Final Report: Principles of Neurotoxicity Risk Assessment; Notice ENVIRONMENTAL PROTECTION AGENCY
Federal RegisterAug 17, 1994
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SUMMARY: The U.S. Environmental Protection Agency is publishing a
document entitled Final Report: Principles of Neurotoxicity Risk
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 Biologics 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, Followup) 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
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 Neurotoxicity
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). Food, Drug, and Cosmetics Act for
food additives; color in
cosmetics; medical devices; animal
drugs of medical and feed
additives.
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 Occupational Safety and Health Act
Administration (OSHA). covers toxic chemicals in the
workplace.
A unit of the Department of Labor ...................................
that regulates workplace
conditions.
Environmental Protection Agency
(EPA).
Independent agency (i.e., not part Toxic Substances Control Act
of a Cabinet department); requires premanufacture evaluation
administers a number of diverse of all new chemicals (other than
laws concerned with human health foods, food additives, drugs,
and the environment. pesticides, 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
dumping.
Consumer Product Safety Commission
(CPSC).
Regulates a variety of consumer Federal Hazardous Substances Act
products including household covers ``toxic'' household
chemicals and fabric treatments. products.
Consumer Product Safety Act covers
dangerous consumer products.
Poison Prevention Packaging Act
covers packaging of dangerous
children's products.
Lead-Based Paint Poison Prevention
Act covers use of lead paint in
federally assisted housing.
------------------------------------------------------------------------
Table 1-2.--Authorities for Toxicity Testing
--------------------------------------------------------------------------------------------------------------------------------------------------------
Authorities
-----------------------------------------------------------------
Agency Law Coverage Premarketing Testing by
approval manufacturer Reporting of data
--------------------------------------------------------------------------------------------------------------------------------------------------------
FDA......................... Food, Drug, and Cosmetics Drugs and foods............ x x x
Act.
Food additives and x x ....................
cosmetics.
EPA......................... Federal Insecticide, Pesticides................. x x x
Fungicide, and Rodenticide
Act.
Toxic Substances Control Industrial chemicals....... \1\x x x
Act.
Clean Air Act.............. Air pollutants............. .................... .................... ....................
Resource Conservation and Industrial waste........... x x
Recovery Act.
OSHA........................ Occupational Safety and Occupational exposure...... .................... .................... x
Health Act.
CPSC........................ Federal Hazardous Consumer products.......... x ....................
Substances Act.
Consumer Product Safety Act Consumer products.......... 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 Biologics 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 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 g/dcl whole blood has
been recommended (CDC, 1991). Lead exposure in the United States has
decreased significantly during the last several years.
Table 2-1.--Human Neurotoxic Exposures
------------------------------------------------------------------------
Year(s) Location Substance Comments
------------------------------------------------------------------------
370 B.C......... Greece......... Lead........... Lead toxicity
recognized in
mining industry.
1st century A.D. Rome........... Lead........... Vapors recognized as
toxic.
1837............ Scotland....... Manganese...... Chronic manganese
poisoning
described.
1924............ United States Tetraethyl lead Workers suffer
(New Jersey). neurologic
symptoms.
1930............ United States Tri-o- Chemical contaminant
(Southeast). cresylphosphat added to Ginger
e (TOCP). Jake, an alcoholic
beverage
substitute; more
than 5,000
paralyzed, 20,000
to 100,000
affected.
1930's.......... Europe......... Apiol.......... Drug containing TOCP
causes 60 cases of
neuropathy.
1932............ United States Thallium....... Contaminated barley
(California). laced with thallium
sulfate poisons
family, causing
neurologic
symptoms.
1937............ South Africa... TOCP........... Paralysis develops
after use of
contaminated
cooking oil.
1946............ England........ Tetraethyl lead Neurologic effects
observed in people
cleaning gasoline
tanks.
1950's.......... Japan (Mina-... Methylmercury.. Fish and shellfish
mata).......... contaminated with
mercury are
ingested, causing
neurotoxicity.
1950's.......... France......... Organotin...... Medication
(Stalinon)
containing
diethyltin diiodide
results in
poisoning.
1950's.......... Morocco........ Manganese...... Miners suffer
chronic manganese
intoxication.
1950's.......... Guam........... Cycad.......... Ingestion of plants
associated with
amyortrophic
lateral sclerosis
and Parkinson-like
syndrome.
1956............ Turkey......... Hexachlorobenze Hexachlorobenzene
ne. causes poisoning.
1956............ Japan.......... Clioquinol..... Drug causes
neuropathy.
1959............ Morocco........ TOCP........... Cooking oil
contaminated with
lubricating oil
causes poisoning.
1960............ Iraq........... Methylmercury.. Mercury-treated seed
grain causes
neurotoxicity.
1964............ Japan.......... Methylmercury.. Methylmercury
neurotoxicity.
1968............ Japan.......... PCBs........... Polychlorinated
biphenyls are
leaked into rice
oil, causing
neurotoxicity.
1969............ Japan.......... n-Hexane....... Neuropathy due to n-
hexane exposure.
1969............ United States Methylmercury.. Fungicide-treated
(New Mexico). grain results in
alkyl mercury
poisoning.
1971............ United States.. Hexachlorophene Hexachlorophene-
containing
disinfectant is
found to be toxic
to nervous system.
1971............ Iraq........... Methylmercury.. Methylmercury used
as fungicide to
treat seed grain
causes poisoning.
1972............ France......... Hexachlorophene Hexachlorophene
poisoning of
children.
1973............ United States Methyl n- Fabric production
(Ohio). butylketone. plant employees
exposed to MnBK
solvent suffer
polyneuropathy.
1974-1975....... United States Chlordecone Chemical plant
(Virginia). (Keptone). employees exposed
to insecticide
suffer severe
neurologic
problems.
1976............ United States Leptophos At least nine
(Texas). (Phosvel). employees suffer
serious neurologic
problems after
exposure to
insecticide.
1977............ United States Dichloropropene People hospitalized
(California). (Telone II). after exposure to
pesticide.
1979-1980....... United States 2-t-Butylazo-2- Employees of
(Texas). hydroxy-5- manufacturing plant
methylhexane experience serious
(BHMH) (Lucel- neurologic
7). problems.
1980's.......... United States.. Methylphenyltet Impurity in
rahydropyridin synthesis of
e (MPTP). illicit drug causes
Parkinson's disease-
like effects.
1981............ Spain.......... Toxic oil...... People ingesting
toxic substance in
oil suffer severe
neuropathy.
1983-84......... United States.. Vitamin B6..... Excessive intake,
causes sensory
neuropathy,
numbness,
parathesia, and
motor dysfunction.
1985............ United States Aldicarb....... People experience
and Canada. neuromuscular
deficits after
ingestion of
contaminated
melons.
1987............ Canada......... Domoic acid.... Ingestion of mussels
contaminated with
domoic acid causes
illnesses.
1988............ India.......... TOCP........... Ingestion of
adulterated
rapeseed oil cause
polyneuritis.
1989............ United States.. L-tryptophan- Ingestion of a
containing chemical
products. contaminant
associated with the
manufacture of L-
tryptophan results
in eosinophilia-
myalgia syndrome.
1991............ Nigeria........ Scopoletin..... Natural component of
gari caused
neuropathy
associated with
optic atrophy 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). 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 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(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. paspali-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 Claviceps-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, NIOSH, 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 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 all
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. The 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-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 (Boettcher et al., 1987; Lim, 1986;
Bhattacharyya and Dayal, 1984).
The nervous system is a highly complex and integrated organ. It is
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 m
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 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 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 Cadividedivide 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 Cadividedivide 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; 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. 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
neuroanatomic 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
are 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 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
------------------------------------------------------------------------
Neurobehavioral function Test
------------------------------------------------------------------------
Sensation.......................... Flicker Fusion.
Lanthony (color
vision).
Motor/Dexterity.................... Pursuit Aiming.
Finger Tapping.
Postural Stability.
Reaction Time.
Santa Ana Peg Board.
Cognition.......................... Benton Visual Retention.
Continuous Performance Task.
Digit-Symbol.
Digit Span.
Dual Tasks.
Paired-Associate.
Symbol-Digit Task.
Wechsler Adult Intelligence Scale--
Revised> (Components).
Wechsler Memory Scale.>
Affect............................. Profile of Mood States>
(POMS).
------------------------------------------------------------------------
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 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 NO2, 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 PNS 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 (EEG), visually evoked
response (VER), somatosensory evoked potential (SEP), and the brainstem
auditory evoked response (BAER). The EEG 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 EEG changes in
chemically exposed (especially solvents and styrene) individuals have
been noted (Seppalainen and Harkonen, 1976). EEG 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 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
pharmacokinetic 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 EEG 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; 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 g/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) 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 due 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/
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 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 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.
The 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 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 humans 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 epidemiologi
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