled from a population of persons
with and without exposure and the
comparison group is selected from the
same population; the relative
distribution of the potential risk factor
(exposure) in both groups is evaluated
by computing an odds ratio that serves
as an estimate of the strength of the
association between the disease and the
potential risk factor. The statistical
significance of the ratio is determined
by calculating a p-value and is used to
approximate relative risk.
The case-control approach to the
study of potential neurotoxins in the
environment has provided a great deal
of information. In his recent text,
Valciukas (1991) notes that the case-
control approach is the strategy of
choice when no other environmental or
biological indicator of neurotoxic
exposure is available. He further states:
"Considering the fact that for the vast
majority of neurotoxic chemical
compounds, no objective biological
indicators of exposure are available (or
if they are, their half-life is too short to
be of any practical value), the case-
control paradigm is a widely accepted
strategy for the assessment of toxic
causation." The case-control study
design, however, can be very
susceptible to bias. The potential
sources of bias are numerous and can be
specific to a particular study, and will
be discussed only briefly here. Many of
these biases also can be present in cross-
sectional studies. For example, recall
bias or faulty recall of information by
study subjects in a questionnaire-based
study can distort the results of the
study. Analysis of the case-comparison
study design assumes that the selected
cases are representative persons with
the disease—either all cases with the
disease or a representative sample of
them have been ascertained. It further
assumes that the control or comparison
group is representative of the
nondiseased population (or that the
prevalence of the characteristic under
study is the same in the control group
as in general population). Failure to
satisfy these assumptions may result in
selection bias, but violation of
assumptions does not necessarily
invalidate the study results.
An additional source of bias in case-
control studies is the presence of
confounding variables, i.e., factors
known to be associated with the
exposure and causally related to the
disease under study. These must be
controlled either in the design of the
study by matching cases to controls on
the basis of the confounding factor or in
the analysis of the data by using
statistical techniques such as
stratification or regression. Matching
requires time to identify an adequate
number of potential controls to
distinguish those with the proper
characteristics, while statistical control
of confounding requires a larger study.
The definition of exposure is critical
in epidemiologic studies. In
occupational settings, exposure
assessment is based on the job
assignment of the study subjects, but
can be more precise if detailed company
records allow the development of
exposure profiles.
3.4.1.4. Prospective (cohort, followup)
studies.
In a prospective study design, a
healthy group of people is assembled
and followed forward in time and
observed for the development of
disease. Such studies are invaluable for
determining the time course for
development of disease (e.g., followup
studies performed in various cities on
the effects of lead on child
development). This approach allows the
direct estimate of risks attributed to a
particular exposure since disease
incidence rates in the cohort are
determined and allows the study of
chronic effects of exposure. One major
strength of the cohort design is that it
allows the calculation of rates to
determine the excess risk associated
with an exposure. Also, biases are
reduced by obtaining information before
the disease develops. This approach,
however, can be very time-consuming
and costly.
In cohort studies information bias can
be introduced when individuals provide
distorted information about their health
because they know their exposure status
and may have been told of the expected
health effects of the exposure under
study.
A special type of cohort study is the
retrospective cohort study in which the
investigator goes back in time to select
the study groups and traces them over
time, often to the present. The studies
usually involve specially exposed
groups and have provided much
assistance in estimating risks clue to
occupational exposures. Occupational
retrospective cohort studies rely on
company records of past and current
employees that include information on
the dates of employment, age at
employment, date of departure, and
whether diseased (or dead in the case of
mortality studies). Workers can then be
classified by duration and degree of
exposure. A retrospective cohort study
was performed in which a cohort of
1,790 bricklayers and 2,601 men
exposed to paint solvents was
retrospectively identified and, if a
disability pension had been awarded,
the subjects were examined for evidence
of presenile dementia. This study found
a rate ratio of 3.4 for presenile dementia
among the painters as compared with
the bricklayers (Johnson, 1987).
3.4.2. Human Laboratory Exposure
Studies
Neurotoxicity assessment has an
advantage not afforded the evaluation of
other toxic endpoints, such as cancer or
reproductive toxicity, in that the effects
of some chemicals are short in duration
and reversible. Under certain
circumstances, it is ethically possible to
perform human laboratory exposure
studies and obtain data relevant to the
risk assessment process. Information
from experimental human exposure
studies has been used to set
occupational exposure limits, mostly for
organic solvents that can be inhaled.
Laboratory exposure studies have
contributed to risk assessment and the
setting of exposure limits for several
solvents and other chemicals with acute
reversible effects. These chemicals
include methylene chloride,
perchloroethylene, trichloroethylene,
and p-xylene (Dick and Johnson, 1986).
Human exposure studies offer
advantages over epidemiologic field
studies. Combined with appropriate
biological sampling (breath or blood), it
is possible to calculate body
concentrations, to examine
toxicokinetics, and identify metabolites.
Bioavailability, elimination, dose-
related changes in metabolic pathways,
individual variability, time course of
effects, interactions between chemicals,
interactions between chemical and
environmental/biobehavioral factors
(stressors, workload/respiratory rate) are
some processes that can be evaluated in
laboratory studies.
Other goals of laboratory studies
include the indepth characterization of
effects, the development of new
assessment methods, and the
examination of the sensitivity,
specificity, and reliability of
neurobehavioral assessment methods
across chemical classes.
The laboratory is the most appropriate
setting for the study of environmental
and biobehavioral variables that affect
the action of chemical agents. The
effects of ambient temperature, task
difficulty, the rate of ongoing behavior,
conditioning variables, tolerance/
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sensitization, sleep deprivation,
motivation, etc., can be studied.
3.4.2.1. Methodologic aspects.
From a methodologic standpoint,
human laboratory studies can be
divided into two categories—between-
subjects and within-subjects designs. In
the former, the neurobehavioral
performance of exposed volunteers is
compared with that of nonexposed
participants. In the latter, preexposure
performance is compared with
neurobehavioral function under the
influence of the chemical or drug.
Within-subjects designs have the
advantage of requiring fewer
participants, eliminating individual
differences as a source of variability,
and controlling for chronic mediating
variables, such as caffeine use and
educational achievement. A
disadvantage of the within-subjects
design is that neurobehavioral tests
must be administered more than once.
Practice on many neurobehavioral tests
often leads to improved performance
that may confound the effect of the
chemical/drug. It is important to allow
a sufficient number of test sessions in
the preexposure phase of the study to
allow performance on all tests to
achieve a relatively stable baseline level.
3.4.2.2. Human subject selection factors.
Participants in laboratory exposure
studies may be recruited from
populations of persons already exposed
to the chemical/drug or from naive
populations. Although the use of
exposed volunteers has ethical
advantages, can militate against novelty
effects, and allows evaluation of
tolerance/sensitization, finding an
accessible exposed population in
reasonable proximity to the laboratory is
difficult. Naive participants are more
easily recruited, but may differ
significantly in important characteristics
from a representative sample of exposed
persons. Naive volunteers are often
younger, healthier, and better educated
than the populations exposed
environmentally, in the workplace, or
pharmacotherapeutically. For example,
phase I drug trial data from relatively
young and healthy volunteers may not
adequately predict the incidence of
neurotoxic side effects in older persons
with chronic health problems.
3.4.2.3. Exposure conditions and
chemical classes.
Compared with workplace and
environmental exposures, laboratory
exposure conditions can be controlled
more precisely, but exposure periods are
much shorter. Generally only one or two
relatively pure chemicals are studied for
several hours while the population of
interest may be exposed to multiple
chemicals containing impurities for
months or years. Laboratory studies are
therefore better at identifying and
characterizing effects with acute onset
and the selective effects of pure agents.
Most laboratory studies of
neurobehavioral function have
employed individual solvents,
combinations of two solvents, or very
low concentrations of chemicals
released from household and office
materials (volatile organic compounds).
This selection is primarily because
solvent effects are reversible, because
there are wide margins of safety for
acute effects of solvents, because
solvents can be administered via
inhalation methods that allow
calculation of body concentrations by
breath sampling methods that do not
require needle sticks, because over 1
million workers may have occupational
solvent exposure, and because of the
extensive use of solvents in household
products. Chemicals studied in the
laboratory over the past 40 years have
included ozone, NO2, CO, styrene, lead,
anesthetic gases, pesticides, irritants,
chlorofluorocarbon compounds, and
propylene glycol dinitrite. Caffeine,
diazepam, and ethanol have been used
in laboratory studies as positive control
substances.
3.4.2.4. Test methods.
Neurobehavioral test methods may be
selected according to several strategies.
A test battery that examines multiple
neurobehavioral functions may be more
useful for screening and the initial
characterization of acute effects.
Selected neurobehavioral tests that
measure a more limited number of
functions in multiple ways may be more
useful for elucidating mechanisms or
validating specific effects.
3.4.2.5. Controls.
Both chemical and behavioral control
procedures are valuable for examining
the specificity of the effects. A
concordant effect among different
measures of the same neurobehavioral
function (e.g., reaction time) and a lack
of effect on some other measures of
psychomotor function (e.g., untimed
manual dexterity) would increase the
confidence in a selective effect on motor
speed and not on attention or on
nonspecific motor function. Likewise,
finding concordant effects among
similar chemical or drug classes along
with different effects from dissimilar
classes would support the specificity of
chemical effect. For example, finding
that the effects of a solvent were similar
to those of ethanol but not caffeine
would support the specificity of solvent
effects on a given measure of
neurotoxicity.
3.4.2.6. Ethical issues.
Most human exposure studies in the
laboratory have been justified on the
basis of data indicating that the
chemical or drug exposure produces
only temporary and reversible
functional effects. The use of
occupationally, environmentally, or
therapeutically exposed populations as
a source of participants also makes the
risks from research exposure small
relative to nonlaboratory sources of risk.
Protection of human subjects is also
provided by the informed consent
process; the health risks (known and
unknown) and benefits of the research
are thoroughly explained to each
participant, who may terminate
participation in the study at any time.
Despite safeguards, several chemicals
and drugs thought at the time of the
exposure study to produce only
temporary neurobehavioral effects are
now (20 years later) suspected of being
potential human carcinogens on the
basis of animal and human data (e.g.,
methylene chloride, perchloroethylene).
Other chemicals, however, are now
thought to be less carcinogenic or
otherwise less toxic in humans than
once believed. Rapid advances in all
areas of toxicology make it difficult to
communicate, to potential subjects,
reliable information about the
likelihood of long-term, latent, or
delayed adverse effects on health
subsequent to the study. The
communication of uncertainty about
potential long-term effects to research
participants is essential if human
exposure studies are to be conducted
ethically and are to continue their
contributions to neurotoxicology and
risk assessment.
3.5. Assessment of Developmental
Neurotoxicity
3.5.1. Developmental Deficits
While adult neurotoxicology
evaluates the effects of chemical
exposure on relatively stable nervous
system structure and function,
developmental neurotoxicology
addresses the special vulnerabilities of
the young and the old. Neurobehavioral
assessment of chemical neurotoxicity is
complicated by having to measure
functional impairment within a
sequential progression of emergence,
maturation, and gradual decline of
nervous system capabilities. Methods in
developmental neurotoxicity assessment
must reflect the diversity of
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neurobehavioral functions, from
neonates to the elderly.
Exposure of pregnant women to
alcohol, drugs of abuse, therapeutic
drugs, nicotine, and environmental
chemicals may result in the immediate
or delayed appearance of
neurobehavioral impairment in children
(Kimmel, 1988; Nelson, 1991a).
Postnatal exposure of children to
chemical agents in the environment,
such as lead, also may impair IQ and
other indices of neurobehavioral
function (Needleman et al., 1979).
Neurotoxic effects may impair speech
and language, attention, general
intelligence, "state" regulation and
responsiveness to external stimulation,
learning and memory, sensory and
motor skills, visuospatial processing,
affect and temperament, and
responsiveness to nonverbal social
stimuli. Chemical neurotoxicity may be
manifested as decreases in functional
capabilities or delays in normative
developmental progression.
Neurotoxic effects are not limited to
direct exposure of the fetus or child to
the chemical. Animal studies suggest
that altered neurobehavioral
development in offspring may result
from exposure of males (Joffe and
Soyka, 1981) and females to chemical
substances prior to conception. In this
case, altered postnatal development may
reflect chemical influences on
mechanisms of inheritance, copulatory
behavior, nutritional status, hormonal
status, or the uterine environment. In
animals and humans, chemical
exposure of parents may indirectly
impair postnatal development through
changes in milk composition, parenting
behaviors, and other aspects of the
environment.
In older adults the normal aging
process alters the response to
neurotoxicants. Both pharmacodynamic
and pharmacokinetic changes may
underlie altered sensitivities to the
neurotoxic effects of drugs and
chemicals. An example well known in
geriatric medicine is the apparent
increase in sensitivity of the elderly to
the toxic effects of anxiolytics (Salzman,
1981). Decreases in biotransformation
rate and renal elimination of parent
drug and active metabolites, not related
to disease processes, may partially
account for the increased vulnerability
(Friedel, 1978). Chronic disease states in
older persons may result in decreased
functional capabilities and increased
vulnerability to neurotoxic effects.
Chronic diseases also may prompt
pharmacotherapy that may impair
neurobehavioral function.
Cardiovascular, psychopharmacologic,
and antineoplastic medications may
result in patterns of neurobehavioral
impairment not typically seen in
younger individuals.
3.5.2. Methodologic Considerations
Standardized methods are being
developed for pediatric neurotoxicity
assessment. Neurobehavioral functions
emerge during developmental phases
from neonatal stage through secondary
school, and nervous system insult may
be reflected not only in impairment of
emergent functions, but also as delays in
the appearance of new functions. Both
the severity and type of deficit are
affected by the dose and duration of
exposure (Nelson, 1991b), and different
sensitivities to chemical effects may be
exhibited at different stages of nervous
system development. Early episodes of
exposure may produce structural
damage to the nervous system that may
not be developmentally expressed in
behavior for several months or years.
The selection of appropriate testing
methods and conditions is more
important when assessing children
because of shorter attention spans and
increased dependence on parental and
environmental supports. In addition,
because of the increasing complexity of
functional capabilities during early
development, only a few tests
appropriate for infants can be validly
readministered to older children. Given
the complexity of these variables, the
task of devising sensitive, reliable, and
valid assessment instruments or
batteries for pediatric populations will
be challenging.
Assessment methods in older adults
must be capable of distinguishing
chemical and drug effects from the
effects of aging processes and chronic
disease states (Crook et al., 1983).
Assessment methods must be valid and
reliable with repeated administration
across a significant portion of the
lifespan, and take into consideration the
time (days, months, or years) that may
intervene between exposure/insult and
the expression of neurotoxicity as
functional impairment. Research on
nonexposed populations to develop age-
appropriate normative scores for
neurobehavioral functions will be
important for the interpretation of
assessment instruments.
Environmental exposure to neurotoxic
chemicals and drugs is correlated with
socioeconomic and ethnic status.
Assessment methods will therefore have
to be adapted to diverse ethnic, cultural,
and language groups. While gender
differences in early development have
been noted, differential responses of
males and females to neurotoxicants
have been less well explored and should
receive attention.
3.6. Issues in Human Neurotoxicology
Test Methods
3.6.1. Risk Assessment Criteria for
Neurobehavioral Test Methods
The value of human neurobehavioral
test methods for quantitative risk
assessment is related to the number of
the following criteria that can be met:
a. Demonstrate sensitivity to the kinds
of neurobehavioral impairment
produced by chemicals; that is, able to
detect a difference between exposed and
nonexposed populations in field studies
or between exposure and nonexposure
periods in human laboratory research or
within exposed populations over time.
b! Show specificity for neurotoxic
chemical effects and not be unduly
responsive to a host of other
nonchemical factors, and show
specificity for the neurobehavioral
function believed to be measured by the
test method.
c. Demonstrate adequate reliability
(consistency of measurement over time)
and validity (concordance with other
behavioral, physiologic, biochemical, or
anatomic measures of neurotoxicity).
d. Show graded amounts of
neurobehavioral change as a function of
exposure parameter, absorbed dose, or
body burden along some ordinal or
continuous metric (dose response).
e. For representative classes or
subclasses of CNS/PNS-active
chemicals, identify single effects or
patterns of impairment across several
tests or functional domains that are
reasonably consistent from study to
study (structure-activity).
f. Be amenable to the development of
a procedurally similar counterpart that
can be used to assess homologous
behaviors in animals.
g. Whenever it is relevant, care must
be taken to insure to the extent possible
that subjects are blind to the variate of
interest (Benignus, 1993).
3.6.1.1. Sensitivity.
Individual neurobehavioral tests and
test batteries have detected differences
between exposed and nonexposed
populations in epidemiologic studies
and in laboratory studies. Effects have
been detected by neurobehavioral
methods at concentrations thought by
other kinds of evaluation not to produce
neurotoxicity. Workplace exposure
limits to many chemicals have been set
on the basis of neurobehavioral studies.
While the overall sensitivity of
neurobehavioral methods is sufficient to
be useful in neurotoxicology risk
assessment, some methods are notably
insensitive across several chemical
classes while the sensitivity of other
neurobehavioral tests varies according
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to the spectrum of neurotoxic effects of
the chemical or drug.
Sensitivity is sometimes negatively
correlated with reliability; selecting for
tests that show little change over time
may also select for tests that are not
sensitive to neurotoxic insult.
Having more control over the testing
environment and using a repeated
measures design may decrease
variability and increase statistical
power, but these tactics may introduce
other problems. There is some
suggestion that experience in highly
structured laboratory environments with
explicit stimulus conditions may reduce
the sensitivity of humans and animals to
the effects of drugs and chemicals, and
the sensitivity of neurobehavioral
measures to impairment by a chemical
or drug may depend on neurobehavioral
training history (Terrace, 1963; Brady
and Barrett, 1986). Sensitivity may also
be decreased if baseline behaviors are
stable and well practiced or an escape/
avoidance procedure is employed.
The systematic introduction of
stimulus or response changes to induce
transitional behaviors, such as in a
transitional state or repeated learning
paradigms, may be one way to retain the
advantage of a stable baseline, have
sufficient sensitivity, and avoid practice
effects (Anger and Setzer, 1979).
3.6.1.2. Specificity.
There are two kinds of specificity in
neurobehavioral assessment of chemical
or drug neurotoxicity. Chemical
specificity refers to the ability of a test
to reflect chemical or drug effects and to
be relatively resistant to the influence of
nonchemical variables. The second type
of specificity refers to the ability of a
test method to measure changes in a
single neurobehavioral function (e.g.,
dexterity) or a restricted number of
functions, rather than a broad range of
functions (attention, reasoning,
dexterity, and vision).
The neurobehavioral expression of
neurotoxic chemical or drug effects is a
function of the joint interaction of
ongoing nervous system processes with
the chemical substance and with
biopsychosocial variables that also
influence nervous system activity. In
laboratory exposure studies numerous
environmental, behavioral, and biologic
variables can influence the type or
magnitude of neurotoxic effects of
chemical agents and drugs (MacPhail,
1990). These variables include ambient
temperature, physical workload, task
difficulty, the social and tangible reward
characteristics of the laboratory setting,
redundancy of stimuli, the rate and form
of the behavioral response, conditioning
factors, and the interoceptive stimulus
properties of the chemicals.
Tne laboratory research participant's
history and habits outside the laboratory
also may affect chemical-
neurobehavioral interactions by
influencing the baseline level of
performance on neurobehavioral tests or
directly affecting the response of the
CNS to the exposure. Age, gender,
educational level, intellectual
functioning, economic status, acute and
chronic health conditions (including
developmental or current neurologic
conditions), alcohol/drug/tobacco
effects or withdrawal, emotional status
or significant life events, sleep
deprivation, fatigue, and cultural factors
are only a few of the variables that may
affect performance in laboratory studies
(Williamson, 1991; Cassitto et al., 1990).
The influence of these selection and
biopsychosocial variables on the
neurobehavioral effects of workplace
chemicals is poorly understood,
although their effects on drug-behavior
interactions have been more thoroughly
explored. Controlling or understanding
chemical and nonchemical variables
will be important for ensuring adequate
specificity for risk assessment purposes.
3.6.1.3. Reliability and validity.
Reliability refers to the ability of a
given test to produce closely similar
results when administered more than
once over a period of time or in similar
populations. Reliability is meaningful
only with respect to the measurement of
functions that would not be expected to
change significantly over the time
period. Test-retest reliability coefficients
are between 0.6 and 0.9 (Beaumont,
1990) for most of the tests in the NCTB.
With notable exceptions, other
neurobehavioral tests have similar
reliabilities. Reliabilities in the 0.8 to
0.9 range are usually thought
acceptable. As reliability decreases,
measurement error is more likely to
mask neurotoxic chemical effects.
The validity of a given neurotoxicity
test relies on evidence that it adequately
measures the domain of interest and is
not highly correlated with tests that are
believed to measure unrelated
functions. These convergent and
divergent aspects of validity are
frequently divided into construct,
content, and criterion subcategories.
Construct validity refers to the ability of
a given test to measure the intended
function or construct (e.g., attention),
content to how well the test measures
the major aspects of the function, and
criterion to how highly the test
correlates with other tests of the same
function or predicts neurotoxic
impairment after similar insult.
Many neurobehavioral tests purport to
measure the same or similar cognitive,
sensory, or motor functions, but
correlations between these tests under
chemical exposure or control conditions
can be disappointingly low. This is not
surprising given the procedural
differences that exist among
neurobehavioral tests. Tests intended to
measure the same function often have
different presentation and response
modalities (visual, verbal, manual), have
differing numbers of trials or a different
time limit, and have different methods
for scoring the results. Many tests have
such large procedural differences that
direct comparison is difficult.
Assessment of validity for
neurobehavioral tests of specific
constructs, such as attention, is further
complicated in that sensory input, other
cognitive processes, and motor
responses are unavoidable contributors
to the test result.
3.6.1.4. Dose response.
Dose in this discussion refers to the
measurement of chemical or metabolite
concentrations in the body and to
estimations of exposure. Both exposure
assessment and biologic concentrations
should be measured whenever possible.
Dose-response relationships have been
observed both in field and laboratory
studies. Two recent human solvent
exposure studies used lower exposure
concentration that resulted in mucosal
membrane effects reported by subjects
as odors or irritation (Dick et al., 1992;
Hjelm et al., 1990). Neurobehavioral
impairment was not detected in these
studies. A review of over 50 organic
solvent human exposure experiments
found that neurobehavioral impairment
generally occurred at mean
concentrations higher than those
associated with irritation, although
there was often overlap among the
irritant and impairment concentration
ranges (Dick, 1988). Defining neurotoxic
dose-response relationships in humans
decreases the uncertainties of
extrapolation from animal data and
allows a more accurate risk assessment.
Recent human solvent exposure
studies have employed low
concentrations under which
neurobehavioral impairment was not
observed. Rather, these studies have
primarily detected the effects of solvents
on mucosal membranes reported by
subjects as odors or irritation (Dick,
unpublished observation). While these
data may be relevant to setting
workplace and environmental exposure
limits, they can be expected to provide
little information about the
neurobehavioral impairment that occurs
at higher concentrations. The
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relationship between irritant/odor
concentration-effect functions and
neurobehavioral impairment
concentration-effect functions is not
known, but it is probably not linear.
Dose-dependent mechanisms of toxic
effect can be expected to complicate risk
extrapolation across the dose-response
range in humans.
A further complication in dose-
response extrapolation is that low
concentrations of chemicals may appear
to improve performance as measured by
neurobehavioral tests, while higher
doses are more likely to impair
performance. Improved performance
does not necessarily indicate the
absence of neurotoxicity; both increases
and decreases in neurobehavioral
performance may result from
deleterious chemical interactions with
neurons. Dose-response extrapolation is
further complicated by the observation
that facilitative or impairment effects
within a given dosage range may occur
at some parameters of the test stimulus
or aspects of the response (response
rate-dependent) but not at others
(Altmann et al., 1991). Therefore, dose
extrapolations are more difficult when
there is uncertainty about the shape of
the dose-response function (biphasic,
linear, etc.) at the relevant test stimulus
and response parameters.
The risk assessment process with
animal data involves extrapolation from
the effects of high doses in animals to
predict the effects of chronic low-dose
exposure in humans. With data from
laboratory studies of hurnans in a risk
assessment, however, the extrapolation
is in the other direction, from very low-
dose laboratory exposure to predict the
effects of chronic exposure at higher
(but still low) concentrations in the
environment and workplace. Low- to
high-dose extrapolation within the same
species may require different
assumptions and risk assessment
procedures. Although high-dose human
exposures have occurred in accidents,
those data are primarily descriptive in
nature and cannot easily be plugged into
a quantitative risk extrapolation process.
Low dose laboratory data may be
combined with data from epidemiologic
studies of persons exposed to higher
concentrations.
3.6.1.5. Structure-activity.
Structure-activity relationships for
well-known chemicals have largely been
established by clinical methods (and
animal studies) and verified by
neurobehavioral and neurophysiologic
testing. Although an area of active
research, neurobehavioral testing of
humans has not yet been able to identify
reliable patterns of impairment among
chemical classes. This endeavor has
been hampered by most laboratory
research having been limited to the
evaluation of low concentrations of
solvents and a few other reversible
toxicants and by the exposure
uncertainties, biases, and confounding
variables found in cross-sectional or
cohort field studies.
3.6.2. Other Considerations in Risk
Assessment
3.6.2.1. Mechanisms of action
Uncovering behavioral and
neurophysiologic mechanisms of action
is a potential contribution of human
laboratory exposure studies to
neurotoxicity risk assessment. For
example, Stewart et al. (1972)
demonstrated that methylene chloride
was metabolized to carbon monoxide in
humans, and further studies (Putz et al.,
1979) found that CO production could
account for some of the neurobehavioral
impairment observed with that
chemical. Recent human laboratory
studies of solvents employed low
concentrations that produced mucosal
irritation and strong odor, but little
neurobehavioral impairment (Dick,
unpublished observation). The
mechanisms of action that produce
mucosal irritation and the neurotoxic
mechanisms that are expressed in
neurobehavioral impairment may be
quite different. Data on mucosal
irritation and odor may therefore
provide limited information for a
neurotoxicity risk assessment.
3.6.2.2. Exposure duration
A criticism of extrapolation from
animal studies to human exposure
conditions is that the effects of short-
term exposure (months to 1-2 years) in
animals may not accurately predict the
effects of chronic exposure (>10 years)
in humans. Laboratory studies rarely
expose human subjects to solvents for
more than 4-6 hours per day for 2-5
days while environmental and
workplace exposures of concern involve
6-8 hours of exposure per day for years.
The uncertainties of extrapolating from
relatively acute exposures to predict the
risks from chronic exposure will not be
eliminated by using human laboratory
exposure data in risk assessment.
3.6.2.3. Time-dependent effects
The acute exposures that are possible
in human laboratory studies may
provide little information on chronic
time-dependent neurobehavioral effects.
The effects of initial exposure may
remain the same, decrease (tolerance),
or increase (sensitization) with
continued or repeated exposure to the
chemical. All effects will not change in
unison; tolerance and sensitization may
be observed simultaneously on different
measures of neurobehavioral function.
The multiple toxicodynamic effects of
chemical exposure (neurobehavioral
and other) seem to follow individual
time courses suggestive of multiple
mechanisms of action. In addition, the
processes of tolerance and sensitization
can be influenced by testing conditions
and the nature of the behavioral task.
One also must be concerned about
latent effects that do not appear for
some time after a brief exposure and
"silent" cumulative neurotoxic effects
that are not observable in acute human
studies. Latent and silent effects not
only bring up the possibility of
unknown risks for human subjects, but
also make more difficult the
extrapolation of chronic neurotoxic
risks on the basis of acute exposures.
Therefore, the acute exposure
conditions possible in human laboratory
studies may provide us with very
limited information about the long-term
effects of chronic exposure.
3.6.2.4. Multiple exposures
In the environment and the
workplace, persons are seldom exposed
to only a single chemical. Rather, they
are most often exposed to complex
mixtures of chemicals, the relative
concentrations of which may vary over
time. For example, one farmer had more
than 50 different chemical products
(pesticides, herbicides, solvents, metals,
gases) with nervous system effects that
he used, prepared, or stored in his work
shed. Chemicals used in industrial
processes may also contain impurities
or contaminants that may produce
neurotoxic effects or alter the
neurotoxicity of the more abundant
chemical species. Chemical mixtures
may have additive or potentiating
effects not predictable from studies of
single chemicals (Strong and Garruto,
1991). Human laboratory exposure
studies traditionally have employed one
highly purified chemical or
combinations of two chemicals (usually
solvents) and thus may produce a
spectrum of neurotoxic effects different
from environmental and occupational
exposures.
Recently volatile organic compounds
(VOCs) have been used in human
exposure studies (Otto and Hudnell,
1991). VOCs consist of multiple volatile
compounds administered at
concentrations commonly found in
indoor air from emissions by laminates,
carpet, plastics, and other building and
decorating materials. Although VOCs
are thought to produce primarily
mucosal irritation and odors, reports of
"sick building syndrome" and
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individual sensitivity to indoor air
contaminants suggest that other
neurobehavioral mechanisms also may
be operating.
3.6.2.5. Generalizability and individual
differences
The results of field studies and
laboratory exposure studies are most
valuable when they can be extrapolated
to the general population. Studies
conducted in male workers or in young,
healthy volunteers may have limited
applicability to women or to people in
other age ranges. It therefore is
important to conduct studies that
include males and females of different
ages and ethnic heritage. Culture-
sensitive neurobehavioral test methods
are being developed and validated in
the United States and other countries.
While it is important to increase the
generalizability of results, it is equally
important to know when results cannot
be generalized. Studies should be
specifically directed toward identifying
subsets of individuals who are more or
less sensitive to neurotoxic insult or
differ in mode of expression. There are
many examples of individual
differences that alter response to
chemicals and drugs: phenylketonurics
are more sensitive to dietary tyramine
and persons with variants of plasma
pseudocholinesterase are more affected
by some neuromuscular blocking agents.
3.6.2.6. Veracity of neurobehavioral test
results
In most epidemiologic and human
laboratory studies, research volunteers
are highly motivated to perform well on
tests of neurobehavioral function. Under
voluntary conditions, actual
neurobehavioral performance may serve
as a reasonable index of nervous system
capabilities. Some studies, however, are
conducted in response to complaints of
symptoms thought to be related to
workplace, environmental, or
therapeutic exposure to chemicals and
drugs. The performance of research
participants with symptoms and
complaints may be significantly affected
(consciously or unconsciously) by
monetary rewards, emotional relief, or
social gains from the validation of their
complaints. Under these conditions,
performance may or may not accurately
reflect the capabilities of the nervous
system and may lead to inaccurate
conclusions about the magnitude of
nervous system dysfunction or about
putative chemical or drug etiologies.
In addition to suboptimal
performance engendered by potential
reinforcers or rewards, research
participants involved in disputes over
suspected neurotoxic exposures or in
litigation for monetary damages are
likely to be experiencing significant
emotional and behavioral reactions from
situational sources that can alter the
outcome of neurobehavioral assessment.
Anxiety, depression, sleep disturbances,
fatigue, worry, obsessive thoughts, and
distractibility may contribute to less
than optimal performance on motor and
cognitive neurobehavioral tasks,
especially where speed and sustained
concentration are important. Under
stressful conditions, it may be extremely
difficult to differentiate between
neurotoxic and situational sources of
observed functional impairment.
Functional neurobehavioral tests are not
well equipped to distinguish between
impairment from neurotoxicity and
from nonchemical variables. The use of
functional tests in symptomatic
populations requires great care in
interpretation. The development of
validity scales and other control
procedures for assessing nonchemical
influences on performance is greatly
needed.
across species is of paramount
importance for neurotoxicity risk
assessment.
4. Methods to Assess Animal
Neurotoxicity
4.1. Introduction
4.1.1. Role of Animal Models
3.6.3. Cross-Species Extrapolation
Many neurobehavioral tests were
developed according to constructs of
human cognitive processes. The diverse
measures of cognitive, sensory, and
motor performance in humans are
therefore not easily compared with
neurobehavioral function in animals.
While it may be possible to
conceptually relate some animal and
human neurobehavioral tests {e.g., grip
strength or signal detection), many
procedural differences prevent direct
comparison between species.
A more direct extrapolation from
animals to man might be possible if the
tests were chosen on the basis of
procedural similarity rather than on a
conceptual basis (Anger, 1991). Stebbins
and colleagues (1975) were successful in
developing homologous procedures in
nonhuman primates for the
psychophysical evaluation of antibiotic
ototoxicity. Efforts to develop
comparable tests of memory and other
neurobehavioral functions in animals
and humans are under way (Stanton and
Spear, 1990, Paule et al., 1990), and
such efforts may aid in cross-species
extrapolation. Other procedurally
defined methods, such as Pavlovian
conditioning (Solomon and Pendlebury,
1988), operant conditioning (Cory-
Slechta, 1990), signal detection, and
psychophysical scaling techniques
(Stebbins and Coombs, 1975), could also
be used to facilitate interspecies risk
extrapolation. Deriving comparable
neurobehavioral assessment methods in
animals and humans that will allow a
more straightforward extrapolation
Determining the risk posed to human
health from chemicals requires
information about the potential
lexicological hazards and the expected
levels of exposure. Some toxicological
data can be derived directly from
humans. Sources of such information
include accidental exposures to
industrial chemicals, cases of food-
related poisoning, epidemiological
studies, as well as clinical
investigations. While human data are
available from clinical trials for
therapeutics and they provide the most
direct means of determining effects of
potentially toxic substances, for other
categories of substances, it is generally
difficult, expensive, and, in some cases,
unethical to develop this type of
information. Quite often, the nature and
extent of available human toxicological
data are too incomplete to serve as the
basis for an adequate assessment of
potential health hazards. Furthermore,
for a majority of chemical substances
human toxicological data are simply not
available. Consequently, for most
toxicological assessments it is necessary
to rely on information derived from
animal models, usually rats or mice.
One of the primary functions of animal
studies is to predict human toxicity
prior to human exposure. In some cases,
species phylogenetically more similar to
human, such as monkeys or baboons,
are used in neurotoxicological studies.
Biologically, animals resemble
humans in many ways and can serve as
adequate models for toxicity studies
(Russell, 1991). This is particularly true
with regard to the assessment of adverse
effects to the nervous system, whereby
animal models provide a variety of
useful information that helps minimize
exposure of humans to the risk of
neurotoxicity. There are many
approaches to testing for neurotoxicity,
including whole animal (in vivo) testing
and tissue/cell culture (in vitro) testing.
At present, in vivo animal studies
currently serve as the principal
approach to detect and characterize
neurotoxic hazard and to help identify
factors affecting susceptibility to
neurotoxicity. Data from animal studies
are used to supplement or clarify
limited information obtained from
clinical or epidemiological studies in
humans, as well as provide specific
types of information not readily
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42383
obtainable from humans due to ethical
considerations. Frequently, results from
animal studies are used to guide the
design of lexicological studies in
humans.
In vitro tests have been proposed as
a means of complementing whole
animal tests, which could ultimately
reduce the number of animals used in
routine toxicity testing. It also has been
proposed that in vitro testing, when
properly developed, may be less time-
consuming and more cost-effective than
in vivo assessments (Goldberg and
Frazier, 1989; Atterwill and Walum,
1989). By understanding the biological
structures or functions affected by toxic
substances in vitro, it also may be
possible to predict neurotoxicological
effects in the whole animal. An added
advantage of in vitro testing is the
growing availability of human cell lines
that could be used for directly assessing
potential neurotoxic effects on human
tissue. The currently available strategies
for in vitro testing have certain
limitations, including the inability to
model neurobehavioral effects such as
loss of memory or sensory dysfunction
or to evaluate effectively the influence
of organ system interactions (e.g.,
neuronal, endocrinological, and
immunological) on the development
and expression of neurotoxicity.
In using animal models to predict
neurotoxic risk in humans, it is
important to understand that the
biochemical and physiological
mechanisms that underlie human
biological processes, particularly those
involving neurological and
psychological functions, are very
complex and are sometimes difficult, if
not impossible, to model exactly in a
lower species. While this caveat does
not preclude extrapolating the results of
animal studies to humans, it does
highlight the importance of using valid
animal models in well-designed
experimental studies.
4.1.2. Validity of Animal Models
Whether animal tests or methods
actually measure what they are intended
to measure, whether the data from such
tests can be obtained reliably, and
whether such data can be logically
extrapolated to humans are problems for
most disciplines in toxicology. Various
proposals have been made for the
standardization and validation of
methods used in neurotoxicological
research. It is generally agreed that
validation is an ongoing process that
establishes the credibility of a test,
building an increasing level of
confidence in the effective utility of any
model of evaluation. The credibility of
a method, as it applies to testing, is
usually discussed within several
different contexts, including construct
validity, criterion validity, predictive
validity, and detection accuracy.
Construct validity concerns the ability
of a method to measure selectively a
particular biological function and not
other dimensions. Construct validity is
frequently established empirically. For
example, sensory dysfunction such as
hearing loss is reported by humans
exposed to some chemicals, and tests
are designed to detect and quantify
those changes. Such tests are designed
to measure changes in auditory
function, while other sensations are
unaffected (Tilson, 1987; Moser, 1990).
Criterion validity refers to the ability
of a method to measure a characteristic
relative to some standard. For example,
Horvath and Frantik (1973) noted that
the significance of a test measurement
as an index of an actual treatment effect
should be validated relative to the
effects of a defined reference substance
or positive control. Furthermore, each
specific test or type of effect may require
an appropriate reference substance for
which the given type of effect is a
determining factor of the toxicity. Use of
reference agents has obvious advantages
in the assessment of unknown
chemicals.
Predictive validity refers to the ability
of a method to predict effects from an
incomplete or partial data set. An
animal model of neurotoxicity with
good predictive validity would reliably
predict neurotoxicity in humans, i.e.,
the animal to human extrapolation
would be good. There are several
examples in neurotoxicology where
animal models have been developed
based on neurotoxicological reports
from humans. Presumably, the
predictive validity of such models
would enable detecting similar kinds of
effects produced by uncharacterized
chemicals having a similar mechanism
of action.
It has been proposed (Tilson and
Cabe, 1978) that the most logical
approach to validate animal methods in
neurotoxicology is to evaluate chemicals
with and without known neurotoxicity
in humans in tests designed for animals
(predictive validity). By using such an
approach, it is possible to generate a
profile of effects characteristic of each
type of neurotoxicant (criterion
validity). This profile could then be
used to assess the construct validity of
various tests. That is, procedures
assumed to measure the same
neurobiological dimension should show
similar effects; measures designed to
detect changes in other functions should
not be affected. This approach to test
validation has been described as the
multitrait-multimethod process of
validation (Campbell and Fiske, 1959).
Of particular importance in
establishing the credibility of a method
is the accuracy of detecting a treatment-
related effect (Gad, 1989). Accuracy is a
function of two interacting elements,
specificity and sensitivity. Specificity is
the ability of a test to respond positively
only when the toxic endpoint of interest
is present. Sensitivity is the ability to
detect a change when present. This
aspect depends on the inherent design
of the procedure and experiment.
Increasing the specificity of a test may
reduce the possibility of classifying a
chemical as neurotoxic when, in fact, it
is not (false positive), but it may
increase the probability of missing a
true neurotoxicant (false negative).
Increasing sensitivity of a test may
reduce the possibility of false negatives,
but may increase the probability of false
positives.
4.1.3. Special Considerations in Animal
Models
4.1.3.1. Susceptible populations.
Like most other measures of
lexicological effect, neurotoxic
endpoints are subject to a number of
experimental variables that may affect
susceptibility to the biological effects of
toxicants. In this regard, genetic
variation (Festing, 1991) is a particularly
important issue in neurotoxicology. For
example, most neurotoxicological
assessments are carried out with only
one or two species. This may pose
problems, however, since species may
differ in sensitivity to neurotoxicants.
For example, nonhuman primates are
more sensitive than rats (Boyce et al.,
1984) or mice (Heikkila et al., 1984) to
the neurodegenerative effects of MPTP,
a byproduct in the illicit synthesis of a
meperidine analog (Langston et al.,
1983). In the assessment of delayed
neuropathology produced by some
cholinesterase inhibitors, it is well
known that hens are much more
sensitive than rodents (Cavanagh, 1954;
Abou-Donia, 1981,1983). In addition,
rat strains also may be differentially
sensitive to some neurotoxicants (Moser
et al., 1991). Although it is preferred
that more than one species be tested, the
cost required for routine multispecies
testing must be considered. Whenever
possible, the choice of animal models
should take into account differences in
species with regard to
pharmacodynamic, genetic composition
and sensitivity to neurotoxic agents.
In addition to species, other factors
such as gender of the test animal must
be taken into consideration. Some toxic
substances may have a greater
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neurotoxicological effect in one gender
(Squibb et al.( 1981; Matthews et al.,
1990). Thus, screening evaluations
frequently require both male and female
animals. Another important variable is
the age of the animal (Veronesi et al.,
1990). Whether a chemical produces
neurotoxicity may depend on the
maturations! stage of the organism
(Rodier, 1986). Most preliminary
assessments are designed to provide
information on adults, which have the
greatest probability of being exposed.
However, populations undergoing rapid
maturation or aged individuals may be
especially vulnerable to neurotoxic
agents. Longitudinal studies that assess
both genders at any stage of
development address many of the
problems associated with differentially
sensitive populations.
4.1.3.2. Dosing scenario.
The dosing strategy used in
experimental studies is an important
variable in the development and
expression of neurotoxicity (WHO,
1986). Some neurotoxicants can
produce neurotoxicity following a single
exposure, while others require repeated
dosing. Repeated dosing represents the
typical pattern of human exposure to
many chemical substances. Significant
differences in response may occur when
an acutely toxic quantity of material is
administered over different exposure
periods. For some neurotoxicants the
onset of neurotoxicity can occur
immediately after dosing, while others
may require time after exposure for the
toxicity to develop. Effects of repeated
exposure may result in a progressive
alteration in nervous system function or
structure, while latent or residual effects
may be discovered only in association
with age-related changes or after
suitable environmental or
pharmacological challenge (Zenick,
1983; MacPhail et al., 1983). To ensure
adequate assessment of neurotoxicity,
study designs should include multiple
dosing regimens, e.g., repeated
exposure, with appropriate dose-to-
response intervals of testing. Conduct of
neurotoxicological evaluations in
studies utilizing excessively toxic doses
should be avoided.
4.1.3.3. Other factors.
There are a number of other factors
that should be considered in the design
and interpretation of studies using
animal models (WHO, 1986). Design
factors include such issues as using
properly trained personnel to conduct
the studies, the use of appropriate
numbers of animals per group to
achieve reliable statistical significance,
and controlling the time-of-day
variability. Time of testing relative to
exposure is also important for assessing
neurotoxic endpoints such as behavior,
and experiments should be designed to
generate a time course of effects,
including recovery of function, if any.
Housing is an important environmental
design factor, because animals housed
individually and animals housed in
groups can respond differently to toxic
agents. Temperature, as an experimental
variable, may also affect the outcome of
neurotoxicological studies. The
responsiveness to some chemicals (e.g.,
triethyltin, methamphetamine) varies
with ambient temperature (Dyer and
Howell, 1982; Bowyer et al., 1992).
Some neurobiological endpoints, such
as sensory evoked potentials, can be
influenced by the endogenous
temperature of the animal (Dyer, 1987).
Therefore, changes in body temperature,
whether due to fluctuations in ambient
temperature or to some chemically
induced effect such as inhibition of
sweating, can confound the
interpretation of measures such as
evoked responses unless proper controls
are included in the experimental design.
Because a variety of other
physiological changes can influence
neuronal functions, it is important to
recognize that chemical-related
neurotoxicity could result from
treatment-induced physiological
changes, such as altered nutritional state
(WHO, 1986). As part of a
neurotoxicological profile, correlative
measures, such as relative and absolute
organ weights, food and water
consumption, and body weight and
weight gain, may be signs of
physiological change associated with
systemic toxicity and may be useful in
determining the relative contribution of
general toxicity.
4.1.3.4. Statistical considerations.
Experimental designs for
neurotoxicological studies are
frequently complex, with two or more
major variables (e.g., gender, time of
testing) varying in any single
experiment. In addition, such studies
typically generate varying types of data,
including continuous, dichotomous,
and rank-order data. Knowledge and
experience in experimental design and
statistical analyses are important. There
are several key statistical concepts that
should be understood in
neurotoxicological studies (WHO, 1986;
Gad, 1989). The power, or probability,
of a study to detect a true effect is
dependent on the size of the study
group, the frequency of the outcome
variable in the general population, and
the magnitude of effect to be identified.
Statistical evaluation of a treatment-
related effect involves the consideration
of two factors or types of errors to be
avoided. A Type I error refers to the
attribution of an exposure-related
neurotoxicological effect when none has
occurred (false positive), while a Type
II error refers to the failure to attribute
an effect when an exposure-related
effect has actually occurred (false
negative). In general, the probability of
a Type I error should not exceed 5
percent and the probability of a Type II
error should not exceed 20 percent.
Power is defined as one minus the
probability of a Type II error.
Determination of power also requires
knowledge of the difference in
magnitude of outcome measures
observed between exposed and control
groups and the variability of the
outcome measure among subjects. The
sample size required to achieve a given
level of statistical power increases as
variability increases or the difference
between groups decreases.
Continuous data (i.e., magnitude, rate,
amplitude), if found to be normally
distributed, can be analyzed with a
general linear model using a grouping
factor of dose and, if necessary, repeated
measures across time. Post hoc
comparisons between control and other
treatment groups can be made following
tests for overall significance. In the case
of multiple endpoints within a series of
evaluations, correction for multiple
observations (e.g., Bonferroni's) might
be necessary.
Descriptive data (categorical) and rank
data can be analyzed using standard
nonparametric techniques. In some
cases, if it is believed that the data fit
the linear model, the categorical data
modeling procedure can be used for
weighted least-squares estimation of
parameters for a wide range of general
linear models, including repeated
measures analyses. The weighted least-
squares approach to categorical and
rank data allows computation of
statistics for testing the significance of
sources of variation as reflected by the
model.
4.2. Tiered Testing in Neurotoxicology
The utility of tiered testing as an
efficient and cost-effective approach to
evaluate chemical toxicity, including
neurotoxicity, has been recognized
(NRC, 1975). Briefly, first-tier tests are
designed to determine the presence or
absence of neurotoxicity, while second-
tier tests characterize the neurotoxic
effect (NRC, 1992). There are at least
two aspects of tiered testing, one
involving the type of test used (Tilson,
1990a) and the other involving the
dosing regimen (Goldberg and Frazier
1989).
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42385
4.2.1. Type of Test
Tests designed to measure the
presence or absence of an effect are
usually different from those used to
assess the degree of toxicity or the
lowest exposure level required to
produce an effect (Tilson, 1990a).
Screening procedures are first-tier tests
that typically permit the testing of many
groups of animals. Such procedures may
not require extensive resources and are
usually simple to perform. However,
these techniques may be labor intensive,
provide subjective measures, yield
semiquantitative data, and may not be
as sensitive to subtle effects as those
designed to characterize neurotoxic
effects or second-tier tests. Specialized
tests are usually more sensitive and
employed in studies concerning
mechanisms of action or the estimation
of the lowest effective dose. Such testing
procedures are usually referred to as
secondary tests and may require special
equipment and more extensive
resources. Secondary tests are usually
quantitative and yield graded or
continuous data amenable to routine
parametric statistical analyses.
Testing at the first tier is used to
determine if a chemical might produce
neurotoxicity following exposure, i.e.,
hazard detection. In this case, there may
be little existing information concerning
the neurotoxic potential of an agent.
Examples of first-tier tests include
functional observational batteries (FOB),
including an evaluation of motor
activity and routine
neurohistopathology. For some
chemicals or types of chemicals, there
may be a specific interest in screening
for a particular presumed mechanism of
toxicity (e.g., inhibition of
cholinesterase or neurotoxic esterase) or
neurobiological response (e.g., a site-
specific neuronal degeneration). In these
cases, specific neurochemical or
neuropathological endpoints can be
used in conjunction with first-tier tests.
It is desirable that tests selected for use
in hazard detection provide a suitable
level of sensitivity using the smallest
number of animals necessary.
A decision to test at the next tier is
based on data suggesting that an agent
produces neurotoxicity. The
information used to make a decision to
test a chemical at the secondary level
can come from a variety of sources,
including neurotoxicological data
already in the literature, structure-
activity relationships, data from first-tier
testing, or following reports of specific
neurotoxic effects in humans exposed to
the agent. Testing at the secondary level
includes detailed neuropathological
evaluation as well as specific behavioral
tests, e.g., procedures to assess learning
and memory, or sensory function. Tests
at the second tier usually measure the
most sensitive endpoints of
neurotoxicity, and are the most suitable
for determining the no observable
adverse effect level or benchmark dose.
At this stage of testing, the use of a
second species is considered to address
the issue of cross-species extrapolation.
At the present time, tiered testing
approaches in neurotoxicology rely
heavily on functional endpoints. It is
possible that future testing protocols
will employ a different strategy as more
information concerning neurotoxic
mechanisms of action become available
and biologically based dose-response
models are developed.
4.2.2. Dosing Regimen
Goldberg and Frazier (1989) have
indicated that first-tier evaluations
identify effects of substances following
acute or repeated exposure over a wide
range of doses. Measures are simple,
focused on detection of effects, and
results are used to help establish
parameters for the second tier of testing.
The subsequent stage(s) of tier testing
are designed to characterize more fully
the toxicity of repeated dosing. In this
case, animals are exposed repeatedly or
continuously to define the scope of
toxicity, including latent or delayed
effects, development of tolerance, and
the reversibility of adverse effects. The
subsequent stage(s) of testing also
provide information about specific
effects or study mechanisms of
neurotoxicity. This tier uses methods
appropriate to characterize the effects
observed in the first tier of testing.
4.3. Endpoints of Neurotoxicity
4.3.1. Introduction
As applied to the safety assessment of
chemical substances, neurotoxicity is
any adverse change in the development,
structure, or function of the central and
peripheral nervous system following
exposure to a chemical agent (Tilson,
1990b). Measures used in animal
neurotoxicological studies are designed
to assess these changes. Neurotoxicity
can be described at multiple levels of
organization, including chemical,
anatomical, physiological, or behavioral
levels. At the chemical level, for
example, a neurotoxic substance might
inhibit protein or transmitter synthesis,
alter the flow of ions across cellular
membranes, or prevent release of
neurotransmitter from nerve terminals.
Anatomical changes may include
destruction of the neuron, axon, or
myelin sheath. At the physiological
level, neuronal responsiveness to
stimulation might be enhanced by a
decrease of inhibitory thresholds in the
nervous system. Chemical-induced
effects at the behavioral level can
involve a variety of alterations in motor,
sensory, or cognitive function, including
increases or decreases in frequency or
accuracy of responding. Although
behavioral and neurophysiological
endpoints may be very sensitive
indicators of neurotoxicity, they can be
influenced by other factors. The
uncertainties associated with data from
functional endpoints can be reduced if
interpreted within the context of other
neurotoxicological measures
(neurochemical or neuropathological)
and systemic toxicity endpoints,
particularly if such measures are taken
concurrently. Behavioral effects that
reflect an indirect effect secondary to
systemic toxicities may also be
considered adverse. Table 4-1 provides
examples of potential endpoints of
neurotoxicity at the behavioral,
physiological, chemical, and structural
levels.
TABLE 4-1 .—EXAMPLES OF POTENTIAL
ENDPOINTS OF NEUROTOXICITY
Behavioral Endpoints:
Absence or altered occurrence, magnitude,
or latency of sensorimotor reflex
Altered magnitude of neurological meas-
urements, such as grip strength or
hindlimb splay
Increases or decreases in motor activity
Changes in rate or temporal patterning of
schedule-controlled behavior
Changes in motor coordination, weakness,
paralysis, abnormal movement or pos-
ture, tremor, ongoing performance
Changes in touch, sight, sound, taste, or
smell sensations
Changes in learning and memory
Occurrence of seizures
Altered temporal development of behaviors
or reflex responses
Autonomic signs
Neurophysiological Endpoints:
Change in velocity, amplitude, or refractory
period of nerve conduction
Change in latency or amplitude of sensory-
evoked potential
Change in EEG pattern or power spectrum
Neurochemical Endpoints:
Alterations in synthesis, release, uptake,
degradation of neurotransmitters
Alterations in second messenger associ-
ated signal transduction
Alterations in membrane-bound enzymes
regulating neuronal activity
Decreases in brain AChE
Inhibition of NTE
Altered developmental patterns of neuro-
chemical systems
Altered proteins (c fos, substance P)
Structural Endpoints:
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TABLE 4-1 .—EXAMPLES OF POTENTIAL
ENDPOINTS OF NEUROTOXICITY—
Continued
Accumulation, proliferation, or rearrange-
ment of structural elements
Breakdown of cells
GFAP increases (adult)
Gross changes in morphology, including
brain weight
Discoloration of nerve tissue
Hemorrhage in nerve tissue
4.3.2. Behavioral Endpoints
Neurotoxicants produce a wide array
of functional deficits, including motor,
sensory, and learning or memory
dysfunction (WHO, 1986; Tilson and
Mitchell, 1984). Many procedures have
been devised to assess overt as well as
relatively subtle changes in those
functions; hence their applicability to
the detection of neurotoxicity and to
hazard characterization. Many of the
behavioral tests have been developed
and validated with well-characterized
neurotoxicants. Behavioral tests and
agents that affect them have been
reviewed recently (WHO, 1986; Cory-
Slechta, 1989). Examples of such tests,
the nervous system function being
measured, and neurotoxicants known to
affect these measures are listed in Table
4-2.
TABLE 4-2. EXAMPLES OF SPECIALIZED TESTS TO MEASURE NEUROTOXICITY
Function
Procedure
Representative-agents
Neuromuscular:
Weakness
Incoordination
Tremor
Grip strength; swimming endurance; suspension from rod; discriminative motor
function; hindlimb splay.
Rotorod, gait measurements
Rating scale, spectral analysis .".'"!.'"!"!".'"".'.'."""."".'
methyl butylketone,
Myoclonia, spasms ....
Sensory:
Auditory
Visual toxicity
Somatosensory tox-
icity.
Pain sensitivity
Olfactory toxicity
Learning/Memory:
Habituation
Classical conditioning
Operant or instrumen-
tal conditioning.
Rating scale, spectral analysis
Discriminated conditioning Reflex modification
Discriminated conditioning
Discriminated conditioning
Discriminated conditioning (titration); functional observational battery
Discriminated conditioning
Startle reflex
Nictitating membrane
Conditioned flavor aversion
Passive avoidance I.""!!!""!!!!!"!'
Olfactory conditioning
One-way avoidance """.""!
Two-way avoidance
Y-maze avoidance
Biel water maze
Morris water maze
Radial arm maze
Delayed matching to sample .
Repeated acquisition
Visual discrimination learning
n-hexane,
carbaryl.
3-acetylpyridine, ethanol.
Chlordecone, Type I pyrethroids,
DDT.
DDT, Type II pyrethroids.
Toluene, trimethyltin.
Methyl mercury.
Acrylamide.
Parathion.
3-methylindole methylbromide.
Diisopropyl-flurophosphate (DFP).
Aluminum.
Carbaryl.
Trimethyltin, IDPN.
Neonatal trimethyltin.
Chlordecone.
Neonatal lead.
Hypervitaminosis A.
Styrene.
DFP.
Trimethyltin.
DFP.
Carbaryl.
Lead.
4.3.2.1. Functional observational
batteries.
Functional observational batteries are
first-tier tests designed to detect and
quantify major overt behavioral,
physiological, and other neurotoxic
effects (Moser, 1989). A number of
batteries have been used (Tilson and
Moser, 1992), each consisting of tests
generally intended to evaluate various
aspects of sensorimotor function. Most
FOB are similar to clinical neurological
examinations that rate presence or
absence and, in some cases, the relative
degree of neurological signs. A typical
TABLE 4-3.—SUMMARY OF MEASURES IN THE FUNCTIONAL OBSERVATIONAL
PRODUCED BY EACH
FOB, as summarized in Table 4-3,
evaluates several functional domains,
including neuromuscular (i.e.,
weakness, incoordination, gait, and
tremor), sensory (i.e., audition, vision,
and somatosensory), and autonomic
(i.e., pupil response and salivation)
function.
BATTERY AND THE TYPE OF DATA
Home cage and open field
Posture (D)
Convulsions, tremors (D)
Palpebral closure (R)
Lacrimation (R)
Piloerection (Q)
Salivation (R)
Vocalizations (Q)
Rearing (C)
Manipulative
Ease of removal (R)
Handling reactivity (R)
Approach response (R)
Click response (R)
Touch response (R)
Tail pinch response (R)
Righting reflex (R)
Physiologic
Body temperature (I)
Body weight (I)
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TABLE 4-3.—SUMMARY OF MEASURES IN THE FUNCTIONAL OBSERVATIONAL BATTERY AND THE TYPE OF DATA
PRODUCED BY EACH—Continued
Urination (C)
Defecation (C)
Gait (D, R)
Arousal (R)
Mobility (R)
Stereotypy (D)
Bizarre behavior (D)
Landing foot splay (I)
Forelimb grip strength (I)
Hindlimb grip strength (I)
Pupil response (Q)
D = descriptive data; R = rank order data; Q
I = interval data; C = count data
The major advantages of FOB tests are
that they can be administered within the
context of other ongoing toxicological
tests and provide some indication of the
possible neurological alterations
produced by exposure. Potential
problems include insufficient
interobserver reliability, difficulty in
defining certain endpoints, and the
tendency toward observer bias. The
latter can be controlled by using
observers unaware of the actual
treatment of the subjects. Some FOB
tests may not be very sensitive to agent-
induced sensory loss (i.e., vision,
audition) or alterations in cognitive or
integrative processes such as learning
and memory. FOB data may be used to
trigger experiments performed at the
next tier of testing.
FOB data may be interval, ordinal, or
continuous (Creason, 1989). The
relevance of statistically significant test
results from an FOB is judged according
to the number of signs affected, the
dose(s) at which neurotoxic signs are
observed, and the nature, severity, and
persistence of the effects. Data from the
FOB may provide presumptive evidence
of adverse effects and neurotoxicity. If
only a few unrelated measures in the
FOB are affected or the effects are
unrelated to dose, there is less concern
about neurotoxic potentials of a
chemical. If dose is associated with
other overt signs of toxicity, including
systemic toxicity, large decreases in
body weight, or debilitation, the data
must be interpreted carefully. In cases
where several related measures in a
battery of tests are affected and the
effects appear to be dose dependent, the
level of concern about the potential of
a chemical is higher.
4.3.2.2. Motor activity.
Movement within a defined
environment is a naturally occurring
response and can be affected by
environmental agents. Motor activity
represents a broad class of behaviors
involving coordinated participation of
sensory, motor, and integrative
: quanta! data;
processes. Motor activity measurements
are noninvasive and can be used to
evaluate the effects of acute and
repeated exposure to chemicals
(MacPhail et al., 1989). Motor activity
measurements have also been used in
humans to evaluate disease states,
including disorders of the nervous
system (Goldstein and Stein, 1985). The
assessment of motor activity is often
included in first-tier evaluations, either
as part of the FOB or as a separate
quantitated measurement.
There are many different types of
activity measurement devices, differing
in size, shape, and method of movement
detection (MacPhail et al., 1989).
Because of the accuracy and ease of
calibration, devices with photocells are
widely used. In general, situating the
apparatus to minimize extraneous noise,
movements, or lights usually requires
that the recording devices be placed in
light- and sound-attenuating chambers
during the testing period. A number of
different factors, including age, gender,
and time of day, can affect motor
activity, and should be controlled or
counterbalanced. Different strains of
animals may have significantly different
basal levels of activity, making
comparisons across studies difficult. A
major factor in activity studies is the
duration of the testing session. Motor
activity levels are generally highest at
the beginning of the session and
decrease to a low level throughout the
session. The rate of decline during the
test session is frequently termed
"habituation."
Motor activity measurements are
typically included as part of a battery of
tests to detect or characterize
neurotoxicity. Agent-induced alterations
in motor activity associated with overt
signs of toxicity (e.g., loss of body
weight, systemic toxicity) or occurring
in non-dose-related fashion are of less
concern than changes that are dose
dependent, related to structural or other
functional changes in the nervous
system, or occur in the absence of life-
threatening toxicity and are generally
convincing evidence of neurotoxicity.
4.3.2.3. Neuromotor 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: weakness,
incoordination, tremor, and myoclonia
or spasms. Chemical-induced changes
in motor function can be determined
with relatively simple techniques such
as the FOB. More specialized tests to
assess weakness include measures of
grip strength, swimming endurance,
suspension from a hanging rod,
discriminitive motor function, and
hindlimb splay. Rotarod and gait
assessments measure incoordination,
while rating scales and spectral analysis
techniques quantify tremor and other
abnormal movements (Tilson and
Mitchell, 1984).
An example of a second-tier
procedure to assess motor function 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 that 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). The accuracy of performance may
reflect neuromotor function and is
sensitive to the debilitating effects of
many psychoactive drugs (Walker et al.,
1981; Newland, 1988). Gait, an index of
coordination, has been measured in rats
under standardized conditions and can
be a sensitive indication of specific
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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 been
used extensively in neurotoxicology.
Most require preexposure training
(including alterations of motivational
state) of experimental animals, but such
tests might be useful, in as much as
similar procedures are often used in
assessing humans.
4.3.2.4. 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, body temperature, and other
physiological endpoints. Furthermore,
many tests assess the behavioral
response of an animal to a specific
sensory stimulus; such responses are
usually motor movements 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 sensory stimulus may
have been related to agent-induced
motor dysfunction.
Several first-tier testing procedures
have been devised to screen for overt
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). Screening tests for sensory
deficits are typically not suitable to
characterize chemical-induced changes
in acuity or fields of perception. The
characterization of sensory deficits
usually necessitates psychophysical
methods that study the relationship
between the physical dimensions of a
stimulus and the behavioral response it
generates (Maurissen, 1988).
One second-tier approach to the
characterization of sensory function
involves the use of reflex-modification
techniques (Crofton, 1990). Chemical-
induced changes in the stimulus
frequency or threshold required to
inhibit a reflex are taken as possible
changes in sensory function. 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 (i.e.,
discriminated or conditioned
responding). 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 respond
to 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
methylmercury (Merigan, 1979).
Procedures to characterize chemical-
induced sensory dysfunction have been
used often in neurotoxicology. As in the
case of most procedures designed to
characterize nervous system
dysfunction, training and motivational
factors can be confounding factors.
Many tests designed to assess sensory
function for laboratory animals can also
be applied with some adaptation to
humans.
4.3.2.5. Learning and memory.
Learning and memory disorders are
neurotoxic effects of particular
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
exposure to toxic agents may have a role
in the pathogenesis of senile dementia.
Learning can be defined as an
enduring change in the mechanisms of
behavior that results from experience
with environmental events (Domjan and
Burkhard, 1986). Memory is a change
that can be either short-lasting or long-
lasting (Eckerman and Bushnell, 1992).
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 the brain can
inhibit learning. It is also known that
some brain lesions 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; Peele and Vincent,
1989).
One simple index of learning and
memory, which can be measured as a
first-tier endpoint, is habituation.
Habituation 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 needed to produce
response decrements (Overstreet, 1977).
Although habituation is a very simple
form of learning, it can also be
perturbed by a number of chemical
effects not related to learning.
A more complicated 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 is the
unconditioned stimulus. With repeated
pairings of the two stimuli, the
conditioned stimulus comes to elicit a
response similar to the response elicited
by 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
(Yokel, 1983). Other classically
conditioned responses known to be
affected by psychoactive or neurotoxic
agents are conditioned taste aversion
(Riley and Tuck, 1985) and conditioned
suppression (Chiba and Ando, 1976).
Second-tier procedures to assess
learning or memory typically involve
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42389
r j 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
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. Examples of
instrumental learning procedures used
in neurotoxicology are repeated
acquisition (Schrot et al., 1984). passive
and active avoidance, Y-maze
avoidance, spatial mazes (radial-arm
maze), and delayed matching to sample
(Heise, 1984; WHO, 1986; Tilson and
Mitchell, 1984).
4.3.2.6. Schedule-controlled behavior.
Another type of second-tier procedure
is schedule-controlled operant behavior
(SCOB), which involves the
maintenance of behavior (performance)
by response-dependent reinforcement
(Rice, 1988). 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 with appropriate experimental
design may be useful for studying
chemical-induced effects on motor,
sensory, and cognitive function.
The primary endpoints 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 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 the rate and require analysis
of the distribution of responses relative
to 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 (Paule and
McMillan, 1984; MacPhail 1985; Cory-
Slechta, 1989; 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 great potential for use in
second-tier assessments.
4.3.3. Neurophysiological Endpoints of
Neurotoxicity
Neurophysiological studies are those
that assess function either directly
through measurements of the electrical
activity of the nervous system
(electrophysiology) or indirectly
through measurements of peripheral
organ functions controlled or modulated
by the nervous system (general
physiology) (Dyer, 1987). When
performed properly, neurophysiological
techniques provide information on the
integrity of defined portions of the
nervous system. Many of the endpoints
used in animals have also been used in
humans to determine chemical-induced
alterations in neurophysiological
function.
The term "electrophysiology" refers
to the set of neurophysiological
procedures that study neural function
through the direct measurement of the
electrical activity generated by the
nervous system (Dyer, 1987). A variety
of electrophysiological procedures are
available for application to
neurotoxicological problems, which
range in scale from procedures that
employ microelectrodes to study the
function of single nerve cells or
restricted portions of them, to
procedures that employ macroelectrodes
to perform simultaneous recordings of
the summed activity of many cells. The
latter types of procedures have
historically been used in studies to
detect or characterize the potential
neurotoxicity of agents of regulatory
interest. Several macroelectrode
procedures are discussed below.
4.3.3.1. Nerve conduction studies.
Nerve conduction studies are
generally performed on peripheral
nerves and can be useful in
investigations of possible peripheral
neuropathy. Most peripheral nerves
contain mixtures of both individual
sensory and motor nerve fibers, which
may or may not be differentially
sensitive to neurotoxicants. It is possible
to distinguish sensory from motor
effects in peripheral nerve studies by
measuring activity in purely sensory
nerves such as the sural to study
sensory effects or by measuring the
muscle response evoked by nerve
stimulation to measure motor effects.
While a number of endpoints can be
recorded, the most commonly used
variables are (1) Nerve conduction
velocity, and (2) response amplitude. In
well-controlled studies, decreases in
nerve conduction velocity typically are
evidence of neurotoxicity (Dyer, 1987).
While a decrease in nerve conduction
velocity is a reliable measure of
demyelination, it frequently occurs
rather late in the course of axonal
degradation because normal conduction
velocity may be maintained for some
time in the face of axonal degeneration.
For this reason, a measurement of
normal nerve conduction velocity does
not necessarily rule out peripheral
axonal degeneration if other signs of
peripheral nerve dysfunction are
present. Increases in conduction
velocity of adult organisms following
treatment with neurotoxic compounds,
in the absence of hypothermia, are
atypical responses and may, in fact,
reflect experimental or statistical errors.
Decreases in response amplitude reflect
a loss of active nerve fibers, and may
occur prior to decreases in conduction
velocity in the course of peripheral
neuropathy. Hence changes in response
amplitude may be more sensitive
measurements of axonal degeneration
than conduction velocity.
Measurements of response amplitude,
however, are more variable and require
careful experimental techniques, a
larger sample size, and greater statistical
power than measurements of velocity to
detect changes. Alterations in peripheral
nerve function are associated with
abnormal peripheral sensations such as
numbness, tingling, or burning or with
motor impairments such as weakness.
Examples of compounds that alter
peripheral nerve function in humans or
experimental animals at some level of
exposure include acrylamide, carbon
disulfide, hexacarbons, lead, and some
organophosphates.
4.3.3.2. Sensory evoked potentials.
Sensory evoked potentials are
electrophysiological procedures that
involve measuring the response elicited
by the presentation of a defined sensory
stimulus such as a tone, a light, or a
brief electrical pulse to the skin.
Sensory evoked potentials reflect
sensory function, and can be used to
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investigate visual, auditory, or
somatosensory (body sensation) systems
(Rebert, 1983; Mattsson and Albee,
1988). The data are in the form of a
voltage record over time, which can be
quantified in several ways. Commonly,
the positive and negative voltage peaks
are identified and measured as to their
latency (time from stimulus onset) and
amplitude (voltage).
Changes in peak amplitudes or
equivalent measures reflect changes in
the magnitude of the neural population
that is responsive to stimulation. Both
increases and decreases in amplitude
are possible following exposure to
neurotoxicants because (1) The brain
normally operates in a careful balance
between excitatory and inhibitory
systems, and disruption of this balance
can produce either positive or negative
shifts in the voltages recorded in evoked
potential experiments, and (2) excitatory
or inhibitory neural activity is translated
into a positive or negative deflection in
the sensory evoked potential depending
on the physical orientation of the
electrode with respect to the tissue
generating the response, which is
frequently unknown. Within any given
sensory system, the neural circuits that
generate the different evoked potential
peaks differ as a function of peak
latency. In general, early latency peaks
reflect the transmission of afferent
sensory information, and changes in
either the latency or amplitude of these
peaks generally indicate a neurotoxic
change that is likely to be reflected in
deficits in sensory perception. The later
latency peaks, in general, reflect not
only the sensory input, but also the
more nonspecific factors such as the
behavioral state of the subject including
such factors as arousal level,
habituation, or sensitization. Thus, the
neurotoxicological significance of
changes in later latency evoked
potential peaks must be interpreted in
light of the behavioral status of the
subject.
4.3.3.3. Convulsions.
Observable behavioral convulsions in
animals may be indicative of central
nervous system seizure activity.
However, behavioral convulsions that
occur only at lethal or near lethal dose
levels may reflect an indirect effect
secondary to systemic toxicity and not
directly on the nervous system.
Convulsions occurring at dose levels
that are clearly sublethal, and in the
absence of apparent systemic toxicity,
are more likely due to a direct effect on
the nervous system. In such cases,
neurophysiological recordings of
electrical activity in the brain that are
indicative of seizures may provide
additional evidence of direct
neurotoxicity. In addition to producing
seizures, chemicals may also affect
seizure susceptibility, altering the
frequency, severity, duration, or
threshold for eliciting seizures produced
through other means. Such changes can
occur after acute exposure or after
repeated exposure to dose levels below
the acute threshold, and are considered
neurotoxic. Agents that produce
convulsions include lindane, DDT,
pyrethroids, and trimethyltin (WHO,
1986). Some agents, including many
solvents, act to raise the threshold for
eliciting seizures through other means
or otherwise act to reduce the severity
or duration of the elicited convulsions.
These agents are difficult to classify as
neurotoxic based on such data, but
frequently have other effects on which
a determination of neurotoxic potential
can be based.
4.3.3.4. Electroencephalography (EEC)
EEC analysis is used widely in
clinical settings for the diagnosis of
neurological disorders and less often for
the detection of subtle toxicant-induced
dysfunction (WHO, 1986; Eccles, 1988).
The basis for the use of EEC in either
setting is the relationship between
specific patterns of EEC waveforms and
specific behavioral states. Because states
of alertness and the stages of sleep are
associated with distinct patterns of
electrical activity in the brain, it is
generally thought that arousal level can
be evaluated by monitoring the EEC.
Dissociation of EEC activity and
behavior can, however, occur after
exposure to certain chemicals. Normal
patterns of transition between sleep
stages or between sleeping and waking
states are known to remain disturbed for
prolonged periods of time following
exposure to certain chemical classes
(e.g., organophosphates). Changes in the
pattern of the EEC can be elicited by
stimuli producing arousal (e.g., lights,
sounds) and neuroactive drugs. In
studies with toxicants, changes in EEC
pattern can sometimes precede
alterations in other objective signs of
neurotoxicity. EEC experiments must be
done under highly controlled
conditions, and the neurotoxicological
significance of chemical-induced
changes in the EEC in the absence of
other signs of neurotoxicity must be
considered on a case-by-case basis.
Many chemicals, including metals,
solvents, and pesticides, would be
expected to affect the EEC.
4.3.3.5. Electromyography (EMG).
EMG involves making electrical
recordings from muscle and has been
used extensively in human clinical
studies in the diagnosis of certain
diseases of the muscle (WHO, 1986).
Changes in the EMG include amplitude
and firing frequency of spontaneous
firing; evoked muscle responses to nerve
stimulation can be used to study
alterations in the neuromuscular
junction. EMG has been used to study
toxicant-induced changes in
neuromuscular function, including
organophosphate insecticides, methyl n-
butyl ketone, and botulinum and
tetanus toxin.
4.3.3.6. Spinal reflex excitability.
Segmental spinal monosynaptic and
polysynaptic reflexes are relatively
simple functions in the central nervous
system that can be evaluated by
quantitative techniques (WHO, 1986).
Many of the procedures used in animals
are similar to procedures used clinically
to perform neurological tests in humans.
One approach infers the functional state
of a reflex arc from either the latency
and magnitude of the reflex response
evoked by stimuli of predetermined
intensity or from the stimulus intensity
required to elicit a detectable response
(i.e., the threshold). This approach is
used best in a screening context and the
significance of effects in this test should
be considered on a case-by-case basis.
A second more involved approach
records electrophysiologically the time
required for a stimulus applied to a
peripheral nerve to reach the spinal
cord and return to the site of the original
stimulation. Data from this procedure
can indicate the excitability of the
motoneuron pool, an effect seen with
many volatile solvents. Although this
approach is more invasive and time-
consuming than the noninvasive
procedure, it provides better data
concerning the possible site of action. In
addition, the manner in which the
invasive procedure is carried out (i.e., in
decerebrated animals) precludes
repeated testing on the same animal.
The significance of effects in this
procedure should also be considered on
a case-by-case basis.
4.3.4. Neurochemical Endpoints of
Neurotoxicity
Neuronal function within the nervous
system is dependent on synthesis and
release of specific neurotransmitters and
activation of their receptors in specific
neuronal pathways. With few
exceptions, neurochemical
measurements are invasive and
therefore used infrequently in human
risk assessment. There are many
different neurochemical endpoints that
could be measured in
neurotoxicological studies (Bondy,
1986; Mailman, 1987; Morell and
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42391
Mailman, 1987). Neurotoxicants can
interfere with the ionic balance of a
neuron, act as a cytotoxicant after being
transported into a nerve terminal, block
uptake of neurotransmitter precursors,
act as a metabolic poison, overstimulate
receptors, block transmitter release, and
inhibit transmitter degradation. Table 4-
4 lists several chemicals with known
neurochemical effects. Many
neuroactive agents can increase or
decrease neurotransmitter levels in the
brain. Dose-related changes on these
endpoints may indicate a chemical
effect on the nervous system, but the
neurotoxicological significance of such
changes must be interpreted in the
context of other signs of neurotoxicity.
TABLE 4-4.—NEUROTOXICANTS WITH KNOWN NEUROCHEMICAL MECHANISMS
Site of attack
1 . Neurotoxicants acting on ionic balance
7. Transmitter degradation (ACh) inhibitors
8. Microtubule disrupters
Examples
Tetrodotoxin.
p,p1 — DDT, pyrethroids (I).
Batrachotoxin.
Chlordecone.
MPTP.
Hemicholinium.
Cyanide.
Domoic acid.
Botulinum toxin.
Organophosphates,
carbamates.
Vincristine.
Some chemicals, such as the
organophosphate and carbamate
insecticides, are known to interfere with
a specific enzyme, acetylcholinesterase
(AChE) (Costa, 1988). Inhibition of this
enzyme in brain may be considered
evidence of neurotoxicity, whereas
decreases in AChE in the blood, which
can be easily determined in humans, are
only suggestive of a neurotoxic effect. A
subset of organophosphate agents
produces organophosphate-induced
delayed neuropathy (OPIDN) after acute
or repeated exposure. Neurotoxic
esterase (or neuropathy target enzyme,
NTE) has been associated with agents
that produce OPIDN (Johnson, 1990).
The ultimate functional significance
of many biochemical changes is not
known; therefore it may be difficult to
determine if a specific biochemical
change can be considered adverse or
convincing evidence of neurotoxicity.
Any such change, however, is
potentially adverse and each
determination of adversity requires a
judgment to be made. Likewise, the
absence of specific biochemical testing
protocols does not mean biochemical
changes are of no concern, but instead
reflects a lack of understanding of the
significance of changes at the
biochemical level.
4.3.5. Structural Endpoints of
Neurotoxicity
The central nervous system (brain and
spinal cord) comprises nerve cells or
neurons, which consist of a neuronal
body, axon, and dendritic processes.
Various types of neuropathological
lesions may be classified according to
their nature or the site where they are
found (WHO, 1986; Krinke. 1989;
Griffin, 1990). Lesions may be classified
as neuropathy (changes in the neuronal
body), axonopathy (changes in the
axons), myelinopathy (changes in the
myelin sheaths), neurodegeneration
(changes in the nerve terminals), and
peripheral neuropathy (changes in the
peripheral nerves). For axonopathies, a
more precise location of the changes
should be described (i.e., proximal,
central, or distal axonopathy). In some
cases, agents produce neuropathic
conditions that resemble naturally
occurring neurodegenerative disorders
in humans (WHO, 1986). Table 4-5 lists
examples of such chemicals, their
known site of action, the type of
neuropathology produced, and the
disease or condition that each typifies.
TABLE 4-5.—EXAMPLES OF KNOWN NEUROPATHIC AGENTS
Site of attack
Neuropathology
Neuronopathy
Neurodegeneration ....
Myelinopathy
Distal axonopathy
Central axonopathy ...
Proximal axonopathy .
Corresponding
neurotoxicant
Methylmercury ..
A ETT
Quinolinic acid ..
3-acetylpridine ..
Aluminum
MPTP
Lead Buckthorn
toxin.
Acrylamide
Hexacarbons
Carbon disul-
fide.
Clioquinol
B.B'-imminodi-
proprionitrile.
Disease or
neurodegenerative condi-
tion
Minamata disease.
Ceroid lipofuscinoses.
Huntington's disease.
Cerebellar ataxia.
Alzheimer's disease.
Parkinson's disease.
Neuropathy of metachro-
matic leukodystrophy.
Vitamin deficiency.
Subacute myelooptico-neu-
ropathy.
Motor neuron disease.
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In general, chemical effects lead to
two types of primary cellular alteration:
(1) the accumulation, proliferation, or
rearrangement of structural elements
(e.g., intermediate filaments,
microtubules) or organelles
(mitochondria) and (2) the breakdown of
cells, in whole or in part. The latter can
be associated with regenerative
processes that may occur during
chemical exposure. Such changes are
considered to be neurotoxic.
While most neurotoxic damage is
evident at the microscopic level, gross
changes in morphology can be reflected
by a significant change in the weight of
the brain. Weight changes (absolute or
relative to body weight), discoloration,
discrete or massive cerebral
hemorrhage, or obvious lesions in nerve
tissue are generally considered
neurotoxic effects.
Chemical-induced injury to the
central nervous system is associated
with astrocytic hypertrophy at the site
of damage. Assays of glial fibrillary
acidic protein (GFAP), the major
intermediate filament protein of
astrocytes, has been proposed as a
biomarker of this response (O'Callaghan,
1988). A number of chemicals known to
injure the central nervous system,
including trimethyltin, methylmercury,
cadmium, 3-acetylpyridine, and MPTP,
have been shown to increase GFAP. In
addition, increases in GFAP may be
seen at dosages below those necessary to
produce cytopathology as determined
by Nissl-based stains used in standard
neuropathological examinations.
Because increases in GFAP may be an
early indicator of neuronal injury in the
adult, exposure level-dependent
increases in GFAP should be viewed
with concern.
Chemical-induced alterations in the
structure of the nervous system are
generally considered neurotoxic effects.
To ensure reliable data, it is important
that neuropathological studies minimize
fixation artifacts and potential
differences in the section(s) of the
nervous system sampled and control for
variability due to the age, sex, and body
weight of the subject (WHO, 1986).
4.3.6. Developmental Neurotoxicity
Exposure to chemicals during
development can result in effects other
than death, gross structural abnormality,
or altered growth. There are several
instances in which functional
alterations have resulted from exposure
during the period between conception
and sexual maturity (Riley and Vorhees,
1986; Vorhees, 1987). Table 4-6 lists
several examples of chemicals known to
produce developmental neurotoxicity in
experimental animals. Animal models
of developmental neurotoxicity have
been shown to be sensitive to several
environmental chemicals known to
produce developmental toxicity in
humans, including lead, ethanol,
methylmercury, and PCBs (Kimmel et
al., 1990).
TABLE 4-6.—PARTIAL LIST OF AGENTS
BELIEVED TO HAVE DEVELOPMENTAL
NEUROTOXICITY
Alcohols
Antimitotics
Insecticides
Metals
Polyhalogenated hy-
drocarbons
Psychoactive drugs
Solvents
Vitamins
Methanol, ethanol
X-radiation,
azacytidine
DDT, kepone,
organophosphates
Lead, methylmercury,
cadmium
PCB, PBB
Cocaine, phenytoin
Carbon disulfide, tolu-
ene
Vitamin A
Sometimes functional defects are
observed at dose levels below those at
which other indicators of
developmental toxicity are evident
(Rodier, 1986). Such effects may be
transient or reversible in nature, but
generally are considered adverse effects.
Data from postnatal studies, when
available, are considered useful for
further assessment of the relative
importance and severity of findings in
the fetus and neonate. Often, the long-
term consequences of adverse
developmental outcomes noted at birth
are unknown and further data on
postnatal development and function are
necessary to determine the full
spectrum of potential developmental
effects. Useful data also can be derived
from well-conducted multigeneration
studies, although the dose levels used in
these studies may be much lower than
those in studies with shorter-term
exposure.
Much of the early work in
developmental neurotoxicology was
related to behavioral evaluations. Recent
advances in this area have been
reviewed in several publications (Riley
and Vorhees, 1986; Kimmel et al., 1990).
Several expert groups have focused on
the functions that should be included in
a behavioral testing battery, including
sensory systems, neuromotor
development, locomotor activity,
learning and memory, reactivity and
habituation, and reproductive behavior.
No testing battery has fully addressed
all of these functions, but it is important
to include as many as possible, and
several testing batteries have been
developed and evaluated for use in
testing.
Direct extrapolation of functional
developmental effects to humans is
limited in the same way as for other
endpoints of developmental toxicity,
i.e., by the lack of knowledge about
underlying toxicological mechanisms
and their significance. It can be assumed
that functional effects in animal studies
indicate the potential for altered
development in humans, although the
types of developmental effects seen in
experimental animal studies will not
necessarily be the same as those that
may be produced in humans. Thus,
when data from functional
developmental toxicity studies are
encountered for particular agents, they
should be considered in the risk
assessment process.
Agents that produce developmental
neurotoxicity at a dose that is not toxic
to the maternal animal are of special
concern because the developing
organism is affected but toxicity is not
apparent in the adult. More commonly,
however, adverse developmental effects
are produced only at doses that cause
minimal maternal toxicity; in these
cases, the developmental effects are still
considered to represent developmental
toxicity and should not be discounted as
secondary to maternal toxicity. At doses
causing excessive maternal toxicity (that
is, significantly greater than the minimal
toxic dose), information on
developmental effects may be difficult
to interpret and of limited value.
Current information is inadequate to
assume that developmental effects at
maternally toxic doses result only from
maternal toxicity; it may be that the
mother and developing organism are
sensitive to that dose level. Moreover,
whether developmental effects are
secondary to maternal toxicity or not,
the maternal effects may be reversible
while effects on the offspring may be
permanent. These are important
considerations for agents to which
humans may be exposed at minimally
toxic levels either voluntarily or
involuntarily, because several agents are
known to produce adverse
developmental effects at minimally
toxic doses in adult humans (e.g.,
smoking, alcohol).
Although interpretation of functional
developmental neurotoxicity data may
be limited at present, it is clear that
functional effects must be evaluated in
light of other toxicity data, including
other forms of developmental toxicity
(e.g., structural abnormalities, perinatal
death, and growth retardation). The
level of confidence in an adverse effect
may be as important as the type of
change seen, and confidence may be
increased by such factors as replicability
of the effect either in another study of
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the same function or by convergence of
data from tests that purport to measure
similar functions. A dose-response
relationship is considered an important
measure of chemical effect; in the case
of functional effects, both monotonic
and biphasic dose-response curves are
likely, depending on the function being
tested.
4.3.7. Physiological and Neuroendocrine
Endpoints
One of the key roles played by the
nervous system is to orchestrate the
general physiological functions of the
body to help maintain homeostasis. To
this end, the nervous system and many
of the peripheral organ systems are
integrated and functionally
interdependent. For example, specific
neuronal processes are intimately
involved in maintaining or modulating
respiration, cardiovascular function,
body temperature, and gastrointestinal
function. Because many peripheral
organ functions involve neuronal
components, changes in such
physiological endpoints as blood
pressure, heart rate, EKG, body
temperature, respiration, lacrimation, or
salivation may indirectly reflect
possible treatment-related effects on the
functional integrity of the nervous
system. However, since physiological
endpoints also depend on the integrity
of the related peripheral organ itself,
changes in physiological function also
may reflect a systemic toxicity involving
that organ. Consequently, the
neurotoxicological significance of a
physiological change must be
interpreted within the context of other
signs of toxicity. A variety of general
physiological procedures can be applied
to neurotoxicological problems. These
procedures range in scale from simple
measurements, for example, of body
temperature, respiration, lacrimation,
salivation, urination, and defecation,
which may be included in routine
functional observational batteries used
for chemical screening, to more
involved procedures involving
measurements of blood pressure,
endocrine responses, cardiac function,
gastrointestinal function, etc. The latter
would be more appropriate for second-
level tests to characterize the scope of
chemically related toxicity.
The central nervous system also
regulates the outflow of the endocrine
system, which together with the
influence of the autonomic nervous
system, can affect immunologic function
(WHO, 1986). Hormonal balance results
from the integrated action of the
hypothalamus, located in the central
nervous system, and the pituitary,
which regulates activities of endocrine
target organs. Each site is susceptible to
disruption by neurotoxic agents.
Neuroendocrine dysfunction may occur
because of a disturbance in the
regulation and modulation of the
neuroendocrine feedback systems. One
major indicator of neuroendocrine
function is secretions of hormones from
the pituitary. Hormones from the
anterior pituitary are important for
reproduction (follicle-stimulating
hormone, luteinizing hormone), growth
(thyroid-stimulating hormone), and
response to stress (adrenocorticotropic
hormone). Hypothalamic control of
anterior pituitary secretions occurs
through the release of hypothalamic-
hypophysiotropic hormones. Hormones
from the posterior hypothalamus
(prolactin, melanocyte-stimulating
hormone, and growth hormone) are also
involved in a number of important
bodily functions.
Many types of behaviors (e.g.,
reproductive behaviors, sexually
dimorphic behaviors) are dependent on
the integrity of the hypothalamic-
pituitary system, which could represent
an important site for neurotoxic action.
Pituitary secretions arise from a number
of different cell types in this gland and
neurotoxicants could affect these cells
either directly or indirectly.
Morphological changes in follicular
cells, chromophobe cells, somatotropic
cells, prolactin cells, gonadotropic cells,
follicle-stimulating hormone secreting
cells, luteinizing hormone-containing
cells, thyrotropic cells, and cortico cells
might be associated with adverse effects
on the pituitary, which could ultimately
affect behavior and the functioning of
the nervous system.
Biochemical changes in the
hypothalamus also may be used as
indices of potential changes in
neuroendocrine function. However, the
neuroendocrine significance of changes
in hypothalamic neurotransmitters and
neuropcptides is usually only
inferential and data must be considered
on a case-by-case basis.
Most anterior pituitary hormones are
subject to negative feedback control by
peripheral endocrine glands and, if
neurotoxicants modify peripheral
secretions, neuroendocrine changes can
result from this altered feedback.
Modifications in the functioning of
these endocrine secretions could occur
after toxic exposure; a number of agents
have been shown to alter blood levels of
glucocorticoids, thyroxine, estrogen,
corticosterone, and testosterone.
Although such changes are not
necessarily due to direct
neuroendocrine effects, target organ
changes often can be a first indication
of neuroendocrine changes.
4.3.8. Other Considerations
4.3.8.1. Structure-activity relationship.
Because of a general lack of
epidemiologic or toxicologic data on
most chemical substances, attempts
have been made in toxicology to predict
activities based 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, there are relatively
few well-characterized SARs in
neurotoxicology. Since SARs cannot be
used to rule out all neurotoxic activity,
it is not acceptable to use them as a
basis for excluding potential
neurotoxicity. Caution is warranted in
interpreting SARs in anything other
than the most preliminary analyses. Use
of SARs requires detailed knowledge
not only of structure, but also of each
critical step in the pathogenetic
mechanism of neurotoxic injury. Such
knowledge is still generally unavailable.
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
their utility depends on a better
understanding of neurotoxic
mechanisms.
4.3.8.2. In vitro methods.
In vitro procedures for testing have
practical advantages, but studies must
be done to correlate the 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 are likely to
require expertise. 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
endpoint that is not exactly what it
measures (e.g., carcinogenicity rather
than specific aspects of genotoxicity). In
vitro changes can be markers for
toxicity, even when the structural or
functional consequences are not known
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or predicted. In addition, in vitro
methods can examine the more
evolutionarily conserved elements of
the nervous system and improve
neurotoxicity detection and could also
provide suitable systems for studying
developmental neurotoxicity.
A broad range of tissue-culture
systems are available for assessing the
neurologic impact of environmental
agents, including cell lines, dissociated
cell cultures, reaggregate cultures,
explant cultures, and organ cultures
(Veronesi, 1991).
Neuronal and glial cell lines are used
extensively in neurobiology and have
potential for neurotoxicological studies.
They consist of populations of
continuously dividing cells that, when
treated appropriately, stop dividing and
exhibit differentiated neuronal or glial
properties. Neuronal lines can develop
electric excitability, chemosensitivity,
axon formation, neurotransmitter
synthesis and secretion, and synapse
formation. Large quantities of cells can
be generated routinely to develop
extensive dose-response or other
quantitative data.
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
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
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 granular cell culture system,
for example, has been exploited recently
in studies of the mechanism of alkyllead
toxicity (Verity et al., 1990).
A related 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 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.
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.
Organotypic explant cultures also are
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
electrophysiologically. 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. 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,
is grown in vitro. Obviously, only
structures so small that their viability is
not compromised can be treated in this
way.
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.
5. Neurotoxicology Risk Assessment
5.1. Introduction
Risk assessment is an empirically
based process used to estimate the risk
that exposure of an individual or
population to a chemical, physical, or
biological agent will be associated with
an adverse effect. Generally, such effects
can be quantified and the relative
probability of their occurrence can be
calculated. The risk assessment process
usually involves four steps: hazard
identification, dose-response
assessment, exposure assessment, and
risk characterization (NRC, 1983). Risk
management is the process that applies
information obtained through the risk
assessment process to determine
whether the assessed risk should be
reduced and, if so, to what extent (NRC,
1983). In some cases, risk is the only
factor considered in a decision to
regulate exposure to a substance.
Alternatively, the risk posed by a
substance is weighed against social,
ethical, and medical benefits and
economic and technological factors in
formulating a risk management
decision. The risk-balancing approach is
used by some agencies to consider the
benefits as well as the risks associated
with unrestricted or partially restricted
use of a substance. The purpose of this
chapter is to describe the risk
assessment process as it has currently
evolved in neurotoxicology and present
available options for quantitative risk
assessment.
5.2. The Risk Assessment Process
5.2.1. Hazard Identification
Agents that adversely affect the
neurophysiological, neurochemical, or
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42395
structural integrity of the nervous
system or the integration of nervous
system function expressed as modified
behavior may be classified as
neurotoxicants (Tilson, 1990b). For
hazard identification, the best or most
generalizable studies would measure
these changes in humans. With the
exclusion of therapeutic agents,
information on effects in humans is
usually derived from case reports of
accidental exposures and
epidemiological studies. This type of
data affords less certainty regarding
generalizability as well as less specific
exposure information. As discussed in
chapter 4, a common alternative method
of data generation for hazard
identification is the use of animal
models. Animal models that measure
behavioral, neurophysiological,
neurochemical, and structural effects
have been developed and validated.
Studies that employ these models to
evaluate specific potential hazards are
used to predict the outcome of exposure
to the same hazard in humans.
5.2.1.1. Human studies
Information obtained through the
evaluation of human exposure data
provides direct identification of
neurotoxic hazards. This type of
information is generally available from
clinical trials required for the approval
of therapeutic products for human use.
For the purposes of risk assessment of
nontherapeutic substances, data on
effects of exposure to humans come
primarily from two types of studies,
case reports and epidemiological
(Friedlander and Hearn, 1980) (see
chapter 3). Case studies can supply
evidence of an agent's toxicity, but are
often limited by both the qualitative
nature of the signs and symptoms
reported and the nature of the exposure
data. Epidemiological studies can
provide data on the types of neurotoxic
effects and the possible susceptibilities
of certain populations. Under
appropriate considerations, they can
generally provide convincing and
reliable evidence of potential human
neurotoxicity. As with case studies,
however, often only qualitative
estimates of exposure can be obtained.
Controlled laboratory studies have the
potential to provide adequate exposure
and effects data for accurate hazard
identification, but ethical considerations
place moral and practical restrictions on
such studies except in those instances
where direct benefit to the subjects, as
in the case of therapeutic agents, may be
expected. Excluding instances of
therapeutic product development, most
studies are limited to measuring the
effects of acute, rather than long-term,
exposure. This limits their utility in risk
assessment because the effect of long-
term, low-level exposure to a potentially
toxic agent is often the issue of concern.
Methods available to evaluate
neurotoxicity in humans include
examination of neurophysiological and
behavioral parameters. Specific tests to
measure neuromuscular strength and
coordination, alterations in sensation,
deficits in learning and memory,
changes in mood and personality, and
disruptions of autonomic function are
frequently employed (see chapter 3).
5.2.1.2. Animal studies
As discussed in chapter 4, animal
models for many endpoints of
neurotoxicity are available and widely
used for hazard identification. Data from
animal studies are frequently
extrapolated to humans. For example, if
exposure to an agent produces
neuropathology in an animal model,
damage to a comparable structure in
humans is predicted. Similarly,
biochemical and physiological effects
observed in animals are commonly
extrapolated to humans. Agents that
produce alterations in the levels of
specific enzymes in one animal species
generally have the same effect in other
species, including humans.
Neurophysiological endpoints also tend
to be affected by the same
manipulations across species. Thus, an
agent interfering with nerve conduction
in an animal study is often assumed to
have the same effect in humans.
Behavioral studies in animals are also
applied to human hazard identification,
although the correspondence between
methods employed in animals and
humans is sometimes not as obvious.
For this reason, behavioral methods
developed for neurotoxic hazard
identification need to be considered on
a case-by-case basis.
5.2.1.3. Special issues
5.2.1.3.1. Animal-to-human
extrapolation. The use of animal data to
identify hazard to humans is not
without controversy. Relative sensitivity
across species as well as between sexes
is a constant concern. Overly
conservative risk assessments, based on
the assumption that humans are always
more sensitive than a tested animal
species, can result in poor risk
management decisions. Conversely, an
assumption of equivalent sensitivity in
a case where humans actually are more
sensitive to a given agent can result in
underregulation that might have a
negative impact on human health.
5.2.1.3.2. Susceptible populations. A
related controversy concerns the use of
data collected from adult organisms.
animal or human, to predict hazards in
potentially more sensitive populations,
such as the very young and the elderly,
or in other groups, such as the
chronically ill. In some cases,
identification of neurotoxicity hazard
does not generally include subjects from
either end of the human life span or
from other than healthy subjects.
Uncertainty factors are used to adjust for
more sensitive populations. In addition,
single or multigeneration reproductive
studies in animals may provide a source
of information on neurological
disorders, behavioral changes,
autonomical dysfunction,
neuroanatomical anomalies, and other
signs of neurotoxicity in the developing
animal (chapter 4).
5.2.1.3.3. Reversibility. For the most
part, the basic principles of hazard
identification are the same for
neurotoxicity as for any adverse effect
on health. One notable exception,
however, concerns the issue of
reversibility and the special
consideration that must be given to the
inherent redundancy and plasticity of
the nervous system.
For many health effects, temporary, as
opposed to permanent, effects are
repaired during a true recovery. Damage
to many organ systems, if not severe,
can be spontaneously repaired. For
example, damaged liver cells that may
result in impaired liver function often
can be replaced with new cells that
function normally. The resulting
restoration of liver function can be
viewed as recovery. In the central
nervous system, cells generally do not
recover from severe damage and new
cells do not replace them. When
nervous system recovery is observed, it
may represent compensation requiring
activation of cells that were previously
performing some other function,
reactive synaptogenesis, or recovery of
moderately injured cells. While a
damaged liver may recover due to the
addition of new cells, severe damage to
nervous system cells results in a net loss
of cells. This loss of compensatory
capacity may not be noticed for many
years and, when it does appear, it may
be manifest in a way seemingly
unrelated to the original neurotoxic
event. Lack of ability to recover from a
neurotoxic event later in life or
premature onset of signs of normal aging
may result. It is therefore important to
consider the possibility that significant
damage to the nervous system may have
occurred in experiments where effects
appear to be reversible.
5.2.1.3.4. Weight of evidence.
A "weight of evidence" approach to
identifying an agent as a neurotoxic
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hazard is almost always necessary. With
the exception of therapeutic products, a
single, complete, controlled study of an
agent's effects on the nervous system,
conducted in an appropriate
representative sample of humans, is
rarely, if ever, possible. Rather, those
individuals charged with identifying
hazard are usually confronted with a
collection of imperfect studies, often
providing conflicting data (Barnes and
Dourson, 1988).
There are several possible approaches,
depending on the quality of the
evidence. Two examples are the use of
data from only the most sensitive
species tested and the use of data from
only species responding most like the
human for any given endpoint. In
assessing neurotoxicity of therapeutic
products, when human data are
available and neurotoxic endpoints
detected in animals can be clinically
measured, the human findings
supersede those of the nonclinical data
base. Assuming that all available
evidence is to be included,
considerations necessary for formulating
a conclusion include the relative
weights that should be given to positive
and negative studies. Sometimes
positive studies are given more weight
than negative ones, even when the
quality of the studies is comparable.
Experimental design factors such as the
species tested, the number and gender
of subjects evaluated, and the duration
of the test are given different weights
when data from different studies are
combined. The route of exposure in a
given study and its relevance to
expected routes of human exposure are
often a weighted factor. The issue of
statistical significance is frequently
debated. Some argue that an effect
occurring at a statistically insignificant
level may nevertheless represent a
biologically or toxicologicnlly
significant event, and should be
afforded the same weight as if the
finding were statistically significant. In
general, however, only statistically
significant measures should be
considered in hazard identification. The
power of various statistical measures is
also considered.
5.2.2. Dose-Response Assessment
In the second step of the risk
assessment process, the dose-response
assessment, the relationship between
the extent of damage or toxicity and
dose of a toxic substance for various
conditions of exposure is determined.
Because several different kinds of
responses may be elicited by a single
agent, more than one dose-response
relationship may need to be developed
(e.g., neurochemical and morphological
parameters).
When quantitative human dose-effect
data are not available for a sufficient
range of exposures, other methods must
be used to estimate exposure levels
likely to produce adverse effects in
humans. In the absence of human data,
the dose-response assessment may be
based on tests performed in laboratory
animals. Evidence for a dose-response
relationship is an important criterion in
assessing neurotoxicity, although this
may be based on limited data from
standard studies that often use only
three dose groups and a control group
(Barnes and Dourson, 1988).
The most frequently used approach
for risk assessment of neurotoxicants
and other noncancer endpoints is the
uncertainty- or safety-factor approach
(Barnes and Dourson, 1988; Kimmel,
1990). For example, within the EPA,
this approach involves the
determination of reference doses (RfDs)
by dividing a no observed adverse effect
level (NOAEL) by uncertainty factors
that presumably account for interspecies
differences in sensitivity (Barnes and
Dourson, 1988). Generally, an
uncertainty factor of 10 is used to allow
for the potentially higher sensitivity in
humans than in animals and another
uncertainty factor of 10 is used to allow
for variability in sensitivity among
humans. Hence, the RfD is equal to the
NOAEL divided by 100. If the NOAEL
cannot be established, it is replaced by
the lowest observed adverse effect level
(LOAEL) in the RfD calculation and an
additional uncertainty factor of 10 is
introduced (i.e., the RfD equals the
LOAEL divided by 1000).
If more than one effect is observed in
the animal bioassays, the effect
occurring at the lowest dose in the most
sensitive animal species and gonder is
generally used as this basis for
estimating the RID (OTA, 19<)0).
Sometimes, different RfDs can be
calculated, depending on endpoint or
species selected. Selection of safety
factors may be influenced by several
considerations, including data available
from humans, weight of evidence, type
of toxic insult, and probability of
variations in responses among
susceptible populations (e.g., very
young or very old). Established
guidelines have been accepted by
several agencies that use the safety-
factor approach to account for
intraspecies variability, cross-species
extrapolation, and exposure duration. In
some instances, comparisons between
these predicted values and experimental
data have been conducted and the
results appear comparable for some
selected examples (Dourson and Stara,
1983; McMillan, 1987).
The uncertainty-factor approach is
based on the assumption that a
threshold does exist, that there is a dose
below which an effect does not change
in incidence or severity. The threshold
concept is complicated and
controversial. As described by Sette and
MacPhail (1992), there are several
different ways in which the term
threshold is used. Thresholds are
defined, in part, by the limit of
detection of an assay. As the sensitivity
of the analytical method or bioassay is
improved, the threshold might be
adjusted downward, indicating that the
true threshold had not been previously
determined.
Another problem inherent with an
observation of no discernible effects at
low doses is that it is impossible to
determine whether the risk is actually
zero (i.e., the dose is below a threshold
dose) or whether the statistical resolving
power of a study is inadequate to detect
small risks (Gaylor and Slikker, 1992).
Every study has a statistical limit of
detection that depends on the number of
individuals or animals involved. For
example, it would be relatively unusual
to conduct an experiment on a
neurotoxicant with as many as 100
animals per dose. If no deleterious
effects were observed in 100 animals at
a particular dose, it might be concluded
that this dose level is below the
threshold dose. However, we can only
be 95 percent confident that the true
risk is less than 0.03. That is, if 3
percent of the animals in a population
actually develop a toxic effect at this
dose, there is a 5 percent chance that a
group of 100 animals would not show
any effect. The observation of no toxic
effects in an extremely large sample of
1,000 animals only indicates with 95
percent confidence that the true risk is
loss than 0.003, ok:. Docauso thresholds
cannot be realistically demonstrated,
they are therefore assumed.
The notion of threshold may be useful
in explaining mechanisms associated
with specific types of toxicity. What
little is known about mechanisms of
neurotoxicity suggests that both
threshold and nonthreshold scenarios
are possible (Silbergeld, 1990).
However, for one of the most studied
neurotoxicants, lead, there has been a
steady decline in exposure levels shown
to have effects, suggesting to some that
no threshold dose is apparent (Bondy,
1985). Sette and MacPhail (1992) also
consider the threshold as a
mathematical assumption and as a
population sensitivity and conclude that
"the idea of no threshold seems
experimentally untestable. . . ."
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The RfD approach relies on single
experimental observations (the NOAEL
or LO AEL) instead of complete dose-
response curve data to calculate risk
estimations. Chemical interactions with
biological systems are often specific,
stereoselective, and saturable. Examples
include enzyme-substrate binding
leading to substrate metabolism,
transport, and receptor-binding, any or
all of which may be a requirement of an
agent's effect or toxicity. Therefore, a
chemical's dose-response curve may not
be linear. The certainty of low-dose
extrapolation has been determined to be
markedly affected by the shape of the
dose-response curve (Food and Drug
Administration Advisory Committee on
Protocols for Safety Evaluation, 1971).
Therefore, the appropriate use of dose-
response curve data should enhance the
certainty of risk estimations when
thresholds are not assumed or
determined.
Dose-response models have generated
considerable interest as more
appropriate and quantitative
alternatives to the safety-factor approach
in risk assessment. Rather than
routinely applying a "fixed" safety
factor to the NOAEL (based on a single
dose) to obtain a "safe" dose, another
approach uses data from the entire dose-
response curve.
Two fundamentally different
approaches in the use of dose-response
data to estimate risk have been
developed. Dews and coworkers (Dews,
1986; Glowa and Dews, 1987; Glowa et
al., 1983) and Crump (1984)
demonstrated an approach in which
they used information on the shape of
the dose-response curve to estimate
levels of exposure associated with
relatively small effects (i.e., a 1, 5, or 10
percent change in a biological
endpoint). Both Dews and Crump fit a
mathematical function to the data and
provided an estimate of the variability
in exposure levels associated with a
relatively small effect.
An alternative approach developed by
Gaylor and Slikker (1990) first
establishes a mathematical relationship
between a biological effect and the dose
of a given chemical. The second step
determines the distribution (variability)
of individual measurements of
biological effects about the dose-
response curve. The third step
statistically defines an adverse or
"abnormal" level of a biological effect in
an untreated population. The fourth
step estimates the probability of an
adverse or abnormal level as a function
of dose utilizing the information from
the first three steps. The advantages of
these dose-response models are that
they encourage the generation and use
of data needed to define a complete
dose-response curve.
Although more quantitative dose-
response assessment models have
emerged in recent years, uncertainty
remains as to what biological endpoints
from which species with what dosing
regimen should be analyzed. Within a
species, a given agent may produce a
variety of effects, including
neurochemical, neuropathological, and
behavioral effects. In other instances, a
chemical may produce alterations of one
endpoint but not others (Slikker et al.,
1989). Species selection may also
dramatically affect the outcome of risk
assessments. The Parkinson-like
syndrome produced by single doses of
MPTP in the human or nonhuman
primate is not observed in rats given
comparable MPTP doses (Kopin and
Markey, 1988). Although endpoint and
species selection appear to have a
tremendous effect on the outcome of an
assessment, only a few studies have
systematically investigated the effect on
assessment outcome of varying either
the species or the endpoint within a
species (McMillan, 1987; Hattis and
Shapiro, 1990; Gaylor and Slikker,
1992).
5.2.3. Exposure Assessment
This step of the risk assessment
process determines the source, route,
dose, and duration of human exposure
to an agent. The results of the dose-
response assessment are combined with
an estimate of human exposure to obtain
a quantitative estimate of risk. As either
the effect of or the exposure to an agent
approaches zero, the risk of
neurotoxicity approaches zero. It should
be recognized that exposures to multiple
agents may produce synergistic or
additive effects.
Exposure can occur via many routes,
including ingestion, inhalation, or
contact with skin. Sources of exposure
may include soil, food, air, water, or
intended vehicle (e.g., drug
formulation). The degree of exposure
may be strongly influenced by a number
of factors, for example, the occupation
of the individual involved.
The duration of exposure (i.e., acute
or chronic) and interval of exposure
(i.e., episodic or continuous) are
variables of exposure that are common
to all types of risk assessments,
including carcinogenicity (OSTP, 1985).
Although not routinely used,
biological markers or biomarkers of
exposure could theoretically improve
the exposure assessment process and,
thereby, improve the overall risk
assessment of neurotoxicants. Exposure
biomarkers may include either the
quantitation of exogenous agents or the
complex of endogenous substances and
exogenous agents within the system
(Committee on Biological Markers,
1987). A limited number of examples of
biomarkers of exposure have been
reviewed by Slikker (1991) and include
blood or dentine lead concentrations
(Needleman, 1987), cerebrospinal fluid
concentrations of dopamine metabolites
following MPTP administration (Kopin
and Markey, 1988), cerebrospinal fluid
concentrations of a serotonin metabolite
following MDMA exposure (Ricaurte et
al., 1986), and serum esterase
concentrations following
organophosphate exposure (Levine et
al., 1986). The use of muscarinic
receptor binding in peripheral plasma
lymphocytes has also been described as
a potential biomarker of exposure for
the organophosphates (Costa et al.,
1990). These examples suggest that
biomarkers of exposure are available for
some agents, but more effort will be
required to demonstrate that these
biomarkers can routinely be used to
improve the exposure assessment
process.
5.2.4. Risk Characterization
The final step of the risk assessment
process combines the hazard
identification, the dose-response
assessment, and the exposure
assessment to produce the
characterization of risk. As previously
stated, the current practice is to divide
the NOAEL by the appropriate safety
factor to obtain the RfD. The magnitudes
of the safety factors used to determine
RfDs [interspecies extrapolation (10),
intraspecies extrapolation (10), and
acute vs. chronic exposure (10) = 1000]
are based more on conservative
estimates than on actual data (Sheehan
et al., 1989; McMillan, 1987) and have
been questioned for empirical reasons
(Gaylor and Slikker, 1990). Uncertainty
factors may be decreased as more data
become available. Modifying factors are
also employed under certain
circumstances to account for the
completeness of data sets. Along with
this RfD numerical value, any
uncertainties and assumptions inherent
in the risk assessment should also be
stated (OTA, 1990). Although the RfD
provides a single numerical value, it
does not provide information
concerning the uncertainty of this
number nor does the RfD approach
attempt to estimate the potential risk as
a function of dose or consider the
potential risk at the NOAEL. The risk at
the NOAEL generally is greater than
zero and has been estimated to be as
high as about 5 percent (Crump, 1984;
Gaylor, 1989). Concern has been
expressed that the application of the
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RED approach to all neurotoxicants is
unlikely to be biologically defensible in
light of mechanistic data (NRG, 1992).
Several other quantitative risk
assessment procedures have recently
emerged as alternatives to the RfD
approach (Kimmel and Gaylor, 1988).
Quantitative risk assessment may be
defined as a data-based process that
uses dose-response information and
measurements of human exposure to
arrive at estimates of risk. Assumptions
are required to extrapolate results from
high to low doses, to extrapolate from
animal results to humans, and to
extrapolate across different routes and
durations of exposure.
In a step toward quantitative risk
assessment. Crump (1984) suggested the
use of a benchmark dose defined as "a
statistical lower confidence limit
corresponding to a small increase in
effect over the background level." The
benchmark dose is determined with a
mathematical model and is less affected
by the particular shape of the dose-
response curve. Although the
benchmark approach avoids several
problems inherent in the RfD approach
(e.g., lack of precision in defining the
LOAEL; Kimmel. 1990), the same final
step of dividing by arbitrary safety
factors is obligatory.
Another approach to quantitative risk
assessment is the statistical or curve-
fitting approach. If quantal information
concerning the proportion of response at
a given dose is available but
mechanistic information is lacking,
statistical models can be used to fit
population data (Wyzga, 1990). This
approach has been used to fit various
models to data of lead toxicity. The data
were sufficient to allow discrimination
of several models in terms of goodness
of fit; the nerve-conduction velocity
data from children exposed to
environmental lead as a function of
blood lead concentration fit a "hockey-
stick" type dose-response curve rather
than a logistic or quadratic model
(Schwartz et al., 1988). These statistical
approaches not only provide a method
to extrapolate data to lower exposure
conditions but also can provide
circumstantial evidence to support a
proposed mechanism of action.
The development of quantitative risk
assessment approaches depends, in part,
on the availability of information on the
mechanism of action and
pharmacokinetics of the agent in
question. In the development of a
biologically based, dose-response model
for MDMA neurotoxicity, Slikker and
Gaylor (1990) considered several factors,
including the pharmacokinetics of the
parent chemical, the target tissue
concentrations of the parent chemical or
its bioactivated proximate toxicant, the
uptake kinetics of the parent chemical
or metabolite into the target cell and
membrane interactions, and the
interaction of the chemical or metabolite
with presumed receptor site(s). Because
these theoretical factors contain a
saturable step due to limited amounts of
required enzyme, reuptake, or receptor
site(s), a nonlinear, saturable dose-
response curve was predicted. In this
case of neurochemical effects of MDMA
in the rodent, saturation mechanisms
were hypothesized and indeed
saturation curves provided relatively
good fits to the experimental results.
The conclusion was that use of dose-
response models based on plausible
biological mechanisms provide more
validity to prediction than purely
empirical models. Concomitant with
attempts to develop quantitative risk
assessment procedures, it is imperative
that regulatory policy or risk
management procedures also be
developed to use appropriately the type
of data generated by quantitative risk
assessment. However, until alternative
risk assessment procedures have been
validated, the available RfD approach
with its limitations will most likely
continue to be used.
5.3. Generic Assumptions and
Uncertainty Reduction
The purpose of risk assessment is to
determine the risk associated with
human exposure to a hazard. The
quality of the data from toxicological
studies differs. In the case of therapeutic
products where human effects
information is available, risk
assessments rely primarily on the result
of controlled clinical trials. Even when
clinical trial data are available, however,
conducting a risk assessment is
complicated by many uncertainties. In
the face of these uncertainties,
conservative assumptions are usually
made at several steps in the risk
assessment process. For example, unless
adequate clinical data are available, the
most sensitive experimental species is
frequently used. While conservative
assumptions may lead to a risk
assessment that adequately protects the
human population, this may result in an
increased financial burden on the public
(e.g., manufacturing costs or loss of
jobs); even then it is impossible to be
certain that the total population will be
protected. Conversely, errors leading to
allowable exposure levels that are too
high reduce the safety margin for human
health and increase health care costs.
Thus, there are compelling public
health and economic reasons to obtain
more precise risk assessments; all
assumptions cannot be completely
eliminated, but the degree of
uncertainty associated with certain
specific assumptions can at least be
reduced (Sheehan et al., 1989).
Risk assessment for neurotoxicity
shares many common features with
other noncancer toxicities such as
developmental toxicity and
immunotoxicity. As such, there are
several generic assumptions that apply
to all traditional, noncancer endpoint
risk assessment procedures (Table 5-1).
TABLE 5-1.—GENERAL ASSUMPTIONS
THAT UNDERLIE TRADITIONAL RISK
ASSESSMENTS a-b
1. A threshold dose exists (or noncancer
endpolnts.
2. NOAEL/LOAEL uncertainty- or safety-fac-
tor approaches are reasonable.
3. Variability in the toxic response to the
chemical exposure is not due to a hetero-
geneous population response.
4. Average dose or total dose is a reason-
able measure of exposure when doses are
not equivalent in time, rate, or route of ad-
ministration and the average (or total) dose
is proportional to adverse effect.
5. Structure-activity correlations can be used
to predict human toxicity.
6. The mechanism of action is the same at
all doses for all species.
a This is not intended to be an exhaustive
list.
b Modified from Sheehan et al., 1989.
One approach to reducing some of the
uncertainties is to critically define and
examine the assumptions made in the
risk assessment process. Several of the
more generic of these assumptions are
listed in Table 5-1. Despite their
diversity, these assumptions share the
attribute of being partially replaceable
by factual information. If, for example,
the assumption of 100 percent
absorption of a toxicant from a
contaminated food source is replaced by
data demonstrating that 90 percent of
the toxicant is not biologically available
under human exposure conditions, then
a revised risk assessment could allow a
10-fold greater exposure from that
source; i.e., the former risk assessment
was too conservative by a factor of 10.
As another example, many risk
assessments employ data from two
species.
If experimental animals and humans
absorb or metabolize the same fraction
of a dose, the potency estimate would
not change when extrapolating from
animals to humans. Therefore, it is
necessary to have information on both
human and animal rates before changes
in potency estimates are made. If a
toxicant acts via a reactive intermediate
and humans produce 10-fold more of
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42399
the intermediate than either of the test
species under similar conditions, then
allowable human exposure should be
decreased 10-fold (i.e., the allowable
exposure levels are 10-fold too high) or
an increased danger to human health
exists. These findings could then
replace the "most sensitive species"
principle with facts concerning relevant
human exposure and susceptibility. In
these examples, the identification of the
assumption helps define research needs
or knowledge gaps (Sheehan et al.,
1989).
In general, the knowledge gaps are
many and complex, but some can be
filled with practical solutions. The
combination of ample dose-response
data and a quantitative risk assessment
process can eliminate assumptions 1
(existence of a threshold) and 2
(reasonableness of safety factors) of the
six generic assumptions (Table 5-1).
The uncertainty of assumption 4
(exposure comparisons) could be at least
reduced with the proper application of
appropriate pharmacokinetic data.
Likewise, the uncertainty of generic
assumption 3 (variability of
heterogeneous populations) can
theoretically be reduced with the use of
biomarkers of exposure and biomarkers
of effect, to more accurately define the
relationship between exposure and
biological effect in a large population.
Many assumptions remain, however,
and uncertainty reduction by filling
knowledge gaps will ultimately require
greater understanding of biological
mechanisms underlying neurotoxicity.
A single risk assessment model may not
he adequate for all conditions of
exposure, for all endpoints, or for all
agents. Risk assessment models of the
future may well include biomarkers of
both effect and exposure as well as
biologically based mechanistic
considerations derived from both
epidemiologic and experimental test
system data.
G. General Summary
It is now generally accepted that some
chemicals, including industrial agents,
pesticides, therapeutic agents, drugs of
abuse, food-related chemicals, and
cosmetic ingredients, can have adverse
effects on the structure and function of
the nervous system. It has recently been
proposed that exposure to
neurotoxicants might also be associated
with Parkinsonism and Alzheimer's
disease. Several Federal agencies have
initiated research programs in
neurotoxicology, developed
neurotoxicology testing guidelines, and
used neurotoxic endpoints to regulate
chemicals in the environment and
workplace.
The scientific basis for identifying
and characterizing chemical-induced
neurotoxicity has advanced rapidly
during the last several years. The
manifestation of neurotoxicity depends
on the relationship between exposure
(applied dose) and the dose at the site
of toxic action (delivered or target dose)
and response. Chemical-induced
changes in the structure or function of
the nervous system at the cellular or
molecular level can be observed as
alterations in sensory, motor, or
cognitive function at the level of the
whole organism. Several important
features about the nervous system make
it particularly vulnerable to chemical
insult, including differential
susceptibilities at different stages of
maturation, the presence of blood brain
and nerve barriers that may be the target
of toxic action, high metabolic rate, and
limited regenerative capability
following damage.
Methods devised to detect and
quantify agent-induced changes in
nervous system function in humans
include clinical evaluations and
neurotoxicity testing methods such as
neurobehavioral, neurophysiological,
neurochemical, imaging, and self-
reporting procedures. Experimental
approaches used in human
neurotoxicology include
epidemiological studies and, to a
limited extent, human laboratory
exposure studies. There are several
important unresolved issues in human
neurotoxicology, including the
development of commonly accepted risk
assessment criteria and animal-to-
human extrapolation.
It is generally assumed that if physical
or chemical-induced neurotoxicity is
observed in animal models, then
neurotoxicity will be produced in
humans. Considerable research has been
performed to demonstrate the validity of
many animal models in an experimental
context and to show predictive validity.
Methods in animal neurotoxicology are
frequently used in a tier-testing
framework with simpler, more cost-
effective tests to screen or identify
neurotoxic potential. In hazard
identification, the presence of
neurotoxicity at the first tier is used to
make decisions about subsequent
development of a chemical or about the
need to conduct additional experiments
to define the level at which
neurotoxicity will be observed. A
number of methods have been devised
for studies in animal neurotoxicology,
including neurobehavioral,
neurophysiological, neurochemical, and
neuroanatomical techniques. It is
known that the neuroendocrine system
may be affected adversely by
neurotoxicants and that there are
populations that are differentially
vulnerable to neurotoxic agents.
Considerable research is in progress to
employ structure-activity relationships
to predict neurotoxicity and newly
developed in vitro procedures are being
used to augment or complement
currently existing in vivo approaches.
Principles of risk assessment for
neurotoxicity are evolving rapidly. At
the present time, neurotoxicity risk
assessment is generally limited to
qualitative hazard identification.
Neurotoxicological risk assessments
have been generally based on a no
observed adverse effect level and
uncertainty factors. As with other
noncancer endpoints, there is a need to
consider more information about the
shape of the dose-response curve and
mechanisms of effect in quantitative
neurotoxicology risk assessment.
Research is needed to develop dose-
response models that incorporate
biologic information and mechanistic
hypotheses into quantitative
extrapolation of dose-response
relationships across species and from
high to low dose exposures.
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Prepared by
Working Party on Neurotoxicology
Subcommittee on Risk Assessment
Federal Coordinating Council on Science,
Engineering, and Technology
Lawrence W. Ruiter, USEPA, Chair
Hugh A. Tilson, USEPA, Executive Secretary
John Dougherty, NIOSH
G. Jean Harry, NIEHS
Carol J. Jones, OSHA
Suzanne McMaster, USEPA
William Slikkur, NGTR/FDA
Thomas J. Sobotka, FDA
Ad Hoc Inturaguncy Committee on
Neurotoxicology
William Boyes, U.S. Environmental
Protection Agency
Joy Cavagnaro, Food and Drug
Administration
Selene Chou, Aguncy for Toxic Substances
and Disease Registry
Murray Cohn, U.S. Consumer Product Safety
Commission
Joseph F. Contrera, Food and Drug
Administration
Miriam Davis, National Institute of
Environmental Health Sciences, National
Institutes of Health
Joseph DeGeorge, Food and Drug
Administration
Robert Dick, National Institute for
Occupational Safety and Health
John Dougherty, National Institute for
Occupational Safety and Health
Lynda Erinoff, National Institute on Drug
Abuse
Joseph P. Hanig, Food and Drug
Administration
G. Jean Harry, National Institute of
Environmental Health Sciences
David G. Hattan, Food and Drug
Administration
Norman A. Krasnegor, National Institutes of
Health
Robert C. MacPhail, U.S. Environmental
Protection Agency
Suzanne McMaster, U.S. Environmental
Protection Agency
Lakshmi C. Mishra, U.S. Consumer Product
Safety Commission
Andres Negro-Vilar, National Institute of
Environmental Health Sciences
James K. Porter, U.S. Department of
Agriculture/Agricultural Research Service
Lawrence W. Reiler, U.S. Environmental
Protection Agency
Jane Robens, U.S. Department of Agriculture/
Agricultural Research Service
Barry Rosloff, Food and Drug Administration
Harry Salem, U.S. Army Chemical Research,
Development, and Enginooring Center
Bernard A. Schvvetz, National Institute of
Environmental Health Sciences
William F. Sotte, U.S. Environmental
Protection Agency
William Slikker, Jr., National Center for
Toxicological Research
D. Stephen Snyder, National Institute on
Aging
Thomas J. Sobotka, Food and Drug
Administration
Hugh A. Tilson, U.S. Environmental
Protection Agency
Mildred Williams-Johnson, Agency for Toxic
Substances and Disease Registry
|FR Doc. 94-20033 Filed U-1G-94; 8:45 am|
BILLING CODE 6560-50-P
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