Federal Register, Volume 59 Issue 158 (Wednesday, August 17, 1994)

[Federal Register Volume 59, Number 158 (Wednesday, August 17, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-20033]

[[Page Unknown]]

[Federal Register: August 17, 1994]

_______________________________________________________________________

Part III

Environmental Protection Agency

_______________________________________________________________________

Final Report: Principles of Neurotoxicity Risk Assessment; Notice
ENVIRONMENTAL PROTECTION AGENCY

[FRL-5050-9]

Final Report: Principles of Neurotoxicity Risk Assessment

AGENCY: U.S. Environmental Protection Agency.

ACTION: Final Document.

-----------------------------------------------------------------------

SUMMARY: The U.S. Environmental Protection Agency is publishing a
document entitled Final Report: Principles of Neurotoxicity 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 Ca^{dividedivide that can result in
the release and activation of intracellular enzymes (which break down
the cytoskeleton) and in further release of glutamate, both of which
can be cytotoxic (Choi, 1988). Critical to an understanding of the
etiopathology associated with at least some of the neurotoxic
degeneration may be the link that impaired energy metabolism could have
with excitotoxic neuronal death. It is likely that reduced oxidative
metabolism results in the partial depolarization of resting membrane
potential, the activation of ionotropic membrane receptor/channels, and
the influx of Ca^{dividedivide 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 epidemiologic
studies of persons exposed to higher concentrations.
3.6.1.5. Structure-activity.
Structure-activity relationships for well-known chemicals have
largely been established by clinical methods (and animal studies) and
verified by neurobehavioral and neurophysiologic testing. Although an
area of active research, neurobehavioral testing of humans has not yet
been able to identify reliable patterns of impairment among chemical
classes. This endeavor has been hampered by most laboratory research
having been limited to the evaluation of low concentrations of solvents
and a few other reversible toxicants and by the exposure uncertainties,
biases, and confounding variables found in cross-sectional or cohort
field studies.
3.6.2. Other Considerations in Risk Assessment
3.6.2.1. Mechanisms of action
Uncovering behavioral and neurophysiologic mechanisms of action is
a potential contribution of human laboratory exposure studies to
neurotoxicity risk assessment. For example, Stewart et al. (1972)
demonstrated that methylene chloride was metabolized to carbon monoxide
in humans, and further studies (Putz et al., 1979) found that CO
production could account for some of the neurobehavioral impairment
observed with that chemical. Recent human laboratory studies of
solvents employed low concentrations that produced mucosal irritation
and strong odor, but little neurobehavioral impairment (Dick,
unpublished observation). The mechanisms of action that produce mucosal
irritation and the neurotoxic mechanisms that are expressed in
neurobehavioral impairment may be quite different. Data on mucosal
irritation and odor may therefore provide limited information for a
neurotoxicity risk assessment.
3.6.2.2. Exposure duration
A criticism of extrapolation from animal studies to human exposure
conditions is that the effects of short-term exposure (months to 1-2
years) in animals may not accurately predict the effects of chronic
exposure (>10 years) in humans. Laboratory studies rarely expose human
subjects to solvents for more than 4-6 hours per day for 2-5 days while
environmental and workplace exposures of concern involve 6-8 hours of
exposure per day for years. The uncertainties of extrapolating from
relatively acute exposures to predict the risks from chronic exposure
will not be eliminated by using human laboratory exposure data in risk
assessment.
3.6.2.3. Time-dependent effects
The acute exposures that are possible in human laboratory studies
may provide little information on chronic time-dependent
neurobehavioral effects. The effects of initial exposure may remain the
same, decrease (tolerance), or increase (sensitization) with continued
or repeated exposure to the chemical. All effects will not change in
unison; tolerance and sensitization may be observed simultaneously on
different measures of neurobehavioral function. The multiple
toxicodynamic effects of chemical exposure (neurobehavioral and other)
seem to follow individual time courses suggestive of multiple
mechanisms of action. In addition, the processes of tolerance and
sensitization can be influenced by testing conditions and the nature of
the behavioral task.
One also must be concerned about latent effects that do not appear
for some time after a brief exposure and ``silent'' cumulative
neurotoxic effects that are not observable in acute human studies.
Latent and silent effects not only bring up the possibility of unknown
risks for human subjects, but also make more difficult the
extrapolation of chronic neurotoxic risks on the basis of acute
exposures.
Therefore, the acute exposure conditions possible in human
laboratory studies may provide us with very limited information about
the long-term effects of chronic exposure.
3.6.2.4. Multiple exposures
In the environment and the workplace, persons are seldom exposed to
only a single chemical. Rather, they are most often exposed to complex
mixtures of chemicals, the relative concentrations of which may vary
over time. For example, one farmer had more than 50 different chemical
products (pesticides, herbicides, solvents, metals, gases) with nervous
system effects that he used, prepared, or stored in his work shed.
Chemicals used in industrial processes may also contain impurities or
contaminants that may produce neurotoxic effects or alter the
neurotoxicity of the more abundant chemical species. Chemical mixtures
may have additive or potentiating effects not predictable from studies
of single chemicals (Strong and Garruto, 1991). Human laboratory
exposure studies traditionally have employed one highly purified
chemical or combinations of two chemicals (usually solvents) and thus
may produce a spectrum of neurotoxic effects different from
environmental and occupational exposures.
Recently volatile organic compounds (VOCs) have been used in human
exposure studies (Otto and Hudnell, 1991). VOCs consist of multiple
volatile compounds administered at concentrations commonly found in
indoor air from emissions by laminates, carpet, plastics, and other
building and decorating materials. Although VOCs are thought to produce
primarily mucosal irritation and odors, reports of ``sick building
syndrome'' and individual sensitivity to indoor air contaminants
suggest that other neurobehavioral mechanisms also may be operating.
3.6.2.5. Generalizability and individual differences
The results of field studies and laboratory exposure studies are
most valuable when they can be extrapolated to the general population.
Studies conducted in male workers or in young, healthy volunteers may
have limited applicability to women or to people in other age ranges.
It therefore is important to conduct studies that include males and
females of different ages and ethnic heritage. Culture-sensitive
neurobehavioral test methods are being developed and validated in the
United States and other countries.
While it is important to increase the generalizability of results,
it is equally important to know when results cannot be generalized.
Studies should be specifically directed toward identifying subsets of
individuals who are more or less sensitive to neurotoxic insult or
differ in mode of expression. There are many examples of individual
differences that alter response to chemicals and drugs:
phenylketonurics are more sensitive to dietary tyramine and persons
with variants of plasma pseudocholinesterase are more affected by some
neuromuscular blocking agents.
3.6.2.6. Veracity of neurobehavioral test results
In most epidemiologic and human laboratory studies, research
volunteers are highly motivated to perform well on tests of
neurobehavioral function. Under voluntary conditions, actual
neurobehavioral performance may serve as a reasonable index of nervous
system capabilities. Some studies, however, are conducted in response
to complaints of symptoms thought to be related to workplace,
environmental, or therapeutic exposure to chemicals and drugs. The
performance of research participants with symptoms and complaints may
be significantly affected (consciously or unconsciously) by monetary
rewards, emotional relief, or social gains from the validation of their
complaints. Under these conditions, performance may or may not
accurately reflect the capabilities of the nervous system and may lead
to inaccurate conclusions about the magnitude of nervous system
dysfunction or about putative chemical or drug etiologies.
In addition to suboptimal performance engendered by potential
reinforcers or rewards, research participants involved in disputes over
suspected neurotoxic exposures or in litigation for monetary damages
are likely to be experiencing significant emotional and behavioral
reactions from situational sources that can alter the outcome of
neurobehavioral assessment. Anxiety, depression, sleep disturbances,
fatigue, worry, obsessive thoughts, and distractibility may contribute
to less than optimal performance on motor and cognitive neurobehavioral
tasks, especially where speed and sustained concentration are
important. Under stressful conditions, it may be extremely difficult to
differentiate between neurotoxic and situational sources of observed
functional impairment. Functional neurobehavioral tests are not well
equipped to distinguish between impairment from neurotoxicity and from
nonchemical variables. The use of functional tests in symptomatic
populations requires great care in interpretation. The development of
validity scales and other control procedures for assessing nonchemical
influences on performance is greatly needed.
3.6.3. Cross-Species Extrapolation
Many neurobehavioral tests were developed according to constructs
of human cognitive processes. The diverse measures of cognitive,
sensory, and motor performance in humans are therefore not easily
compared with neurobehavioral function in animals. While it may be
possible to conceptually relate some animal and human neurobehavioral
tests (e.g., grip strength or signal detection), many procedural
differences prevent direct comparison between species.
A more direct extrapolation from animals to man might be possible
if the tests were chosen on the basis of procedural similarity rather
than on a conceptual basis (Anger, 1991). Stebbins and colleagues
(1975) were successful in developing homologous procedures in nonhuman
primates for the psychophysical evaluation of antibiotic ototoxicity.
Efforts to develop comparable tests of memory and other neurobehavioral
functions in animals and humans are under way (Stanton and Spear, 1990,
Paule et al., 1990), and such efforts may aid in cross-species
extrapolation. Other procedurally defined methods, such as Pavlovian
conditioning (Solomon and Pendlebury, 1988), operant conditioning
(Cory-Slechta, 1990), signal detection, and psychophysical scaling
techniques (Stebbins and Coombs, 1975), could also be used to
facilitate interspecies risk extrapolation. Deriving comparable
neurobehavioral assessment methods in animals and humans that will
allow a more straightforward extrapolation across species is of
paramount importance for neurotoxicity risk assessment.

4. Methods to Assess Animal Neurotoxicity

4.1. Introduction

4.1.1. Role of Animal Models
Determining the risk posed to human health from chemicals requires
information about the potential toxicological hazards and the expected
levels of exposure. Some toxicological data can be derived directly
from humans. Sources of such information include accidental exposures
to industrial chemicals, cases of food-related poisoning,
epidemiological studies, as well as clinical investigations. While
human data are available from clinical trials for therapeutics and they
provide the most direct means of determining effects of potentially
toxic substances, for other categories of substances, it is generally
difficult, expensive, and, in some cases, unethical to develop this
type of information. Quite often, the nature and extent of available
human toxicological data are too incomplete to serve as the basis for
an adequate assessment of potential health hazards. Furthermore, for a
majority of chemical substances human toxicological data are simply not
available. Consequently, for most toxicological assessments it is
necessary to rely on information derived from animal models, usually
rats or mice. One of the primary functions of animal studies is to
predict human toxicity prior to human exposure. In some cases, species
phylogenetically more similar to human, such as monkeys or baboons, are
used in neurotoxicological studies.
Biologically, animals resemble humans in many ways and can serve as
adequate models for toxicity studies (Russell, 1991). This is
particularly true with regard to the assessment of adverse effects to
the nervous system, whereby animal models provide a variety of useful
information that helps minimize exposure of humans to the risk of
neurotoxicity. There are many approaches to testing for neurotoxicity,
including whole animal (in vivo) testing and tissue/cell culture (in
vitro) testing.
At present, in vivo animal studies currently serve as the principal
approach to detect and characterize neurotoxic hazard and to help
identify factors affecting susceptibility to neurotoxicity. Data from
animal studies are used to supplement or clarify limited information
obtained from clinical or epidemiological studies in humans, as well as
provide specific types of information not readily obtainable from
humans due to ethical considerations. Frequently, results from animal
studies are used to guide the design of toxicological studies in
humans.
In vitro tests have been proposed as a means of complementing whole
animal tests, which could ultimately reduce the number of animals used
in routine toxicity testing. It also has been proposed that in vitro
testing, when properly developed, may be less time-consuming and more
cost-effective than in vivo assessments (Goldberg and Frazier, 1989;
Atterwill and Walum, 1989). By understanding the biological structures
or functions affected by toxic substances in vitro, it also may be
possible to predict neurotoxicological effects in the whole animal. An
added advantage of in vitro testing is the growing availability of
human cell lines that could be used for directly assessing potential
neurotoxic effects on human tissue. The currently available strategies
for in vitro testing have certain limitations, including the inability
to model neurobehavioral effects such as loss of memory or sensory
dysfunction or to evaluate effectively the influence of organ system
interactions (e.g., neuronal, endocrinological, and immunological) on
the development and expression of neurotoxicity.
In using animal models to predict neurotoxic risk in humans, it is
important to understand that the biochemical and physiological
mechanisms that underlie human biological processes, particularly those
involving neurological and psychological functions, are very complex
and are sometimes difficult, if not impossible, to model exactly in a
lower species. While this caveat does not preclude extrapolating the
results of animal studies to humans, it does highlight the importance
of using valid animal models in well-designed experimental studies.
4.1.2. Validity of Animal Models
Whether animal tests or methods actually measure what they are
intended to measure, whether the data from such tests can be obtained
reliably, and whether such data can be logically extrapolated to humans
are problems for most disciplines in toxicology. Various proposals have
been made for the standardization and validation of methods used in
neurotoxicological research. It is generally agreed that validation is
an ongoing process that establishes the credibility of a test, building
an increasing level of confidence in the effective utility of any model
of evaluation. The credibility of a method, as it applies to testing,
is usually discussed within several different contexts, including
construct validity, criterion validity, predictive validity, and
detection accuracy.
Construct validity concerns the ability of a method to measure
selectively a particular biological function and not other dimensions.
Construct validity is frequently established empirically. For example,
sensory dysfunction such as hearing loss is reported by humans exposed
to some chemicals, and tests are designed to detect and quantify those
changes. Such tests are designed to measure changes in auditory
function, while other sensations are unaffected (Tilson, 1987; Moser,
1990).
Criterion validity refers to the ability of a method to measure a
characteristic relative to some standard. For example, Horvath and
Frantik (1973) noted that the significance of a test measurement as an
index of an actual treatment effect should be validated relative to the
effects of a defined reference substance or positive control.
Furthermore, each specific test or type of effect may require an
appropriate reference substance for which the given type of effect is a
determining factor of the toxicity. Use of reference agents has obvious
advantages in the assessment of unknown chemicals.
Predictive validity refers to the ability of a method to predict
effects from an incomplete or partial data set. An animal model of
neurotoxicity with good predictive validity would reliably predict
neurotoxicity in humans, i.e., the animal to human extrapolation would
be good. There are several examples in neurotoxicology where animal
models have been developed based on neurotoxicological reports from
humans. Presumably, the predictive validity of such models would enable
detecting similar kinds of effects produced by uncharacterized
chemicals having a similar mechanism of action.
It has been proposed (Tilson and Cabe, 1978) that the most logical
approach to validate animal methods in neurotoxicology is to evaluate
chemicals with and without known neurotoxicity in humans in tests
designed for animals (predictive validity). By using such an approach,
it is possible to generate a profile of effects characteristic of each
type of neurotoxicant (criterion validity). This profile could then be
used to assess the construct validity of various tests. That is,
procedures assumed to measure the same neurobiological dimension should
show similar effects; measures designed to detect changes in other
functions should not be affected. This approach to test validation has
been described as the multitrait-multimethod process of validation
(Campbell and Fiske, 1959).
Of particular importance in establishing the credibility of a
method is the accuracy of detecting a treatment-related effect (Gad,
1989). Accuracy is a function of two interacting elements, specificity
and sensitivity. Specificity is the ability of a test to respond
positively only when the toxic endpoint of interest is present.
Sensitivity is the ability to detect a change when present. This aspect
depends on the inherent design of the procedure and experiment.
Increasing the specificity of a test may reduce the possibility of
classifying a chemical as neurotoxic when, in fact, it is not (false
positive), but it may increase the probability of missing a true
neurotoxicant (false negative). Increasing sensitivity of a test may
reduce the possibility of false negatives, but may increase the
probability of false positives.
4.1.3. Special Considerations in Animal Models
4.1.3.1. Susceptible populations.
Like most other measures of toxicological effect, neurotoxic
endpoints are subject to a number of experimental variables that may
affect susceptibility to the biological effects of toxicants. In this
regard, genetic variation (Festing, 1991) is a particularly important
issue in neurotoxicology. For example, most neurotoxicological
assessments are carried out with only one or two species. This may pose
problems, however, since species may differ in sensitivity to
neurotoxicants. For example, nonhuman primates are more sensitive than
rats (Boyce et al., 1984) or mice (Heikkila et al., 1984) to the
neurodegenerative effects of MPTP, a byproduct in the illicit synthesis
of a meperidine analog (Langston et al., 1983). In the assessment of
delayed neuropathology produced by some cholinesterase inhibitors, it
is well known that hens are much more sensitive than rodents (Cavanagh,
1954; Abou-Donia, 1981, 1983). In addition, rat strains also may be
differentially sensitive to some neurotoxicants (Moser et al., 1991).
Although it is preferred that more than one species be tested, the cost
required for routine multispecies testing must be considered. Whenever
possible, the choice of animal models should take into account
differences in species with regard to pharmacodynamic, genetic
composition and sensitivity to neurotoxic agents.
In addition to species, other factors such as gender of the test
animal must be taken into consideration. Some toxic substances may have
a greater neurotoxicological effect in one gender (Squibb et al., 1981;
Matthews et al., 1990). Thus, screening evaluations frequently require
both male and female animals. Another important variable is the age of
the animal (Veronesi et al., 1990). Whether a chemical produces
neurotoxicity may depend on the maturational stage of the organism
(Rodier, 1986). Most preliminary assessments are designed to provide
information on adults, which have the greatest probability of being
exposed. However, populations undergoing rapid maturation or aged
individuals may be especially vulnerable to neurotoxic agents.
Longitudinal studies that assess both genders at any stage of
development address many of the problems associated with differentially
sensitive populations.
4.1.3.2. Dosing scenario.
The dosing strategy used in experimental studies is an important
variable in the development and expression of neurotoxicity (WHO,
1986). Some neurotoxicants can produce neurotoxicity following a single
exposure, while others require repeated dosing. Repeated dosing
represents the typical pattern of human exposure to many chemical
substances. Significant differences in response may occur when an
acutely toxic quantity of material is administered over different
exposure periods. For some neurotoxicants the onset of neurotoxicity
can occur immediately after dosing, while others may require time after
exposure for the toxicity to develop. Effects of repeated exposure may
result in a progressive alteration in nervous system function or
structure, while latent or residual effects may be discovered only in
association with age-related changes or after suitable environmental or
pharmacological challenge (Zenick, 1983; MacPhail et al., 1983). To
ensure adequate assessment of neurotoxicity, study designs should
include multiple dosing regimens, e.g., repeated exposure, with
appropriate dose-to-response intervals of testing. Conduct of
neurotoxicological evaluations in studies utilizing excessively toxic
doses should be avoided.
4.1.3.3. Other factors.
There are a number of other factors that should be considered in
the design and interpretation of studies using animal models (WHO,
1986). Design factors include such issues as using properly trained
personnel to conduct the studies, the use of appropriate numbers of
animals per group to achieve reliable statistical significance, and
controlling the time-of-day variability. Time of testing relative to
exposure is also important for assessing neurotoxic endpoints such as
behavior, and experiments should be designed to generate a time course
of effects, including recovery of function, if any. Housing is an
important environmental design factor, because animals housed
individually and animals housed in groups can respond differently to
toxic agents. Temperature, as an experimental variable, may also affect
the outcome of neurotoxicological studies. The responsiveness to some
chemicals (e.g., triethyltin, methamphetamine) varies with ambient
temperature (Dyer and Howell, 1982; Bowyer et al., 1992). Some
neurobiological endpoints, such as sensory evoked potentials, can be
influenced by the endogenous temperature of the animal (Dyer, 1987).
Therefore, changes in body temperature, whether due to fluctuations in
ambient temperature or to some chemically induced effect such as
inhibition of sweating, can confound the interpretation of measures
such as evoked responses unless proper controls are included in the
experimental design.
Because a variety of other physiological changes can influence
neuronal functions, it is important to recognize that chemical-related
neurotoxicity could result from treatment-induced physiological
changes, such as altered nutritional state (WHO, 1986). As part of a
neurotoxicological profile, correlative measures, such as relative and
absolute organ weights, food and water consumption, and body weight and
weight gain, may be signs of physiological change associated with
systemic toxicity and may be useful in determining the relative
contribution of general toxicity.
4.1.3.4. Statistical considerations.
Experimental designs for neurotoxicological studies are frequently
complex, with two or more major variables (e.g., gender, time of
testing) varying in any single experiment. In addition, such studies
typically generate varying types of data, including continuous,
dichotomous, and rank-order data. Knowledge and experience in
experimental design and statistical analyses are important. There are
several key statistical concepts that should be understood in
neurotoxicological studies (WHO, 1986; Gad, 1989). The power, or
probability, of a study to detect a true effect is dependent on the
size of the study group, the frequency of the outcome variable in the
general population, and the magnitude of effect to be identified.
Statistical evaluation of a treatment-related effect involves the
consideration of two factors or types of errors to be avoided. A Type I
error refers to the attribution of an exposure-related
neurotoxicological effect when none has occurred (false positive),
while a Type II error refers to the failure to attribute an effect when
an exposure-related effect has actually occurred (false negative). In
general, the probability of a Type I error should not exceed 5 percent
and the probability of a Type II error should not exceed 20 percent.
Power is defined as one minus the probability of a Type II error.
Determination of power also requires knowledge of the difference in
magnitude of outcome measures observed between exposed and control
groups and the variability of the outcome measure among subjects. The
sample size required to achieve a given level of statistical power
increases as variability increases or the difference between groups
decreases.
Continuous data (i.e., magnitude, rate, amplitude), if found to be
normally distributed, can be analyzed with a general linear model using
a grouping factor of dose and, if necessary, repeated measures across
time. Post hoc comparisons between control and other treatment groups
can be made following tests for overall significance. In the case of
multiple endpoints within a series of evaluations, correction for
multiple observations (e.g., Bonferroni's) might be necessary.
Descriptive data (categorical) and rank data can be analyzed using
standard nonparametric techniques. In some cases, if it is believed
that the data fit the linear model, the categorical data modeling
procedure can be used for weighted least-squares estimation of
parameters for a wide range of general linear models, including
repeated measures analyses. The weighted least-squares approach to
categorical and rank data allows computation of statistics for testing
the significance of sources of variation as reflected by the model.

4.2. Tiered Testing in Neurotoxicology

The utility of tiered testing as an efficient and cost-effective
approach to evaluate chemical toxicity, including neurotoxicity, has
been recognized (NRC, 1975). Briefly, first-tier tests are designed to
determine the presence or absence of neurotoxicity, while second- tier
tests characterize the neurotoxic effect (NRC, 1992). There are at
least two aspects of tiered testing, one involving the type of test
used (Tilson, 1990a) and the other involving the dosing regimen
(Goldberg and Frazier, 1989).
4.2.1. Type of Test
Tests designed to measure the presence or absence of an effect are
usually different from those used to assess the degree of toxicity or
the lowest exposure level required to produce an effect (Tilson,
1990a). Screening procedures are first-tier tests that typically permit
the testing of many groups of animals. Such procedures may not require
extensive resources and are usually simple to perform. However, these
techniques may be labor intensive, provide subjective measures, yield
semiquantitative data, and may not be as sensitive to subtle effects as
those designed to characterize neurotoxic effects or second-tier tests.
Specialized tests are usually more sensitive and employed in studies
concerning mechanisms of action or the estimation of the lowest
effective dose. Such testing procedures are usually referred to as
secondary tests and may require special equipment and more extensive
resources. Secondary tests are usually quantitative and yield graded or
continuous data amenable to routine parametric statistical analyses.
Testing at the first tier is used to determine if a chemical might
produce neurotoxicity following exposure, i.e., hazard detection. In
this case, there may be little existing information concerning the
neurotoxic potential of an agent. Examples of first-tier tests include
functional observational batteries (FOB), including an evaluation of
motor activity and routine neurohistopathology. For some chemicals or
types of chemicals, there may be a specific interest in screening for a
particular presumed mechanism of toxicity (e.g., inhibition of
cholinesterase or neurotoxic esterase) or neurobiological response
(e.g., a site-specific neuronal degeneration). In these cases, specific
neurochemical or neuropathological endpoints can be used in conjunction
with first-tier tests. It is desirable that tests selected for use in
hazard detection provide a suitable level of sensitivity using the
smallest number of animals necessary.
A decision to test at the next tier is based on data suggesting
that an agent produces neurotoxicity. The information used to make a
decision to test a chemical at the secondary level can come from a
variety of sources, including neurotoxicological data already in the
literature, structure-activity relationships, data from first-tier
testing, or following reports of specific neurotoxic effects in humans
exposed to the agent. Testing at the secondary level includes detailed
neuropathological evaluation as well as specific behavioral tests,
e.g., procedures to assess learning and memory, or sensory function.
Tests at the second tier usually measure the most sensitive endpoints
of neurotoxicity, and are the most suitable for determining the no
observable adverse effect level or benchmark dose. At this stage of
testing, the use of a second species is considered to address the issue
of cross-species extrapolation. At the present time, tiered testing
approaches in neurotoxicology rely heavily on functional endpoints. It
is possible that future testing protocols will employ a different
strategy as more information concerning neurotoxic mechanisms of action
become available and biologically based dose-response models are
developed.
4.2.2. Dosing Regimen
Goldberg and Frazier (1989) have indicated that first-tier
evaluations identify effects of substances following acute or repeated
exposure over a wide range of doses. Measures are simple, focused on
detection of effects, and results are used to help establish parameters
for the second tier of testing. The subsequent stage(s) of tier testing
are designed to characterize more fully the toxicity of repeated
dosing. In this case, animals are exposed repeatedly or continuously to
define the scope of toxicity, including latent or delayed effects,
development of tolerance, and the reversibility of adverse effects. The
subsequent stage(s) of testing also provide information about specific
effects or study mechanisms of neurotoxicity. This tier uses methods
appropriate to characterize the effects observed in the first tier of
testing.

4.3. Endpoints of Neurotoxicity

4.3.1. Introduction
As applied to the safety assessment of chemical substances,
neurotoxicity is any adverse change in the development, structure, or
function of the central and peripheral nervous system following
exposure to a chemical agent (Tilson, 1990b). Measures used in animal
neurotoxicological studies are designed to assess these changes.
Neurotoxicity can be described at multiple levels of organization,
including chemical, anatomical, physiological, or behavioral levels. At
the chemical level, for example, a neurotoxic substance might inhibit
protein or transmitter synthesis, alter the flow of ions across
cellular membranes, or prevent release of neurotransmitter from nerve
terminals. Anatomical changes may include destruction of the neuron,
axon, or myelin sheath. At the physiological level, neuronal
responsiveness to stimulation might be enhanced by a decrease of
inhibitory thresholds in the nervous system. Chemical-induced effects
at the behavioral level can involve a variety of alterations in motor,
sensory, or cognitive function, including increases or decreases in
frequency or accuracy of responding. Although behavioral and
neurophysiological endpoints may be very sensitive indicators of
neurotoxicity, they can be influenced by other factors. The
uncertainties associated with data from functional endpoints can be
reduced if interpreted within the context of other neurotoxicological
measures (neurochemical or neuropathological) and systemic toxicity
endpoints, particularly if such measures are taken concurrently.
Behavioral effects that reflect an indirect effect secondary to
systemic toxicities may also be considered adverse. Table 4-1 provides
examples of potential endpoints of neurotoxicity at the behavioral,
physiological, chemical, and structural levels.

Table 4-1.--Examples of Potential Endpoints of Neurotoxicity

Behavioral Endpoints:
Absence or altered occurrence, magnitude, or latency of sensorimotor
reflex
Altered magnitude of neurological measurements, such as grip strength
or hindlimb splay
Increases or decreases in motor activity
Changes in rate or temporal patterning of schedule-controlled behavior
Changes in motor coordination, weakness, paralysis, abnormal movement
or posture, tremor, ongoing performance
Changes in touch, sight, sound, taste, or smell sensations
Changes in learning and memory
Occurrence of seizures
Altered temporal development of behaviors or reflex responses
Autonomic signs
Neurophysiological Endpoints:
Change in velocity, amplitude, or refractory period of nerve
conduction
Change in latency or amplitude of sensory-evoked potential
Change in EEG pattern or power spectrum
Neurochemical Endpoints:
Alterations in synthesis, release, uptake, degradation of
neurotransmitters
Alterations in second messenger associated signal transduction
Alterations in membrane-bound enzymes regulating neuronal activity
Decreases in brain AChE
Inhibition of NTE
Altered developmental patterns of neurochemical systems
Altered proteins (c fos, substance P)
Structural Endpoints:
Accumulation, proliferation, or rearrangement of structural elements
Breakdown of cells
GFAP increases (adult)
Gross changes in morphology, including brain weight
Discoloration of nerve tissue
Hemorrhage in nerve tissue

4.3.2. Behavioral Endpoints
Neurotoxicants produce a wide array of functional deficits,
including motor, sensory, and learning or memory dysfunction (WHO,
1986; Tilson and Mitchell, 1984). Many procedures have been devised to
assess overt as well as relatively subtle changes in those functions;
hence their applicability to the detection of neurotoxicity and to
hazard characterization. Many of the behavioral tests have been
developed and validated with well-characterized neurotoxicants.
Behavioral tests and agents that affect them have been reviewed
recently (WHO, 1986; Cory-Slechta, 1989). Examples of such tests, the
nervous system function being measured, and neurotoxicants known to
affect these measures are listed in Table 4-2.

Table 4-2. Examples of Specialized Tests to Measure Neurotoxicity
----------------------------------------------------------------------------------------------------------------
Function Procedure Representative-agents
----------------------------------------------------------------------------------------------------------------
Neuromuscular:
Weakness.................. Grip strength; swimming endurance; suspension n-hexane, methyl butylketone,
from rod; discriminative motor function; carbaryl.
hindlimb splay.
Incoordination............ Rotorod, gait measurements...................... 3-acetylpyridine, ethanol.
Tremor.................... Rating scale, spectral analysis................. Chlordecone, Type I pyrethroids,
DDT.
Myoclonia, spasms......... Rating scale, spectral analysis................. DDT, Type II pyrethroids.
Sensory:
Auditory.................. Discriminated conditioning Reflex modification.. Toluene, trimethyltin.
Visual toxicity........... Discriminated conditioning...................... Methyl mercury.
Somatosensory toxicity.... Discriminated conditioning...................... Acrylamide.
Pain sensitivity.......... Discriminated conditioning (titration); Parathion.
functional observational battery.
Olfactory toxicity........ Discriminated conditioning...................... 3-methylindole methylbromide.
Learning/Memory:
Habituation............... Startle reflex.................................. Diisopropyl-flurophosphate (DFP).
Classical conditioning.... Nictitating membrane............................ Aluminum.
Conditioned flavor aversion..................... Carbaryl.
Passive avoidance............................... Trimethyltin, IDPN.
Olfactory conditioning.......................... Neonatal trimethyltin.
Operant or instrumental One-way avoidance............................... Chlordecone.
conditioning.
Two-way avoidance............................... Neonatal lead.
Y-maze avoidance................................ Hypervitaminosis A.
Biel water maze................................. Styrene.
Morris water maze............................... DFP.
Radial arm maze................................. Trimethyltin.
Delayed matching to sample...................... DFP.
Repeated acquisition............................ Carbaryl.
Visual discrimination learning.................. Lead.
----------------------------------------------------------------------------------------------------------------

4.3.2.1. Functional observational batteries.
Functional observational batteries are first-tier tests designed
to detect and quantify major overt behavioral, physiological, and other
neurotoxic effects (Moser, 1989). A number of batteries have been used
(Tilson and Moser, 1992), each consisting of tests generally intended
to evaluate various aspects of sensorimotor function. Most FOB are
similar to clinical neurological examinations that rate presence or
absence and, in some cases, the relative degree of neurological signs.
A typical FOB, as summarized in Table 4-3, evaluates several functional
domains, including neuromuscular (i.e., weakness, incoordination, gait,
and tremor), sensory (i.e., audition, vision, and somatosensory), and
autonomic (i.e., pupil response and salivation) function.

Table 4-3.--Summary of Measures in the Functional Observational Battery and the Type of Data Produced by Each

Home cage and open field Manipulative Physiologic
Posture (D) Ease of removal (R) Body temperature (I)
Convulsions, tremors (D) Handling reactivity (R) Body weight (I)
Palpebral closure (R)
Lacrimation (R) Approach response (R)
Piloerection (Q) Click response (R)
Salivation (R) Touch response (R)
Vocalizations (Q) Tail pinch response (R)
Rearing (C) Righting reflex (R)
Urination (C) Landing foot splay (I)
Defecation (C) Forelimb grip strength (I)
Gait (D, R) Hindlimb grip strength (I)
Arousal (R) Pupil response (Q)
Mobility (R)
Stereotypy (D)
Bizarre behavior (D)
D = descriptive data; R = rank order data; Q = quantal data;
I = interval data; C = count data

The major advantages of FOB tests are that they can be administered
within the context of other ongoing toxicological tests and provide
some indication of the possible neurological alterations produced by
exposure. Potential problems include insufficient interobserver
reliability, difficulty in defining certain endpoints, and the tendency
toward observer bias. The latter can be controlled by using observers
unaware of the actual treatment of the subjects. Some FOB tests may not
be very sensitive to agent-induced sensory loss (i.e., vision,
audition) or alterations in cognitive or integrative processes such as
learning and memory. FOB data may be used to trigger experiments
performed at the next tier of testing.
FOB data may be interval, ordinal, or continuous (Creason, 1989).
The relevance of statistically significant test results from an FOB is
judged according to the number of signs affected, the dose(s) at which
neurotoxic signs are observed, and the nature, severity, and
persistence of the effects. Data from the FOB may provide presumptive
evidence of adverse effects and neurotoxicity. If only a few unrelated
measures in the FOB are affected or the effects are unrelated to dose,
there is less concern about neurotoxic potentials of a chemical. If
dose is associated with other overt signs of toxicity, including
systemic toxicity, large decreases in body weight, or debilitation, the
data must be interpreted carefully. In cases where several related
measures in a battery of tests are affected and the effects appear to
be dose dependent, the level of concern about the potential of a
chemical is higher.
4.3.2.2. Motor activity.
Movement within a defined environment is a naturally occurring
response and can be affected by environmental agents. Motor activity
represents a broad class of behaviors involving coordinated
participation of sensory, motor, and integrative processes. Motor
activity measurements are noninvasive and can be used to evaluate the
effects of acute and repeated exposure to chemicals (MacPhail et al.,
1989). Motor activity measurements have also been used in humans to
evaluate disease states, including disorders of the nervous system
(Goldstein and Stein, 1985). The assessment of motor activity is often
included in first-tier evaluations, either as part of the FOB or as a
separate quantitated measurement.
There are many different types of activity measurement devices,
differing in size, shape, and method of movement detection (MacPhail et
al., 1989). Because of the accuracy and ease of calibration, devices
with photocells are widely used. In general, situating the apparatus to
minimize extraneous noise, movements, or lights usually requires that
the recording devices be placed in light- and sound-attenuating
chambers during the testing period. A number of different factors,
including age, gender, and time of day, can affect motor activity, and
should be controlled or counterbalanced. Different strains of animals
may have significantly different basal levels of activity, making
comparisons across studies difficult. A major factor in activity
studies is the duration of the testing session. Motor activity levels
are generally highest at the beginning of the session and decrease to a
low level throughout the session. The rate of decline during the test
session is frequently termed ``habituation.''
Motor activity measurements are typically included as part of a
battery of tests to detect or characterize neurotoxicity. Agent-induced
alterations in motor activity associated with overt signs of toxicity
(e.g., loss of body weight, systemic toxicity) or occurring in non-
dose-related fashion are of less concern than changes that are dose
dependent, related to structural or other functional changes in the
nervous system, or occur in the absence of life-threatening toxicity
and are generally convincing evidence of neurotoxicity.
4.3.2.3. Neuromotor function.
Motor dysfunction is a common neurotoxic effect, and many different
types of tests have been devised to measure time- and dose-dependent
effects. Anger (1984) reported 14 motor effects of 89 substances, which
could be classified into four categories: weakness, incoordination,
tremor, and myoclonia or spasms. Chemical-induced changes in motor
function can be determined with relatively simple techniques such as
the FOB. More specialized tests to assess weakness include measures of
grip strength, swimming endurance, suspension from a hanging rod,
discriminitive motor function, and hindlimb splay. Rotarod and gait
assessments measure incoordination, while rating scales and spectral
analysis techniques quantify tremor and other abnormal movements
(Tilson and Mitchell, 1984).
An example of a second-tier procedure to assess motor function has
been described by Newland (1988), who trained squirrel monkeys to hold
a bar within specified limits (i.e., displacement) to receive positive
reinforcement. The bar was also attached to a rotary device, which
allowed measurement of chemical-induced tremor. Spectral analysis was
used to characterize the tremor, which was found to be similar to that
seen in humans exposed to neurotoxicants or with such neurologic
diseases as Parkinson's disease.
Incoordination and performance changes can be assessed with
procedures that measure chemical-induced alterations in force (Fowler,
1987). The accuracy of performance may reflect neuromotor function and
is sensitive to the debilitating effects of many psychoactive drugs
(Walker et al., 1981; Newland, 1988). Gait, an index of coordination,
has been measured in rats under standardized conditions and can be a
sensitive indication of specific damage to the basal ganglia and motor
cortex (Hruska et al., 1979) as well as damage to the spinal cord and
peripheral nervous system.
Procedures to characterize chemical-induced motor dysfunction have
been used extensively in neurotoxicology. Most require preexposure
training (including alterations of motivational state) of experimental
animals, but such tests might be useful, in as much as similar
procedures are often used in assessing humans.
4.3.2.4. Sensory function.
Alterations in sensory processes (e.g., paresthesias and visual or
auditory impairments) are frequently reported signs or symptoms in
humans exposed to toxicants (Anger, 1984). Several approaches have been
devised to measure sensory deficits. Data from tests of sensory
function must be interpreted within the context of changes in body
weight, body temperature, and other physiological endpoints.
Furthermore, many tests assess the behavioral response of an animal to
a specific sensory stimulus; such responses are usually motor movements
that could be directly affected by chemical exposure. Thus, care must
be taken to determine whether proper controls were included to
eliminate the possibility that changes in response to a sensory
stimulus may have been related to agent-induced motor dysfunction.
Several first-tier testing procedures have been devised to screen
for overt sensory deficits. Many rely on orientation or the response of
an animal to a stimulus. Such tests are usually included in the FOB
used in screening (e.g., tail-pinch or click responses). Responses are
usually recorded as being either present, absent, or changed in
magnitude (Moser, 1989; O'Donoghue, 1989). Screening tests for sensory
deficits are typically not suitable to characterize chemical-induced
changes in acuity or fields of perception. The characterization of
sensory deficits usually necessitates psychophysical methods that study
the relationship between the physical dimensions of a stimulus and the
behavioral response it generates (Maurissen, 1988).
One second-tier approach to the characterization of sensory
function involves the use of reflex-modification techniques (Crofton,
1990). Chemical-induced changes in the stimulus frequency or threshold
required to inhibit a reflex are taken as possible changes in sensory
function. Prepulse inhibition has been used only recently in
neurotoxicology (Fechter and Young, 1983) and can be used to assess
sensory function in humans as well as in experimental animals.
Various behavioral procedures require that a learned response occur
only in the presence of a specific stimulus (i.e., discriminated or
conditioned responding). Chemical-induced changes in sensory function
are determined by altering the physical characteristics of the stimulus
(e.g., magnitude or frequency) and measuring the alteration in response
rate or accuracy. In an example of the use of a discriminated
conditional response to assess chemical-induced sensory dysfunction,
Maurissen et al. (1983) trained monkeys to respond to the presence of a
vibratory or electric stimulus applied to the fingertip. Repeated
dosing with acrylamide produced a persistent decrease in vibration
sensitivity; sensitivity to electric stimulation was unimpaired. That
pattern of sensory dysfunction corresponded well to known sensory
deficits in humans. Discriminated conditional response procedures have
been used to assess the ototoxicity produced by toluene (Pryor et al.,
1983) and the visual toxicity produced by methylmercury (Merigan,
1979).
Procedures to characterize chemical-induced sensory dysfunction
have been used often in neurotoxicology. As in the case of most
procedures designed to characterize nervous system dysfunction,
training and motivational factors can be confounding factors. Many
tests designed to assess sensory function for laboratory animals can
also be applied with some adaptation to humans.
4.3.2.5. Learning and memory.
Learning and memory disorders are neurotoxic effects of particular
importance. Impairment of memory is reported fairly often by adult
humans as a consequence of toxic exposure. Behavioral deficits in
children have been caused by lead exposure (Smith et al., 1989), and it
is hypothesized (Calne et al., 1986) that chronic low-level exposure to
toxic agents may have a role in the pathogenesis of senile dementia.
Learning can be defined as an enduring change in the mechanisms of
behavior that results from experience with environmental events (Domjan
and Burkhard, 1986). Memory is a change that can be either short-
lasting or long-lasting (Eckerman and Bushnell, 1992). Alterations in
learning and memory must be inferred from changes in behavior. However,
changes in learning and memory must be separated from other changes in
behavior that do not involve cognitive or associative processes (e.g.,
motor function, sensory capabilities, and motivational factors), and an
apparent toxicant-induced change in learning or memory should be
demonstrated over a range of stimuli and conditions. Before it is
concluded that a toxicant alters learning and memory, effects should be
confirmed in a second learning procedure. It is well known that lesions
in the brain can inhibit learning. It is also known that some brain
lesions can facilitate some types of learning by removing behavioral
tendencies (e.g., inhibitory responses due to stress) that moderate the
rate of learning under normal circumstances. A discussion of learning
procedures and examples of chemicals that can affect learning and
memory have appeared in recent reviews (Heise, 1984; WHO, 1986; Peele
and Vincent, 1989).
One simple index of learning and memory, which can be measured as a
first-tier endpoint, is habituation. Habituation is defined as a
gradual decrease in the magnitude or frequency of a response after
repeated presentations of a stimulus. A toxicant can affect habituation
by increasing or decreasing the number of stimulus presentations needed
to produce response decrements (Overstreet, 1977). Although habituation
is a very simple form of learning, it can also be perturbed by a number
of chemical effects not related to learning.
A more complicated approach to studying the effects of a chemical
on learning and memory involves the pairing of a novel stimulus with a
second stimulus that produces a known, observable, and quantifiable
response (i.e., classical ``Pavlovian'' conditioning). The novel
stimulus is known as the conditioned stimulus, and the second,
eliciting stimulus is the unconditioned stimulus. With repeated
pairings of the two stimuli, the conditioned stimulus comes to elicit a
response similar to the response elicited by the unconditioned
stimulus. The procedure has been used in behavioral pharmacology and,
to a lesser extent, in neurotoxicology. Neurotoxicants that interfere
with learning and memory would alter the number of presentations of the
pair of stimuli required to produce conditioning or learning. Memory
would be tested by determining how long after the last presentation of
the two stimuli the conditioned stimulus would still elicit a response
(Yokel, 1983). Other classically conditioned responses known to be
affected by psychoactive or neurotoxic agents are conditioned taste
aversion (Riley and Tuck, 1985) and conditioned suppression (Chiba and
Ando, 1976).
Second-tier procedures to assess learning or memory typically
involve the pairing of a response with a stimulus that increases the
probability of future response through reinforcement. Response rate can
be increased by using positive reinforcement or removing negative
reinforcement. Learning is usually assessed by determining the number
of presentations or trials needed to produce a defined frequency of
response. Memory can be defined specifically as the maintenance of a
stated frequency of response after initial training. Neurotoxicants may
adversely affect learning by increasing or decreasing the number of
presentations required to achieve the designated criterion. Decrements
in memory may be indicated by a decrease in the probability or
frequency of a response at some time after initial training. Toxicant-
induced changes in learning and memory should be interpreted within the
context of possible toxicant-induced changes in sensory, motor, and
motivational factors. Examples of instrumental learning procedures used
in neurotoxicology are repeated acquisition (Schrot et al., 1984),
passive and active avoidance, Y-maze avoidance, spatial mazes (radial-
arm maze), and delayed matching to sample (Heise, 1984; WHO, 1986;
Tilson and Mitchell, 1984).
4.3.2.6. Schedule-controlled behavior.
Another type of second-tier procedure is schedule-controlled
operant behavior (SCOB), which involves the maintenance of behavior
(performance) by response-dependent reinforcement (Rice, 1988).
Different patterns of behavior and response rates are controlled by the
relationship between response and later reinforcement. SCOB affords a
measure of learned behavior and with appropriate experimental design
may be useful for studying chemical-induced effects on motor, sensory,
and cognitive function.
The primary endpoints for evaluation are agent-induced changes in
response rate or frequency and the temporal pattern of responding.
Response rate is usually related to an objective response, such as
lever press or key peck, and differs according to the schedule of
reinforcement. Response rates are expressed per unit of time. For some
classes of chemicals, the direction of an effect on response rate can
differ between low and high doses. Agent-induced changes in temporal
pattern of responding can occur independently of changes in the rate
and require analysis of the distribution of responses relative to
reinforcement schedule.
SCOB has been used to study the effects of psychoactive drugs on
behavior and is sensitive to many neurotoxicants, including
methylmercury, solvents, pesticides, acrylamides, carbon monoxide, and
organic and inorganic lead (Paule and McMillan, 1984; MacPhail 1985;
Cory-Slechta, 1989; Rice, 1988). The experimental animal often serves
as its own control, and the procedure provides an opportunity to study
a few animals extensively over a relatively long period. SCOB typically
requires motivational procedures, such as food deprivation, and
training sessions are usually required to establish a stable baseline
of responding. Because of its sensitivity to neuroactive chemicals,
SCOB has great potential for use in second-tier assessments.
4.3.3. Neurophysiological Endpoints of Neurotoxicity
Neurophysiological studies are those that assess function either
directly through measurements of the electrical activity of the nervous
system (electrophysiology) or indirectly through measurements of
peripheral organ functions controlled or modulated by the nervous
system (general physiology) (Dyer, 1987). When performed properly,
neurophysiological techniques provide information on the integrity of
defined portions of the nervous system. Many of the endpoints used in
animals have also been used in humans to determine chemical-induced
alterations in neurophysiological function.
The term ``electrophysiology'' refers to the set of
neurophysiological procedures that study neural function through the
direct measurement of the electrical activity generated by the nervous
system (Dyer, 1987). A variety of electrophysiological procedures are
available for application to neurotoxicological problems, which range
in scale from procedures that employ microelectrodes to study the
function of single nerve cells or restricted portions of them, to
procedures that employ macroelectrodes to perform simultaneous
recordings of the summed activity of many cells. The latter types of
procedures have historically been used in studies to detect or
characterize the potential neurotoxicity of agents of regulatory
interest. Several macroelectrode procedures are discussed below.
4.3.3.1. Nerve conduction studies.
Nerve conduction studies are generally performed on peripheral
nerves and can be useful in investigations of possible peripheral
neuropathy. Most peripheral nerves contain mixtures of both individual
sensory and motor nerve fibers, which may or may not be differentially
sensitive to neurotoxicants. It is possible to distinguish sensory from
motor effects in peripheral nerve studies by measuring activity in
purely sensory nerves such as the sural to study sensory effects or by
measuring the muscle response evoked by nerve stimulation to measure
motor effects. While a number of endpoints can be recorded, the most
commonly used variables are (1) Nerve conduction velocity, and (2)
response amplitude. In well-controlled studies, decreases in nerve
conduction velocity typically are evidence of neurotoxicity (Dyer,
1987). While a decrease in nerve conduction velocity is a reliable
measure of demyelination, it frequently occurs rather late in the
course of axonal degradation because normal conduction velocity may be
maintained for some time in the face of axonal degeneration. For this
reason, a measurement of normal nerve conduction velocity does not
necessarily rule out peripheral axonal degeneration if other signs of
peripheral nerve dysfunction are present. Increases in conduction
velocity of adult organisms following treatment with neurotoxic
compounds, in the absence of hypothermia, are atypical responses and
may, in fact, reflect experimental or statistical errors. Decreases in
response amplitude reflect a loss of active nerve fibers, and may occur
prior to decreases in conduction velocity in the course of peripheral
neuropathy. Hence changes in response amplitude may be more sensitive
measurements of axonal degeneration than conduction velocity.
Measurements of response amplitude, however, are more variable and
require careful experimental techniques, a larger sample size, and
greater statistical power than measurements of velocity to detect
changes. Alterations in peripheral nerve function are associated with
abnormal peripheral sensations such as numbness, tingling, or burning
or with motor impairments such as weakness. Examples of compounds that
alter peripheral nerve function in humans or experimental animals at
some level of exposure include acrylamide, carbon disulfide,
hexacarbons, lead, and some organophosphates.
4.3.3.2. Sensory evoked potentials.
Sensory evoked potentials are electrophysiological procedures that
involve measuring the response elicited by the presentation of a
defined sensory stimulus such as a tone, a light, or a brief electrical
pulse to the skin. Sensory evoked potentials reflect sensory function,
and can be used to investigate visual, auditory, or somatosensory (body
sensation) systems (Rebert, 1983; Mattsson and Albee, 1988). The data
are in the form of a voltage record over time, which can be quantified
in several ways. Commonly, the positive and negative voltage peaks are
identified and measured as to their latency (time from stimulus onset)
and amplitude (voltage).
Changes in peak amplitudes or equivalent measures reflect changes
in the magnitude of the neural population that is responsive to
stimulation. Both increases and decreases in amplitude are possible
following exposure to neurotoxicants because (1) The brain normally
operates in a careful balance between excitatory and inhibitory
systems, and disruption of this balance can produce either positive or
negative shifts in the voltages recorded in evoked potential
experiments, and (2) excitatory or inhibitory neural activity is
translated into a positive or negative deflection in the sensory evoked
potential depending on the physical orientation of the electrode with
respect to the tissue generating the response, which is frequently
unknown. Within any given sensory system, the neural circuits that
generate the different evoked potential peaks differ as a function of
peak latency. In general, early latency peaks reflect the transmission
of afferent sensory information, and changes in either the latency or
amplitude of these peaks generally indicate a neurotoxic change that is
likely to be reflected in deficits in sensory perception. The later
latency peaks, in general, reflect not only the sensory input, but also
the more nonspecific factors such as the behavioral state of the
subject including such factors as arousal level, habituation, or
sensitization. Thus, the neurotoxicological significance of changes in
later latency evoked potential peaks must be interpreted in light of
the behavioral status of the subject.
4.3.3.3. Convulsions.
Observable behavioral convulsions in animals may be indicative of
central nervous system seizure activity. However, behavioral
convulsions that occur only at lethal or near lethal dose levels may
reflect an indirect effect secondary to systemic toxicity and not
directly on the nervous system. Convulsions occurring at dose levels
that are clearly sublethal, and in the absence of apparent systemic
toxicity, are more likely due to a direct effect on the nervous system.
In such cases, neurophysiological recordings of electrical activity in
the brain that are indicative of seizures may provide additional
evidence of direct neurotoxicity. In addition to producing seizures,
chemicals may also affect seizure susceptibility, altering the
frequency, severity, duration, or threshold for eliciting seizures
produced through other means. Such changes can occur after acute
exposure or after repeated exposure to dose levels below the acute
threshold, and are considered neurotoxic. Agents that produce
convulsions include lindane, DDT, pyrethroids, and trimethyltin (WHO,
1986). Some agents, including many solvents, act to raise the threshold
for eliciting seizures through other means or otherwise act to reduce
the severity or duration of the elicited convulsions. These agents are
difficult to classify as neurotoxic based on such data, but frequently
have other effects on which a determination of neurotoxic potential can
be based.
4.3.3.4. Electroencephalography (EEG)
EEG analysis is used widely in clinical settings for the diagnosis
of neurological disorders and less often for the detection of subtle
toxicant-induced dysfunction (WHO, 1986; Eccles, 1988). The basis for
the use of EEG in either setting is the relationship between specific
patterns of EEG waveforms and specific behavioral states. Because
states of alertness and the stages of sleep are associated with
distinct patterns of electrical activity in the brain, it is generally
thought that arousal level can be evaluated by monitoring the EEG.
Dissociation of EEG activity and behavior can, however, occur after
exposure to certain chemicals. Normal patterns of transition between
sleep stages or between sleeping and waking states are known to remain
disturbed for prolonged periods of time following exposure to certain
chemical classes (e.g., organophosphates). Changes in the pattern of
the EEG can be elicited by stimuli producing arousal (e.g., lights,
sounds) and neuroactive drugs. In studies with toxicants, changes in
EEG pattern can sometimes precede alterations in other objective signs
of neurotoxicity. EEG experiments must be done under highly controlled
conditions, and the neurotoxicological significance of chemical-induced
changes in the EEG in the absence of other signs of neurotoxicity must
be considered on a case-by-case basis. Many chemicals, including
metals, solvents, and pesticides, would be expected to affect the EEG.
4.3.3.5. Electromyography (EMG).
EMG involves making electrical recordings from muscle and has been
used extensively in human clinical studies in the diagnosis of certain
diseases of the muscle (WHO, 1986). Changes in the EMG include
amplitude and firing frequency of spontaneous firing; evoked muscle
responses to nerve stimulation can be used to study alterations in the
neuromuscular junction. EMG has been used to study toxicant-induced
changes in neuromuscular function, including organophosphate
insecticides, methyl n-butyl ketone, and botulinum and tetanus toxin.
4.3.3.6. Spinal reflex excitability.
Segmental spinal monosynaptic and polysynaptic reflexes are
relatively simple functions in the central nervous system that can be
evaluated by quantitative techniques (WHO, 1986). Many of the
procedures used in animals are similar to procedures used clinically to
perform neurological tests in humans. One approach infers the
functional state of a reflex arc from either the latency and magnitude
of the reflex response evoked by stimuli of predetermined intensity or
from the stimulus intensity required to elicit a detectable response
(i.e., the threshold). This approach is used best in a screening
context and the significance of effects in this test should be
considered on a case-by-case basis.
A second more involved approach records electrophysiologically the
time required for a stimulus applied to a peripheral nerve to reach the
spinal cord and return to the site of the original stimulation. Data
from this procedure can indicate the excitability of the motoneuron
pool, an effect seen with many volatile solvents. Although this
approach is more invasive and time-consuming than the noninvasive
procedure, it provides better data concerning the possible site of
action. In addition, the manner in which the invasive procedure is
carried out (i.e., in decerebrated animals) precludes repeated testing
on the same animal. The significance of effects in this procedure
should also be considered on a case-by-case basis.
4.3.4. Neurochemical Endpoints of Neurotoxicity
Neuronal function within the nervous system is dependent on
synthesis and release of specific neurotransmitters and activation of
their receptors in specific neuronal pathways. With few exceptions,
neurochemical measurements are invasive and therefore used infrequently
in human risk assessment. There are many different neurochemical
endpoints that could be measured in neurotoxicological studies (Bondy,
1986; Mailman, 1987; Morell and Mailman, 1987). Neurotoxicants can
interfere with the ionic balance of a neuron, act as a cytotoxicant
after being transported into a nerve terminal, block uptake of
neurotransmitter precursors, act as a metabolic poison, overstimulate
receptors, block transmitter release, and inhibit transmitter
degradation. Table 4-4 lists several chemicals with known neurochemical
effects. Many neuroactive agents can increase or decrease
neurotransmitter levels in the brain. Dose-related changes on these
endpoints may indicate a chemical effect on the nervous system, but the
neurotoxicological significance of such changes must be interpreted in
the context of other signs of neurotoxicity.

Table 4-4.--Neurotoxicants With Known Neurochemical Mechanisms
------------------------------------------------------------------------
Site of attack Examples
------------------------------------------------------------------------
1. Neurotoxicants acting on ionic
balance
A. Inhibit sodium entry....... Tetrodotoxin.
B. Block closing of sodium p,p\1\--DDT, pyrethroids (I).
channel.
C. Increase permeability to Batrachotoxin.
sodium.
D. Increase intracellular Chlordecone.
calcium.
2. Cytotoxicants--depend on uptake MPTP.
into nerve terminal.
3. Uptake blockers................ Hemicholinium.
4. Metabolic poisons.............. Cyanide.
5. Receptor hyperactivators....... Domoic acid.
6. Transmitter release (ACh) Botulinum toxin.
blockers.
7. Transmitter degradation (ACh) Organophosphates, carbamates.
inhibitors.
8. Microtubule disruptors......... Vincristine.
------------------------------------------------------------------------

Some chemicals, such as the organophosphate and carbamate
insecticides, are known to interfere with a specific enzyme,
acetylcholinesterase (AChE) (Costa, 1988). Inhibition of this enzyme in
brain may be considered evidence of neurotoxicity, whereas decreases in
AChE in the blood, which can be easily determined in humans, are only
suggestive of a neurotoxic effect. A subset of organophosphate agents
produces organophosphate-induced delayed neuropathy (OPIDN) after acute
or repeated exposure. Neurotoxic esterase (or neuropathy target enzyme,
NTE) has been associated with agents that produce OPIDN (Johnson,
1990).
The ultimate functional significance of many biochemical changes is
not known; therefore it may be difficult to determine if a specific
biochemical change can be considered adverse or convincing evidence of
neurotoxicity. Any such change, however, is potentially adverse and
each determination of adversity requires a judgment to be made.
Likewise, the absence of specific biochemical testing protocols does
not mean biochemical changes are of no concern, but instead reflects a
lack of understanding of the significance of changes at the biochemical
level.
4.3.5. Structural Endpoints of Neurotoxicity
The central nervous system (brain and spinal cord) comprises nerve
cells or neurons, which consist of a neuronal body, axon, and dendritic
processes. Various types of neuropathological lesions may be classified
according to their nature or the site where they are found (WHO, 1986;
Krinke, 1989; Griffin, 1990). Lesions may be classified as neuropathy
(changes in the neuronal body), axonopathy (changes in the axons),
myelinopathy (changes in the myelin sheaths), neurodegeneration
(changes in the nerve terminals), and peripheral neuropathy (changes in
the peripheral nerves). For axonopathies, a more precise location of
the changes should be described (i.e., proximal, central, or distal
axonopathy). In some cases, agents produce neuropathic conditions that
resemble naturally occurring neurodegenerative disorders in humans
(WHO, 1986). Table 4-5 lists examples of such chemicals, their known
site of action, the type of neuropathology produced, and the disease or
condition that each typifies.

Table 4-5.--Examples of Known Neuropathic Agents
----------------------------------------------------------------------------------------------------------------
Corresponding Disease or neurodegenerative
Site of attack Neuropathology neurotoxicant condition
----------------------------------------------------------------------------------------------------------------
Neuron cell body........... Neuronopathy.............. Methylmercury....... Minamata disease.
A.E.T.T............. Ceroid lipofuscinoses.
Quinolinic acid..... Huntington's disease.
3-acetylpridine..... Cerebellar ataxia.
Aluminum............ Alzheimer's disease.
Nerve terminal............. Neurodegeneration......... MPTP................ Parkinson's disease.
Schwann cell myelin........ Myelinopathy.............. Lead Buckthorn toxin Neuropathy of metachromatic
leukodystrophy.
Central-peripheral distal Distal axonopathy......... Acrylamide Vitamin deficiency.
axon. Hexacarbons Carbon
disulfide.
Central axons.............. Central axonopathy........ Clioquinol.......... Subacute myelooptico-neuropathy.
Proximal axon.............. Proximal axonopathy....... B,B'-imminodi- Motor neuron disease.
proprionitrile.
----------------------------------------------------------------------------------------------------------------

In general, chemical effects lead to two types of primary cellular
alteration: (l) the accumulation, proliferation, or rearrangement of
structural elements (e.g., intermediate filaments, microtubules) or
organelles (mitochondria) and (2) the breakdown of cells, in whole or
in part. The latter can be associated with regenerative processes that
may occur during chemical exposure. Such changes are considered to be
neurotoxic.
While most neurotoxic damage is evident at the microscopic level,
gross changes in morphology can be reflected by a significant change in
the weight of the brain. Weight changes (absolute or relative to body
weight), discoloration, discrete or massive cerebral hemorrhage, or
obvious lesions in nerve tissue are generally considered neurotoxic
effects.
Chemical-induced injury to the central nervous system is associated
with astrocytic hypertrophy at the site of damage. Assays of glial
fibrillary acidic protein (GFAP), the major intermediate filament
protein of astrocytes, has been proposed as a biomarker of this
response (O'Callaghan, 1988). A number of chemicals known to injure the
central nervous system, including trimethyltin, methylmercury, cadmium,
3-acetylpyridine, and MPTP, have been shown to increase GFAP. In
addition, increases in GFAP may be seen at dosages below those
necessary to produce cytopathology as determined by Nissl-based stains
used in standard neuropathological examinations. Because increases in
GFAP may be an early indicator of neuronal injury in the adult,
exposure level-dependent increases in GFAP should be viewed with
concern.
Chemical-induced alterations in the structure of the nervous system
are generally considered neurotoxic effects. To ensure reliable data,
it is important that neuropathological studies minimize fixation
artifacts and potential differences in the section(s) of the nervous
system sampled and control for variability due to the age, sex, and
body weight of the subject (WHO, 1986).

4.3.6. Developmental Neurotoxicity

Exposure to chemicals during development can result in effects
other than death, gross structural abnormality, or altered growth.
There are several instances in which functional alterations have
resulted from exposure during the period between conception and sexual
maturity (Riley and Vorhees, 1986; Vorhees, 1987). Table 4-6 lists
several examples of chemicals known to produce developmental
neurotoxicity in experimental animals. Animal models of developmental
neurotoxicity have been shown to be sensitive to several environmental
chemicals known to produce developmental toxicity in humans, including
lead, ethanol, methylmercury, and PCBs (Kimmel et al., 1990).

Table 4-6.--Partial List of Agents Believed to Have Developmental
Neurotoxicity

Alcohols Methanol, ethanol
Antimitotics X-radiation, azacytidine
Insecticides DDT, kepone, organophosphates
Metals Lead, methylmercury, cadmium
Polyhalogenated hydrocarbons PCB, PBB
Psychoactive drugs Cocaine, phenytoin
Solvents Carbon disulfide, toluene
Vitamins Vitamin A

Sometimes functional defects are observed at dose levels below
those at which other indicators of developmental toxicity are evident
(Rodier, 1986). Such effects may be transient or reversible in nature,
but generally are considered adverse effects. Data from postnatal
studies, when available, are considered useful for further assessment
of the relative importance and severity of findings in the fetus and
neonate. Often, the long-term consequences of adverse developmental
outcomes noted at birth are unknown and further data on postnatal
development and function are necessary to determine the full spectrum
of potential developmental effects. Useful data also can be derived
from well-conducted multigeneration studies, although the dose levels
used in these studies may be much lower than those in studies with
shorter-term exposure.
Much of the early work in developmental neurotoxicology was related
to behavioral evaluations. Recent advances in this area have been
reviewed in several publications (Riley and Vorhees, 1986; Kimmel et
al., 1990). Several expert groups have focused on the functions that
should be included in a behavioral testing battery, including sensory
systems, neuromotor development, locomotor activity, learning and
memory, reactivity and habituation, and reproductive behavior. No
testing battery has fully addressed all of these functions, but it is
important to include as many as possible, and several testing batteries
have been developed and evaluated for use in testing.
Direct extrapolation of functional developmental effects to humans
is limited in the same way as for other endpoints of developmental
toxicity, i.e., by the lack of knowledge about underlying toxicological
mechanisms and their significance. It can be assumed that functional
effects in animal studies indicate the potential for altered
development in humans, although the types of developmental effects seen
in experimental animal studies will not necessarily be the same as
those that may be produced in humans. Thus, when data from functional
developmental toxicity studies are encountered for particular agents,
they should be considered in the risk assessment process.
Agents that produce developmental neurotoxicity at a dose that is
not toxic to the maternal animal are of special concern because the
developing organism is affected but toxicity is not apparent in the
adult. More commonly, however, adverse developmental effects are
produced only at doses that cause minimal maternal toxicity; in these
cases, the developmental effects are still considered to represent
developmental toxicity and should not be discounted as secondary to
maternal toxicity. At doses causing excessive maternal toxicity (that
is, significantly greater than the minimal toxic dose), information on
developmental effects may be difficult to interpret and of limited
value. Current information is inadequate to assume that developmental
effects at maternally toxic doses result only from maternal toxicity;
it may be that the mother and developing organism are sensitive to that
dose level. Moreover, whether developmental effects are secondary to
maternal toxicity or not, the maternal effects may be reversible while
effects on the offspring may be permanent. These are important
considerations for agents to which humans may be exposed at minimally
toxic levels either voluntarily or involuntarily, because several
agents are known to produce adverse developmental effects at minimally
toxic doses in adult humans (e.g., smoking, alcohol).
Although interpretation of functional developmental neurotoxicity
data may be limited at present, it is clear that functional effects
must be evaluated in light of other toxicity data, including other
forms of developmental toxicity (e.g., structural abnormalities,
perinatal death, and growth retardation). The level of confidence in an
adverse effect may be as important as the type of change seen, and
confidence may be increased by such factors as replicability of the
effect either in another study of the same function or by convergence
of data from tests that purport to measure similar functions. A dose-
response relationship is considered an important measure of chemical
effect; in the case of functional effects, both monotonic and biphasic
dose-response curves are likely, depending on the function being
tested.

4.3.7. Physiological and Neuroendocrine Endpoints

One of the key roles played by the nervous system is to orchestrate
the general physiological functions of the body to help maintain
homeostasis. To this end, the nervous system and many of the peripheral
organ systems are integrated and functionally interdependent. For
example, specific neuronal processes are intimately involved in
maintaining or modulating respiration, cardiovascular function, body
temperature, and gastrointestinal function. Because many peripheral
organ functions involve neuronal components, changes in such
physiological endpoints as blood pressure, heart rate, EKG, body
temperature, respiration, lacrimation, or salivation may indirectly
reflect possible treatment-related effects on the functional integrity
of the nervous system. However, since physiological endpoints also
depend on the integrity of the related peripheral organ itself, changes
in physiological function also may reflect a systemic toxicity
involving that organ. Consequently, the neurotoxicological significance
of a physiological change must be interpreted within the context of
other signs of toxicity. A variety of general physiological procedures
can be applied to neurotoxicological problems. These procedures range
in scale from simple measurements, for example, of body temperature,
respiration, lacrimation, salivation, urination, and defecation, which
may be included in routine functional observational batteries used for
chemical screening, to more involved procedures involving measurements
of blood pressure, endocrine responses, cardiac function,
gastrointestinal function, etc. The latter would be more appropriate
for second-level tests to characterize the scope of chemically related
toxicity.
The central nervous system also regulates the outflow of the
endocrine system, which together with the influence of the autonomic
nervous system, can affect immunologic function (WHO, 1986). Hormonal
balance results from the integrated action of the hypothalamus, located
in the central nervous system, and the pituitary, which regulates
activities of endocrine target organs. Each site is susceptible to
disruption by neurotoxic agents. Neuroendocrine dysfunction may occur
because of a disturbance in the regulation and modulation of the
neuroendocrine feedback systems. One major indicator of neuroendocrine
function is secretions of hormones from the pituitary. Hormones from
the anterior pituitary are important for reproduction (follicle-
stimulating hormone, luteinizing hormone), growth (thyroid-stimulating
hormone), and response to stress (adrenocorticotropic hormone).
Hypothalamic control of anterior pituitary secretions occurs through
the release of hypothalamic-hypophysiotropic hormones. Hormones from
the posterior hypothalamus (prolactin, melanocyte-stimulating hormone,
and growth hormone) are also involved in a number of important bodily
functions.
Many types of behaviors (e.g., reproductive behaviors, sexually
dimorphic behaviors) are dependent on the integrity of the
hypothalamic-pituitary system, which could represent an important site
for neurotoxic action. Pituitary secretions arise from a number of
different cell types in this gland and neurotoxicants could affect
these cells either directly or indirectly. Morphological changes in
follicular cells, chromophobe cells, somatotropic cells, prolactin
cells, gonadotropic cells, follicle-stimulating hormone secreting
cells, luteinizing hormone-containing cells, thyrotropic cells, and
cortico cells might be associated with adverse effects on the
pituitary, which could ultimately affect behavior and the functioning
of the nervous system.
Biochemical changes in the hypothalamus also may be used as indices
of potential changes in neuroendocrine function. However, the
neuroendocrine significance of changes in hypothalamic
neurotransmitters and neuropeptides is usually only inferential and
data must be considered on a case-by-case basis.
Most anterior pituitary hormones are subject to negative feedback
control by peripheral endocrine glands and, if neurotoxicants modify
peripheral secretions, neuroendocrine changes can result from this
altered feedback. Modifications in the functioning of these endocrine
secretions could occur after toxic exposure; a number of agents have
been shown to alter blood levels of glucocorticoids, thyroxine,
estrogen, corticosterone, and testosterone. Although such changes are
not necessarily due to direct neuroendocrine effects, target organ
changes often can be a first indication of neuroendocrine changes.
4.3.8. Other Considerations
4.3.8.1. Structure-activity relationship.
Because of a general lack of epidemiologic or toxicologic data on
most chemical substances, attempts have been made in toxicology to
predict activities based on chemical structure. The basis for inference
from structure-activity relationships (SARs) can be either comparison
with structures known to have biologic activity or knowledge of
structural requirements of a receptor or macromolecular site of action.
However, given the complexity of the nervous system and the lack of
information on biologic mechanisms of neurotoxic action, there are
relatively few well-characterized SARs in neurotoxicology. Since SARs
cannot be used to rule out all neurotoxic activity, it is not
acceptable to use them as a basis for excluding potential
neurotoxicity. Caution is warranted in interpreting SARs in anything
other than the most preliminary analyses. Use of SARs requires detailed
knowledge not only of structure, but also of each critical step in the
pathogenetic mechanism of neurotoxic injury. Such knowledge is still
generally unavailable.
SAR approaches are more successful when the range of possible sites
of action or mechanisms of action is narrow. Thus, SARs have had more
use in relation to carcinogenicity and mutagenicity than in other kinds
of toxicity. The SAR approaches used in the development of novel
neuropharmacologic structures deserve consideration in neurotoxicology,
but their utility depends on a better understanding of neurotoxic
mechanisms.
4.3.8.2. In vitro methods.
In vitro procedures for testing have practical advantages, but
studies must be done to correlate the results with responses in whole
animals. One advantage of validated in vitro tests is that they
minimize the use of live animals. Some of the more developed in vitro
tests might be simple and might not have to be conducted by highly
trained personnel, but, as with many in vivo tests, the analysis and
interpretation of results are likely to require expertise. Experience
with the Ames test for mutagenesis confirms the advantages of in vitro
procedures, but also illustrates the problems that arise when an assay
is used to predict an endpoint that is not exactly what it measures
(e.g., carcinogenicity rather than specific aspects of genotoxicity).
In vitro changes can be markers for toxicity, even when the structural
or functional consequences are not known or predicted. In addition, in
vitro methods can examine the more evolutionarily conserved elements of
the nervous system and improve neurotoxicity detection and could also
provide suitable systems for studying developmental neurotoxicity.
A broad range of tissue-culture systems are available for assessing
the neurologic impact of environmental agents, including cell lines,
dissociated cell cultures, reaggregate cultures, explant cultures, and
organ cultures (Veronesi, 1991).
Neuronal and glial cell lines are used extensively in neurobiology
and have potential for neurotoxicological studies. They consist of
populations of continuously dividing cells that, when treated
appropriately, stop dividing and exhibit differentiated neuronal or
glial properties. Neuronal lines can develop electric excitability,
chemosensitivity, axon formation, neurotransmitter synthesis and
secretion, and synapse formation. Large quantities of cells can be
generated routinely to develop extensive dose-response or other
quantitative data.
When neural tissue, typically from fetal animals, is dissociated
into a suspension of single cells, and the suspension is inoculated
into tissue-culture dishes, the neurons and glia survive, grow, and
establish functional neuronal networks. Such preparations have been
made from most regions of the CNS and exhibit highly differentiated,
site-specific properties that constitute an in vitro model of different
portions of the CNS. Most of the neuronal transmitter and receptor
phenotypes can be demonstrated, and a variety of synaptic interactions
can be studied. Glial cells are also present, and neuroglial
interactions are a prominent feature of the cultures. A substantial
battery of assays (neurochemical and neurophysiologic) is now available
to assess the development of the cultures and to indicate toxic effects
of test agents added to the culture medium. Relatively pure populations
of different cell types can be isolated and cultured, so that effects
on specific cell types can be assessed independently. Pure glial cells
or neurons, or even specific neural categories, can be prepared in this
way and studied separately, or interaction between neurons and glial
cells can be studied at high resolution. The neurobiologic measures
used to assess the effect of any agent can be very specific (for
example, activity of neurotransmitter-related enzyme or binding of a
receptor ligand) or global (for example, neuron survival or
concentration of glial fibrillary acidic protein). The two-dimensional
character of the preparations makes them particularly suited for
morphologic evaluation, and detailed electrophysiologic studies are
readily performed. The toxic effects and mechanisms of anticonvulsants,
excitatory amino acids, and various metals and divalent cations have
been assessed with these preparations. The cerebellar granular cell
culture system, for example, has been exploited recently in studies of
the mechanism of alkyllead toxicity (Verity et al., 1990).
A related preparation made from single-cell suspensions of neural
tissue is the reaggregate culture. Instead of being placed in culture
dishes and allowed to settle onto the surface of the dishes, the cells
are kept in suspension by agitation; under appropriate conditions, they
stick to one another and form aggregates of controllable size and
composition. Typically, the cells in an aggregate organize and exhibit
intercellular relations that are a function of, and bear some
resemblance to, the brain region that was the source of the cells. The
cells establish a three-dimensional, often laminated structure.
Reaggregate cultures lend themselves to large-scale, quantitative
experiments in which neurobiologic variables can be examined, although
morphologic and ligand-binding studies are performed less readily than
with surface cultures.
Organotypic explant cultures also are closely related to the intact
nervous system. Small pieces or slices of neural tissue are placed in
culture and can be maintained for long periods with substantial
maintenance of structural and cell-cell relations of intact tissue.
Specific synaptic relations develop and can be maintained and
evaluated, both morphologically and electrophysiologically. Because all
regions of the nervous system are amenable to this sort of preparation,
it is possible to analyze toxic agents that are active only in specific
regions of the central or peripheral nervous system. Explants can be
made from relatively thin slices of neural tissue, so detailed
morphologic and intracellular electrophysiologic studies are possible.
Their anatomic integrity is such that they capture many of the cell-
cell interactions characteristic of the intact nervous system while
allowing a direct, continuing evaluation of the effects of a
potentially neurotoxic compound added to the culture medium. The
process of myelination has been studied extensively in explant
cultures, and considerable neurotoxicologic information has been
gained. A preparation similar to an explant culture is the organ
culture, in which an entire organ, such as the inner ear or a ganglion,
rather than slices or fragments, is grown in vitro. Obviously, only
structures so small that their viability is not compromised can be
treated in this way.
In general, the technical ease of maintaining a culture varies
inversely with the degree to which it captures a spectrum of in vivo
characteristics of nervous system behavior. The problem of
biotransformation of potentially neurotoxic compounds is shared by all,
although the more complete systems (explant or organ cultures) might
alleviate this problem in specific instances. In many culture systems,
complex and ill-defined additives--such as fetal calf serum, horse
serum, and human placental serum--are used to promote cell survival. A
number of thoroughly described synthetic media are now available,
however, and such fully defined culture systems can be used where
necessary.

5. Neurotoxicology Risk Assessment

5.1. Introduction

Risk assessment is an empirically based process used to estimate
the risk that exposure of an individual or population to a chemical,
physical, or biological agent will be associated with an adverse
effect. Generally, such effects can be quantified and the relative
probability of their occurrence can be calculated. The risk assessment
process usually involves four steps: hazard identification, dose-
response assessment, exposure assessment, and risk characterization
(NRC, 1983). Risk management is the process that applies information
obtained through the risk assessment process to determine whether the
assessed risk should be reduced and, if so, to what extent (NRC, 1983).
In some cases, risk is the only factor considered in a decision to
regulate exposure to a substance. Alternatively, the risk posed by a
substance is weighed against social, ethical, and medical benefits and
economic and technological factors in formulating a risk management
decision. The risk-balancing approach is used by some agencies to
consider the benefits as well as the risks associated with unrestricted
or partially restricted use of a substance. The purpose of this chapter
is to describe the risk assessment process as it has currently evolved
in neurotoxicology and present available options for quantitative risk
assessment.

5.2. The Risk Assessment Process

5.2.1. Hazard Identification
Agents that adversely affect the neurophysiological, neurochemical,
or structural integrity of the nervous system or the integration of
nervous system function expressed as modified behavior may be
classified as neurotoxicants (Tilson, 1990b). For hazard
identification, the best or most generalizable studies would measure
these changes in humans. With the exclusion of therapeutic agents,
information on effects in humans is usually derived from case reports
of accidental exposures and epidemiological studies. This type of data
affords less certainty regarding generalizability as well as less
specific exposure information. As discussed in chapter 4, a common
alternative method of data generation for hazard identification is the
use of animal models. Animal models that measure behavioral,
neurophysiological, neurochemical, and structural effects have been
developed and validated. Studies that employ these models to evaluate
specific potential hazards are used to predict the outcome of exposure
to the same hazard in humans.
5.2.1.1. Human studies
Information obtained through the evaluation of human exposure data
provides direct identification of neurotoxic hazards. This type of
information is generally available from clinical trials required for
the approval of therapeutic products for human use. For the purposes of
risk assessment of nontherapeutic substances, data on effects of
exposure to humans come primarily from two types of studies, case
reports and epidemiological (Friedlander and Hearn, 1980) (see chapter
3). Case studies can supply evidence of an agent's toxicity, but are
often limited by both the qualitative nature of the signs and symptoms
reported and the nature of the exposure data. Epidemiological studies
can provide data on the types of neurotoxic effects and the possible
susceptibilities of certain populations. Under appropriate
considerations, they can generally provide convincing and reliable
evidence of potential human neurotoxicity. As with case studies,
however, often only qualitative estimates of exposure can be obtained.
Controlled laboratory studies have the potential to provide adequate
exposure and effects data for accurate hazard identification, but
ethical considerations place moral and practical restrictions on such
studies except in those instances where direct benefit to the subjects,
as in the case of therapeutic agents, may be expected. Excluding
instances of therapeutic product development, most studies are limited
to measuring the effects of acute, rather than long-term, exposure.
This limits their utility in risk assessment because the effect of
long-term, low-level exposure to a potentially toxic agent is often the
issue of concern.
Methods available to evaluate neurotoxicity in humans include
examination of neurophysiological and behavioral parameters. Specific
tests to measure neuromuscular strength and coordination, alterations
in sensation, deficits in learning and memory, changes in mood and
personality, and disruptions of autonomic function are frequently
employed (see chapter 3).
5.2.1.2. Animal studies
As discussed in chapter 4, animal models for many endpoints of
neurotoxicity are available and widely used for hazard identification.
Data from animal studies are frequently extrapolated to humans. For
example, if exposure to an agent produces neuropathology in an animal
model, damage to a comparable structure in humans is predicted.
Similarly, biochemical and physiological effects observed in animals
are commonly extrapolated to humans. Agents that produce alterations in
the levels of specific enzymes in one animal species generally have the
same effect in other species, including humans. Neurophysiological
endpoints also tend to be affected by the same manipulations across
species. Thus, an agent interfering with nerve conduction in an animal
study is often assumed to have the same effect in humans. Behavioral
studies in animals are also applied to human hazard identification,
although the correspondence between methods employed in animals and
humans is sometimes not as obvious. For this reason, behavioral methods
developed for neurotoxic hazard identification need to be considered on
a case-by-case basis.
5.2.1.3. Special issues
5.2.1.3.1. Animal-to-human extrapolation. The use of animal data to
identify hazard to humans is not without controversy. Relative
sensitivity across species as well as between sexes is a constant
concern. Overly conservative risk assessments, based on the assumption
that humans are always more sensitive than a tested animal species, can
result in poor risk management decisions. Conversely, an assumption of
equivalent sensitivity in a case where humans actually are more
sensitive to a given agent can result in underregulation that might
have a negative impact on human health.
5.2.1.3.2. Susceptible populations. A related controversy concerns
the use of data collected from adult organisms, animal or human, to
predict hazards in potentially more sensitive populations, such as the
very young and the elderly, or in other groups, such as the chronically
ill. In some cases, identification of neurotoxicity hazard does not
generally include subjects from either end of the human life span or
from other than healthy subjects. Uncertainty factors are used to
adjust for more sensitive populations. In addition, single or
multigeneration reproductive studies in animals may provide a source of
information on neurological disorders, behavioral changes, autonomical
dysfunction, neuroanatomical anomalies, and other signs of
neurotoxicity in the developing animal (chapter 4).
5.2.1.3.3. Reversibility. For the most part, the basic principles
of hazard identification are the same for neurotoxicity as for any
adverse effect on health. One notable exception, however, concerns the
issue of reversibility and the special consideration that must be given
to the inherent redundancy and plasticity of the nervous system.
For many health effects, temporary, as opposed to permanent,
effects are repaired during a true recovery. Damage to many organ
systems, if not severe, can be spontaneously repaired. For example,
damaged liver cells that may result in impaired liver function often
can be replaced with new cells that function normally. The resulting
restoration of liver function can be viewed as recovery. In the central
nervous system, cells generally do not recover from severe damage and
new cells do not replace them. When nervous system recovery is
observed, it may represent compensation requiring activation of cells
that were previously performing some other function, reactive
synaptogenesis, or recovery of moderately injured cells. While a
damaged liver may recover due to the addition of new cells, severe
damage to nervous system cells results in a net loss of cells. This
loss of compensatory capacity may not be noticed for many years and,
when it does appear, it may be manifest in a way seemingly unrelated to
the original neurotoxic event. Lack of ability to recover from a
neurotoxic event later in life or premature onset of signs of normal
aging may result. It is therefore important to consider the possibility
that significant damage to the nervous system may have occurred in
experiments where effects appear to be reversible.
5.2.1.3.4. Weight of evidence.
A ``weight of evidence'' approach to identifying an agent as a
neurotoxic hazard is almost always necessary. With the exception of
therapeutic products, a single, complete, controlled study of an
agent's effects on the nervous system, conducted in an appropriate
representative sample of humans, is rarely, if ever, possible. Rather,
those individuals charged with identifying hazard are usually
confronted with a collection of imperfect studies, often providing
conflicting data (Barnes and Dourson, 1988).
There are several possible approaches, depending on the quality of
the evidence. Two examples are the use of data from only the most
sensitive species tested and the use of data from only species
responding most like the human for any given endpoint. In assessing
neurotoxicity of therapeutic products, when human data are available
and neurotoxic endpoints detected in animals can be clinically
measured, the human findings supersede those of the nonclinical data
base. Assuming that all available evidence is to be included,
considerations necessary for formulating a conclusion include the
relative weights that should be given to positive and negative studies.
Sometimes positive studies are given more weight than negative ones,
even when the quality of the studies is comparable. Experimental design
factors such as the species tested, the number and gender of subjects
evaluated, and the duration of the test are given different weights
when data from different studies are combined. The route of exposure in
a given study and its relevance to expected routes of human exposure
are often a weighted factor. The issue of statistical significance is
frequently debated. Some argue that an effect occurring at a
statistically insignificant level may nevertheless represent a
biologically or toxicologically significant event, and should be
afforded the same weight as if the finding were statistically
significant. In general, however, only statistically significant
measures should be considered in hazard identification. The power of
various statistical measures is also considered.
5.2.2. Dose-Response Assessment
In the second step of the risk assessment process, the dose-
response assessment, the relationship between the extent of damage or
toxicity and dose of a toxic substance for various conditions of
exposure is determined. Because several different kinds of responses
may be elicited by a single agent, more than one dose-response
relationship may need to be developed (e.g., neurochemical and
morphological parameters).
When quantitative human dose-effect data are not available for a
sufficient range of exposures, other methods must be used to estimate
exposure levels likely to produce adverse effects in humans. In the
absence of human data, the dose-response assessment may be based on
tests performed in laboratory animals. Evidence for a dose-response
relationship is an important criterion in assessing neurotoxicity,
although this may be based on limited data from standard studies that
often use only three dose groups and a control group (Barnes and
Dourson, 1988).
The most frequently used approach for risk assessment of
neurotoxicants and other noncancer endpoints is the uncertainty- or
safety-factor approach (Barnes and Dourson, 1988; Kimmel, 1990). For
example, within the EPA, this approach involves the determination of
reference doses (RfDs) by dividing a no observed adverse effect level
(NOAEL) by uncertainty factors that presumably account for interspecies
differences in sensitivity (Barnes and Dourson, 1988). Generally, an
uncertainty factor of 10 is used to allow for the potentially higher
sensitivity in humans than in animals and another uncertainty factor of
10 is used to allow for variability in sensitivity among humans. Hence,
the RfD is equal to the NOAEL divided by 100. If the NOAEL cannot be
established, it is replaced by the lowest observed adverse effect level
(LOAEL) in the RfD calculation and an additional uncertainty factor of
10 is introduced (i.e., the RfD equals the LOAEL divided by 1000).
If more than one effect is observed in the animal bioassays, the
effect occurring at the lowest dose in the most sensitive animal
species and gender is generally used as the basis for estimating the
RfD (OTA, 1990). Sometimes, different RfDs can be calculated, depending
on endpoint or species selected. Selection of safety factors may be
influenced by several considerations, including data available from
humans, weight of evidence, type of toxic insult, and probability of
variations in responses among susceptible populations (e.g., very young
or very old). Established guidelines have been accepted by several
agencies that use the safety-factor approach to account for
intraspecies variability, cross-species extrapolation, and exposure
duration. In some instances, comparisons between these predicted values
and experimental data have been conducted and the results appear
comparable for some selected examples (Dourson and Stara, 1983;
McMillan, 1987).
The uncertainty-factor approach is based on the assumption that a
threshold does exist, that there is a dose below which an effect does
not change in incidence or severity. The threshold concept is
complicated and controversial. As described by Sette and MacPhail
(1992), there are several different ways in which the term threshold is
used. Thresholds are defined, in part, by the limit of detection of an
assay. As the sensitivity of the analytical method or bioassay is
improved, the threshold might be adjusted downward, indicating that the
true threshold had not been previously determined.
Another problem inherent with an observation of no discernible
effects at low doses is that it is impossible to determine whether the
risk is actually zero (i.e., the dose is below a threshold dose) or
whether the statistical resolving power of a study is inadequate to
detect small risks (Gaylor and Slikker, 1992). Every study has a
statistical limit of detection that depends on the number of
individuals or animals involved. For example, it would be relatively
unusual to conduct an experiment on a neurotoxicant with as many as 100
animals per dose. If no deleterious effects were observed in 100
animals at a particular dose, it might be concluded that this dose
level is below the threshold dose. However, we can only be 95 percent
confident that the true risk is less than 0.03. That is, if 3 percent
of the animals in a population actually develop a toxic effect at this
dose, there is a 5 percent chance that a group of 100 animals would not
show any effect. The observation of no toxic effects in an extremely
large sample of 1,000 animals only indicates with 95 percent confidence
that the true risk is less than 0.003, etc. Because thresholds cannot
be realistically demonstrated, they are therefore assumed.
The notion of threshold may be useful in explaining mechanisms
associated with specific types of toxicity. What little is known about
mechanisms of neurotoxicity suggests that both threshold and
nonthreshold scenarios are possible (Silbergeld, 1990). However, for
one of the most studied neurotoxicants, lead, there has been a steady
decline in exposure levels shown to have effects, suggesting to some
that no threshold dose is apparent (Bondy, 1985). Sette and MacPhail
(1992) also consider the threshold as a mathematical assumption and as
a population sensitivity and conclude that ``the idea of no threshold
seems experimentally untestable. . . .''
The RfD approach relies on single experimental observations (the
NOAEL or LOAEL) instead of complete dose-response curve data to
calculate risk estimations. Chemical interactions with biological
systems are often specific, stereoselective, and saturable. Examples
include enzyme-substrate binding leading to substrate metabolism,
transport, and receptor-binding, any or all of which may be a
requirement of an agent's effect or toxicity. Therefore, a chemical's
dose-response curve may not be linear. The certainty of low-dose
extrapolation has been determined to be markedly affected by the shape
of the dose-response curve (Food and Drug Administration Advisory
Committee on Protocols for Safety Evaluation, 1971). Therefore, the
appropriate use of dose-response curve data should enhance the
certainty of risk estimations when thresholds are not assumed or
determined.
Dose-response models have generated considerable interest as more
appropriate and quantitative alternatives to the safety-factor approach
in risk assessment. Rather than routinely applying a ``fixed'' safety
factor to the NOAEL (based on a single dose) to obtain a ``safe'' dose,
another approach uses data from the entire dose-response curve.
Two fundamentally different approaches in the use of dose-response
data to estimate risk have been developed. Dews and coworkers (Dews,
1986; Glowa and Dews, 1987; Glowa et al., 1983) and Crump (1984)
demonstrated an approach in which they used information on the shape of
the dose-response curve to estimate levels of exposure associated with
relatively small effects (i.e., a 1, 5, or 10 percent change in a
biological endpoint). Both Dews and Crump fit a mathematical function
to the data and provided an estimate of the variability in exposure
levels associated with a relatively small effect.
An alternative approach developed by Gaylor and Slikker (1990)
first establishes a mathematical relationship between a biological
effect and the dose of a given chemical. The second step determines the
distribution (variability) of individual measurements of biological
effects about the dose-response curve. The third step statistically
defines an adverse or ``abnormal'' level of a biological effect in an
untreated population. The fourth step estimates the probability of an
adverse or abnormal level as a function of dose utilizing the
information from the first three steps. The advantages of these dose-
response models are that they encourage the generation and use of data
needed to define a complete dose-response curve.
Although more quantitative dose-response assessment models have
emerged in recent years, uncertainty remains as to what biological
endpoints from which species with what dosing regimen should be
analyzed. Within a species, a given agent may produce a variety of
effects, including neurochemical, neuropathological, and behavioral
effects. In other instances, a chemical may produce alterations of one
endpoint but not others (Slikker et al., 1989). Species selection may
also dramatically affect the outcome of risk assessments. The
Parkinson-like syndrome produced by single doses of MPTP in the human
or nonhuman primate is not observed in rats given comparable MPTP doses
(Kopin and Markey, 1988). Although endpoint and species selection
appear to have a tremendous effect on the outcome of an assessment,
only a few studies have systematically investigated the effect on
assessment outcome of varying either the species or the endpoint within
a species (McMillan, 1987; Hattis and Shapiro, 1990; Gaylor and
Slikker, 1992).
5.2.3. Exposure Assessment
This step of the risk assessment process determines the source,
route, dose, and duration of human exposure to an agent. The results of
the dose-response assessment are combined with an estimate of human
exposure to obtain a quantitative estimate of risk. As either the
effect of or the exposure to an agent approaches zero, the risk of
neurotoxicity approaches zero. It should be recognized that exposures
to multiple agents may produce synergistic or additive effects.
Exposure can occur via many routes, including ingestion,
inhalation, or contact with skin. Sources of exposure may include soil,
food, air, water, or intended vehicle (e.g., drug formulation). The
degree of exposure may be strongly influenced by a number of factors,
for example, the occupation of the individual involved.
The duration of exposure (i.e., acute or chronic) and interval of
exposure (i.e., episodic or continuous) are variables of exposure that
are common to all types of risk assessments, including carcinogenicity
(OSTP, 1985).
Although not routinely used, biological markers or biomarkers of
exposure could theoretically improve the exposure assessment process
and, thereby, improve the overall risk assessment of neurotoxicants.
Exposure biomarkers may include either the quantitation of exogenous
agents or the complex of endogenous substances and exogenous agents
within the system (Committee on Biological Markers, 1987). A limited
number of examples of biomarkers of exposure have been reviewed by
Slikker (1991) and include blood or dentine lead concentrations
(Needleman, 1987), cerebrospinal fluid concentrations of dopamine
metabolites following MPTP administration (Kopin and Markey, 1988),
cerebrospinal fluid concentrations of a serotonin metabolite following
MDMA exposure (Ricaurte et al., 1986), and serum esterase
concentrations following organophosphate exposure (Levine et al.,
1986). The use of muscarinic receptor binding in peripheral plasma
lymphocytes has also been described as a potential biomarker of
exposure for the organophosphates (Costa et al., 1990). These examples
suggest that biomarkers of exposure are available for some agents, but
more effort will be required to demonstrate that these biomarkers can
routinely be used to improve the exposure assessment process.
5.2.4. Risk Characterization
The final step of the risk assessment process combines the hazard
identification, the dose-response assessment, and the exposure
assessment to produce the characterization of risk. As previously
stated, the current practice is to divide the NOAEL by the appropriate
safety factor to obtain the RfD. The magnitudes of the safety factors
used to determine RfDs [interspecies extrapolation (10), intraspecies
extrapolation (10), and acute vs. chronic exposure (10) = 1000] are
based more on conservative estimates than on actual data (Sheehan et
al., 1989; McMillan, 1987) and have been questioned for empirical
reasons (Gaylor and Slikker, 1990). Uncertainty factors may be
decreased as more data become available. Modifying factors are also
employed under certain circumstances to account for the completeness of
data sets. Along with this RfD numerical value, any uncertainties and
assumptions inherent in the risk assessment should also be stated (OTA,
1990). Although the RfD provides a single numerical value, it does not
provide information concerning the uncertainty of this number nor does
the RfD approach attempt to estimate the potential risk as a function
of dose or consider the potential risk at the NOAEL. The risk at the
NOAEL generally is greater than zero and has been estimated to be as
high as about 5 percent (Crump, 1984; Gaylor, 1989). Concern has been
expressed that the application of the RfD approach to all
neurotoxicants is unlikely to be biologically defensible in light of
mechanistic data (NRC, 1992). Several other quantitative risk
assessment procedures have recently emerged as alternatives to the RfD
approach (Kimmel and Gaylor, 1988).
Quantitative risk assessment may be defined as a data-based process
that uses dose-response information and measurements of human exposure
to arrive at estimates of risk. Assumptions are required to extrapolate
results from high to low doses, to extrapolate from animal results to
humans, and to extrapolate across different routes and durations of
exposure.
In a step toward quantitative risk assessment, Crump (1984)
suggested the use of a benchmark dose defined as ``a statistical lower
confidence limit corresponding to a small increase in effect over the
background level.'' The benchmark dose is determined with a
mathematical model and is less affected by the particular shape of the
dose-response curve. Although the benchmark approach avoids several
problems inherent in the RfD approach (e.g., lack of precision in
defining the LOAEL; Kimmel, 1990), the same final step of dividing by
arbitrary safety factors is obligatory.
Another approach to quantitative risk assessment is the statistical
or curve-fitting approach. If quantal information concerning the
proportion of response at a given dose is available but mechanistic
information is lacking, statistical models can be used to fit
population data (Wyzga, 1990). This approach has been used to fit
various models to data of lead toxicity. The data were sufficient to
allow discrimination of several models in terms of goodness of fit; the
nerve-conduction velocity data from children exposed to environmental
lead as a function of blood lead concentration fit a ``hockey-stick''
type dose-response curve rather than a logistic or quadratic model
(Schwartz et al., 1988). These statistical approaches not only provide
a method to extrapolate data to lower exposure conditions but also can
provide circumstantial evidence to support a proposed mechanism of
action.
The development of quantitative risk assessment approaches depends,
in part, on the availability of information on the mechanism of action
and pharmacokinetics of the agent in question. In the development of a
biologically based, dose-response model for MDMA neurotoxicity, Slikker
and Gaylor (1990) considered several factors, including the
pharmacokinetics of the parent chemical, the target tissue
concentrations of the parent chemical or its bioactivated proximate
toxicant, the uptake kinetics of the parent chemical or metabolite into
the target cell and membrane interactions, and the interaction of the
chemical or metabolite with presumed receptor site(s). Because these
theoretical factors contain a saturable step due to limited amounts of
required enzyme, reuptake, or receptor site(s), a nonlinear, saturable
dose-response curve was predicted. In this case of neurochemical
effects of MDMA in the rodent, saturation mechanisms were hypothesized
and indeed saturation curves provided relatively good fits to the
experimental results. The conclusion was that use of dose-response
models based on plausible biological mechanisms provide more validity
to prediction than purely empirical models. Concomitant with attempts
to develop quantitative risk assessment procedures, it is imperative
that regulatory policy or risk management procedures also be developed
to use appropriately the type of data generated by quantitative risk
assessment. However, until alternative risk assessment procedures have
been validated, the available RfD approach with its limitations will
most likely continue to be used.

5.3. Generic Assumptions and Uncertainty Reduction

The purpose of risk assessment is to determine the risk associated
with human exposure to a hazard. The quality of the data from
toxicological studies differs. In the case of therapeutic products
where human effects information is available, risk assessments rely
primarily on the result of controlled clinical trials. Even when
clinical trial data are available, however, conducting a risk
assessment is complicated by many uncertainties. In the face of these
uncertainties, conservative assumptions are usually made at several
steps in the risk assessment process. For example, unless adequate
clinical data are available, the most sensitive experimental species is
frequently used. While conservative assumptions may lead to a risk
assessment that adequately protects the human population, this may
result in an increased financial burden on the public (e.g.,
manufacturing costs or loss of jobs); even then it is impossible to be
certain that the total population will be protected. Conversely, errors
leading to allowable exposure levels that are too high reduce the
safety margin for human health and increase health care costs. Thus,
there are compelling public health and economic reasons to obtain more
precise risk assessments; all assumptions cannot be completely
eliminated, but the degree of uncertainty associated with certain
specific assumptions can at least be reduced (Sheehan et al., 1989).
Risk assessment for neurotoxicity shares many common features with
other noncancer toxicities such as developmental toxicity and
immunotoxicity. As such, there are several generic assumptions that
apply to all traditional, noncancer endpoint risk assessment procedures
(Table 5-1).

Table 5-1.--General Assumptions That Underlie Traditional Risk
Assessments^{a,b

1. A threshold dose exists for noncancer endpoints.
2. NOAEL/LOAEL uncertainty- or safety-factor approaches are reasonable.
3. Variability in the toxic response to the chemical exposure is not due
to a heterogeneous population response.
4. Average dose or total dose is a reasonable measure of exposure when
doses are not equivalent in time, rate, or route of administration and
the average (or total) dose is proportional to adverse effect.
5. Structure-activity correlations can be used to predict human
toxicity.
6. The mechanism of action is the same at all doses for all species.
aThis is not intended to be an exhaustive list.
bModified from Sheehan et al., 1989.

One approach to reducing some of the uncertainties is to critically
define and examine the assumptions made in the risk assessment process.
Several of the more generic of these assumptions are listed in Table 5-
1. Despite their diversity, these assumptions share the attribute of
being partially replaceable by factual information. If, for example,
the assumption of 100 percent absorption of a toxicant from a
contaminated food source is replaced by data demonstrating that 90
percent of the toxicant is not biologically available under human
exposure conditions, then a revised risk assessment could allow a 10-
fold greater exposure from that source; i.e., the former risk
assessment was too conservative by a factor of 10. As another example,
many risk assessments employ data from two species.
If experimental animals and humans absorb or metabolize the same
fraction of a dose, the potency estimate would not change when
extrapolating from animals to humans. Therefore, it is necessary to
have information on both human and animal rates before changes in
potency estimates are made. If a toxicant acts via a reactive
intermediate and humans produce 10-fold more of the intermediate than
either of the test species under similar conditions, then allowable
human exposure should be decreased 10-fold (i.e., the allowable
exposure levels are 10-fold too high) or an increased danger to human
health exists. These findings could then replace the ``most sensitive
species'' principle with facts concerning relevant human exposure and
susceptibility. In these examples, the identification of the assumption
helps define research needs or knowledge gaps (Sheehan et al., 1989).
In general, the knowledge gaps are many and complex, but some can
be filled with practical solutions. The combination of ample dose-
response data and a quantitative risk assessment process can eliminate
assumptions 1 (existence of a threshold) and 2 (reasonableness of
safety factors) of the six generic assumptions (Table 5-1). The
uncertainty of assumption 4 (exposure comparisons) could be at least
reduced with the proper application of appropriate pharmacokinetic
data. Likewise, the uncertainty of generic assumption 3 (variability of
heterogeneous populations) can theoretically be reduced with the use of
biomarkers of exposure and biomarkers of effect, to more accurately
define the relationship between exposure and biological effect in a
large population.
Many assumptions remain, however, and uncertainty reduction by
filling knowledge gaps will ultimately require greater understanding of
biological mechanisms underlying neurotoxicity. A single risk
assessment model may not be adequate for all conditions of exposure,
for all endpoints, or for all agents. Risk assessment models of the
future may well include biomarkers of both effect and exposure as well
as biologically based mechanistic considerations derived from both
epidemiologic and experimental test system data.

6. General Summary

It is now generally accepted that some chemicals, including
industrial agents, pesticides, therapeutic agents, drugs of abuse,
food-related chemicals, and cosmetic ingredients, can have adverse
effects on the structure and function of the nervous system. It has
recently been proposed that exposure to neurotoxicants might also be
associated with Parkinsonism and Alzheimer's disease. Several Federal
agencies have initiated research programs in neurotoxicology, developed
neurotoxicology testing guidelines, and used neurotoxic endpoints to
regulate chemicals in the environment and workplace.
The scientific basis for identifying and characterizing chemical-
induced neurotoxicity has advanced rapidly during the last several
years. The manifestation of neurotoxicity depends on the relationship
between exposure (applied dose) and the dose at the site of toxic
action (delivered or target dose) and response. Chemical-induced
changes in the structure or function of the nervous system at the
cellular or molecular level can be observed as alterations in sensory,
motor, or cognitive function at the level of the whole organism.
Several important features about the nervous system make it
particularly vulnerable to chemical insult, including differential
susceptibilities at different stages of maturation, the presence of
blood brain and nerve barriers that may be the target of toxic action,
high metabolic rate, and limited regenerative capability following
damage.
Methods devised to detect and quantify agent-induced changes in
nervous system function in humans include clinical evaluations and
neurotoxicity testing methods such as neurobehavioral,
neurophysiological, neurochemical, imaging, and self-reporting
procedures. Experimental approaches used in human neurotoxicology
include epidemiological studies and, to a limited extent, human
laboratory exposure studies. There are several important unresolved
issues in human neurotoxicology, including the development of commonly
accepted risk assessment criteria and animal-to-human extrapolation.
It is generally assumed that if physical or chemical-induced
neurotoxicity is observed in animal models, then neurotoxicity will be
produced in humans. Considerable research has been performed to
demonstrate the validity of many animal models in an experimental
context and to show predictive validity. Methods in animal
neurotoxicology are frequently used in a tier-testing framework with
simpler, more cost-effective tests to screen or identify neurotoxic
potential. In hazard identification, the presence of neurotoxicity at
the first tier is used to make decisions about subsequent development
of a chemical or about the need to conduct additional experiments to
define the level at which neurotoxicity will be observed. A number of
methods have been devised for studies in animal neurotoxicology,
including neurobehavioral, neurophysiological, neurochemical, and
neuroanatomical techniques. It is known that the neuroendocrine system
may be affected adversely by neurotoxicants and that there are
populations that are differentially vulnerable to neurotoxic agents.
Considerable research is in progress to employ structure-activity
relationships to predict neurotoxicity and newly developed in vitro
procedures are being used to augment or complement currently existing
in vivo approaches.
Principles of risk assessment for neurotoxicity are evolving
rapidly. At the present time, neurotoxicity risk assessment is
generally limited to qualitative hazard identification.
Neurotoxicological risk assessments have been generally based on a no
observed adverse effect level and uncertainty factors. As with other
noncancer endpoints, there is a need to consider more information about
the shape of the dose-response curve and mechanisms of effect in
quantitative neurotoxicology risk assessment. Research is needed to
develop dose-response models that incorporate biologic information and
mechanistic hypotheses into quantitative extrapolation of dose-response
relationships across species and from high to low dose exposures.

7. References

Abou-Donia MB. Organophosphorus ester-induced delayed neurotoxicity.
Ann Rev Pharmacol Toxicol 1981; 21:511-548.
Abou-Donia MB. Toxicokinetics and metabolism of delayed neurotoxic
organophosphorous esters. Neurotoxicology 1983; 4:113-130.
Aibara K. Introduction to the Diagnosis of Mycotoxicosis. In:
Richards JL, Thurston JR, eds. Diagnosis of Mycotoxicoses.
Dordrecht, Netherlands: Martinus Nijhoff Publishers, 1986:3-8.
Altmann J, Schardt H, Wiegand H. Selective effects of acute repeated
solvent exposure on the visual system. Arbete och Halsa 1991; 35:67-
69.
Andersen ME, Clewell HJ, Gargas ML, MacNaughton MG, Reitz RH, Nolan
RJ, McKenna MJ. Physiologically based pharmacokinetic modeling with
dichloromethane, its metabolite, carbon monoxide, and blood
carboxyhemoglobin in rats and humans. Toxicol Appl Pharmacol 1991;
108:14-27.
Anderson-Brown T, Slotkin TA, Seidler FJ. Cocaine acutely inhibits
DNA synthesis in developing rat brain regions: evidence for direct
actions. Brain Res 1990; 537:197-202.
Anger WK. Neurobehavioral testing of chemicals; impact on
recommended standard. Neurobehav Toxicol Teratol 1984; 6:147-153.
Anger WK. Worksite behavioral research: results, sensitive methods,
test batteries and the transition from laboratory data to human
health. Neurotoxicology 1990a; 11:629-720.
Anger WK. Human neurobehavioral toxicology testing. In: Russell RW,
Flattau PE, Pope AM, eds. Behavioral Measures of Neurotoxicity.
Washington, DC: National Academy Press, 1990b.
Anger WK. Animal test systems to study behavioral dysfunctions of
neurodegenerative disorders. Neurotoxicology 1991; 12:403-414.
Anger WK, Setzer JV. Effects of oral and intramuscular carbaryl
administrations on repeated chain acquisition in monkeys. J Toxicol
Environ Health 1979; 5:793-808.
Annau Z, Eccles CU. Prenatal exposure. In: Annau A, ed.
Neurobehavioral Toxicology. Baltimore: Johns Hopkins University
Press, 1986:153-169.
Atterwill CK, Walum E. Neurotoxicology in vitro: model systems and
practical applications. Toxic In Vitro 1989; 3:159-161.
Bacon CW, Bennett RM, Hinton DM. Scanning electron microscopy of
Fusarium moniliforme within asymptomatic corn kernels and kernels
associated with leukoencephalomalacia. Plant Disease 1992; 76:144-
148.
Baker EL, Letz RE, Fidler AT, Shalat S, Plantamura D, Lyndon M. A
computer based neurobehavioral evaluation system for occupational
and environmental epidemiology: methodology and validation studies.
Neurobehav Toxicol Teratol 1985; 7:369-377.
Barnes DG, Dourson M. Reference dose (RfD): description and use in
health risk assessments. Regul Toxicol Pharmacol 1988; 8:471-486.
Beaumont JG. Neurobehavioral tests: problems, potential, and
prospects. In: Russell RW, Flatteau PE, Pope AM, eds. Behavioral
Measures of Neurotoxicity. Washington, DC: National Academy Press,
1990.
Bell, I.R., Millers, C. and Schwartz, G.E. An olfactory-limbic model
of multiple chemical sensitivity syndrome: Possible relationships to
kindling and affective spectrum disorders. Biol. Psychiat. 1992;
32:218-242.
Bellinger D, Leviton A, Waterneaux C, Needleman H, Rabinowitz M.
Longitudinal analyses of prenatal and postnatal lead exposure and
early cognitive development. N Engl J Med 1987; 316:1037-1043.
Benignus, V. Importance of experimenter-blind procedure in
neurotoxicology. Neurotoxicol. Teratol. 1993; 15:45-49.
Berde B, Schield HO. Ergot alkaloids and related compounds. In:
Handbook of Experimental Pharmacology. Berlin, Heidelberg, New York:
Springer-Verlag, 1978:49.
Bhattacharyya TK, Dayal VS. Ototoxicity and noise-drug interaction.
J Otolaryngol 1984; 13:361-366.
Bidstrup PL. Toxicity of Mercury and Its Compounds. Amsterdam:
Elsevier Scientific Publishing Company, 1964.
Boettcher FA, Henderson D, Gratton MA, Danielson RW, Byrne CD.
Synergistic interactions of noise and other ototraumatic agents. Ear
Hear 1987; 8:192-212.
Bondy SC. Special considerations for neurotoxicological research.
CRC Crit Rev Toxicol 1985; 14:381-402.
Bondy SC. The biochemical evaluation of neurotoxic damage. Fund Appl
Toxicol 1986; 6:208-216.
Bove FJ. The Story of Ergot. Basel, Switzerland: S. Karger AG, 1970.
Bowyer JF, Tank AW, Newport GD, Slikker W, Ali SF, Holson RR. The
influence of environmental temperature on the transient levels of
methamphetamine on dopamine levels and dopamine release in rat
striatum. J Pharmacol Exp Ther 1992, in press.
Boyce S, Kelly E, Reavill C, Jenner P, Marsen CD. Repeated
administration of N-methyl-4-phenyl-1,2,5,6-tetra-hydropyridine to
rats is not toxic to striatal dopamine neurons. Biochem Pharmacol
1984; 33:1747-1752.
Brady LS, Barrett JE. Drug-behavior interaction history:
modification of the effects of morphine on punished behavior. J Exp
Anal Behav 1986; 45:221-228.
Calne DB, Eisen A, McGeer E, Spencer PS. Alzheimer's disease,
Parkinson's disease, and motoneurone disease: a biotrophic
interaction between ageing and environment? Lancet 1986;
II(8515):1067-1070.
Cannon SB, Veazsy JM, Jackson RS, Burse VW, Hayes C, Straub WE,
Landrigan PJ, Liddle JA. Epidemic kepone poisoning in chemical
workers. Am J Epidemiol 1978; 107:529-537.
Campbell DT, Fiske DW. Convergent and discriminant validation by the
multitrain-multimethod matrix. Psychol Bull 1959; 56:81-91.
Cassells DAK, Dodds EC. Tetra-ethyl lead poisoning. Brit Med J 1946;
2:4479-4483.
Cassitto MG, Camerino D, Hanninen H, Anger WK. International
collaboration to evaluate the WHO neurobehavioral core test battery.
In: Johnson B, Anger WK, Durao A, Xintaras C, eds. Advances in
Neurobehavioral Toxicology: Applications in Environmental and
Occupational Health. Chelsea: Lewis Publishers, 1990.
Cavanagh JB. The toxic effects of tri-ortho-cresyl phosphate on the
nervous system: an experimental study on hens. J Neurol Neurosurg
Psychiat 1954; 17:163-172.
Centers for Disease Control (CDC). Preventing lead poisoning in
young children. U.S. Department of Health and Human Services,
Centers for Disease Control, Atlanta, GA, 1991.
Centers for Disease Control (CDC). Prevention of leading work-
related diseases and injuries. MMUR, 1986; 35(8).
Chang LW. Mercury. In: Spencer PS, Schaumburg HH, eds. Experimental
and Clinical Neurotoxicology. Baltimore: Williams and Wilkins,
1980:508-526.
Chiba S, Ando K. Effects of chronic administration of kanamycin on
conditioned suppression to auditory stimulus in rats. Jpn J
Pharmacol 1976; 26:41-426.
Choi DW. Glutamate neurotoxicity and diseases of the nervous system.
Neuron 1988; 1:623-634.
Committee on Biological Markers of the National Research Council.
Biological markers in environmental health research. Environ Health
Perspect 1987; 74:3-9.
Cone JE, Reeve GR, Landrigan PJ. Clinical and epidemiological
studies. In: Tardiff RG, Rodricks JV, eds. Toxic Substances and
Human Risk. New York: Plenum Press, 1987.
Cook DG, Fahn S, Brait KA. Chronic manganese intoxication. Arch
Neurol 1974; 30:59-71.
Cory-Slechta DA. Behavioral measures of neurotoxicity.
Neurotoxicology 1989; 10:271-296.
Cory-Slechta DA. Bridging experimental animal and human behavioral
toxicology studies. In: Russell RW, Flattau PE, Pope AM, eds.
Behavioral Measures of Neurotoxicity. Washington, DC: National
Academy Press, 1990.
Costa LG. Interactions of neurotoxicants with neurotransmitter
systems. Toxicology 1988; 49:359-366.
Costa LG, Kaylor G, Murphy SD. In vitro and in vivo modulation of
cholinergic muscarinic receptors in rat lymphocytes and brain by
cholinergic agents. Int J Immunopharmacol 1990; 8:1267-75.
Creason JP. Data evaluation and statistical analysis of functional
observational battery data using a linear models approach. J Am Coll
Toxicol 1989; 8:157-169.
Crofton KM. Reflex modification and the detection of toxicant-
induced auditory dysfunction. Neurotoxicol Teratol 1990; 12:461-468.
Crook T, Ferris S, Bartus R, eds. Assessment in Geriatric
Psychopharmacology. New Canaan, NY: Mark Powley Associates, 1983.
Crump KS. A new method for determining allowable daily intakes. Fund
Appl Toxicol 1984; 4:854-871.
Cushner IM. Maternal behavior and perinatal risks: alcohol, smoking,
and drugs. Ann Rev Pub Health 1981; 2:201-218.
Damstra T, Bondy SC. The current status and future of biochemical
assays for neurotoxicity. In: Spencer PS, Schaumburg HH, eds.
Experimental and Clinical Neurotoxicity. Baltimore: Williams and
Wilkins, 1980:820-833.
Davies PW. The action potential. In: Mountcastle VB, ed. Medical
Physiology, 12th edition. St. Louis: C.V. Mosby, 1968:1094-1120.
Davis CS, Richardson RJ. Organophosphorous compounds. In: Spencer
PS, Schaumburg HH, eds. Experimental and Clinical Neurotoxicology.
Baltimore: Williams and Wilkins, 1980:527-544.
Denny-Brown D, Dawson DM, Tyler HR. Handbook of Neurological
Examination and Case Recording (3rd ed). Cambridge: Harvard
University Press, 1982.
Dews PB. On the assessment of risk. In: Krasnegor N, Gray J,
Thompson T, eds. Developmental Behavioral Pharmacology. Hillsdale,
NJ: Lawrence Erlbaum Associates, 1986:53-65.
Dick, RB. Short duration exposure to organic solvents: the
relationship between neurobehavioral test results and other
indicators. Neurotoxicol. Teratol. 1988; 10:35-50.
Dick RB, Johnson BL. Human experimental studies. In: Annau Z, ed.
Neurobehavioral Toxicology. Baltimore: Johns Hopkins University
Press, 1986.
Dick RB, Bhattacharya A, Shukla R. Use of a computerized postural
sway measurement system for neurobehavioral toxicology. Neurotoxicol
Teratol 1990; 12:1-6.
Dick, RB, Krieg, EF, Setzer, J, Taylor, B. Neurobehavioral effects
from acute exposure to methyl isobutyl ketone and methyl ethyl
ketone. Fund. Appl. Toxicol. 1992; 19:453-473.
Domjan M, Burkhard B. The Principles of Learning and Memory.
Monterey, CA: Cole, 1986.
Dourson ML, Stara JF. Regulatory history and experimental support of
uncertainty (safety) factors. Regul Toxicol Pharmacol 1983; 3:224-
238.
Dyer RS. Macrophysiological assessment of organometal neurotoxicity.
In: Tilson H, Sparber SB, eds. Neurotoxicants and Neurobiological
Function. New York: Wiley, 1987:137-184.
Dyer RS, Howell WE. Triethyltin: ambient temperature alters visual
system toxicity. Neurobehav Toxicol Teratol 1982; 4:267-271.
Eccles CU. EEG correlates of neurotoxicity. Neurotoxicol Teratol
1988; 10:423-428.
Eckerman DA, Bushnell PJ. The neurotoxicology of cognition:
Attention, learning and memory. In: Tilson H, Mitchell CL, eds.
Neurotoxicology. New York: Raven Press, 1992:213-270.
Eckerman DA, Caroll JB, Foree D, Gullion CM, Lansman M, Long ER,
Waller MB, Wallsten TS. An approach to brief field testing for
neurotoxicity. Neurobehav Toxicol Teratol 1985; 7:387-393.
Ecobichon DJ, Joy RM. Pesticides and Neurologic Disease. Boca Raton:
CRC Press, 1982.
Fechter LD, Young JS. Discrimination of auditory from non-auditory
toxicity by reflex modification audiometry: effects of triethyltin.
Toxicol Appl Pharmacol 1983; 70:216-227.
Festing MFW. Genetic factors in neurotoxicology and
neuropharmacology: a critical evaluation of the use of genetics as a
research tool. Experientia 1991; 12:1877-1888.
Fisher F. Neurotoxicology and government regulation of chemicals in
the United States. In: Spencer PS, Schaumburg HH, eds. Experimental
and Clinical Neurotoxicology. Baltimore: Williams and Wilkins,
1980:874-882.
Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluation: Panel on carcinogenesis report on cancer testing
in the safety evaluation of food additives and pesticides. Toxicol
Appl Pharmacol 1971; 20:419-438.
Fowler SC. Force and duration of operant response and dependent
variables in behavioral pharmacology. In: Thompson T, Dews PB, eds.
Advances in Behavioral Pharmacology. New York: Erlbaum, 1987.
Freed DM, Kandel E. Long-term occupational exposure and the
diagnosis of dementia. Neurotoxicology 1988; 9:391-400.
Friedel RO. Pharmacokinetics in the geropsychiatric patient. In:
Lipton, M., Usdin, E., Killam, K., eds. Psychopharmacology: A
Generation of Progress. New York: Raven Press, 1978.
Friedlander BR, Hearn HT. Epidemiologic considerations in studying
neurotoxic disease. In: Spencer PS, Schaumburg HH, eds. Experimental
and Clinical Neurotoxicology. Baltimore: Williams and Wilkins,
1980:650-662.
Fullerton PM. Electrophysiological and histological observations on
peripheral nerves in acrylamide poisoning in man. J Neurol Neurosurg
Psychiat 1969; 32:186.
Gad SC. Principles of screening in toxicology with special emphasis
on applications to neurotoxicology. J Am Coll Toxicol 1989; 8:21-27.
Gallagher RT, Hawkes AD, Steyn PS, Vlleggaar R. Tremorgenic
neurotoxins from perennial ryegrass causing ryegrass staggers
disorders of livestock: structure and elucidation of Lolitrem. Chem
Soc Chem Comm 1984; 18:614-616.
Gaylor DW. Quantitative risk analysis for quantal reproductive and
developmental effects. Environ Health Perspect 1989; 79:243-246.
Gaylor DW, Slikker W Jr. Risk assessment for neurotoxic effects.
Neurotoxicology 1990; 11:211-218.
Gaylor D, Slikker W Jr. Risk assessment for neurotoxicants. In:
Tilson H, Mitchell C, eds. Neurotoxicology. New York: Raven Press,
1992:331-343.
Glowa JR, Dews PB. Behavioral toxicology of volatile organic
solvents. IV. Comparison of the behavioral effects of acetone,
methyl ethyl ketone, ethyl acetate, carbon disulfide, and toluene on
the responding of mice. J Am Coll Toxicol 1987; 6:461-469.
Glowa J, DeWeese J, Natale ME, Holland JJ. Behavioral toxicology of
volatile organic solvents. I. Methods: acute effects. J Am Coll
Toxicol 1983; 2:175-185.
Goldberg AM, Frazier JM. Alternatives to animals in toxicity
testing. Sci Amer 1989; 261:24-30.
Goldstein MK, Stein GH. Ambulatory activity in chronic disease. In:
Tryon WH, ed. Behavioral Assessment in Behavioral Medicine. New
York: Springer Publishing Co., 1985:160-162.
Griffin JW. Basic pathologic processes in the nervous system.
Toxicologic Pathology 1990; 18:83-88.
Hagstadius, S, Orbaek, P, Risberg, J, Lindgren, M. Regional cerebral
blood flow at the time of diagnosis of chronic toxic encephalopathy
induced by organic solvent exposure and after the cessation of
exposure. Scand. J. Work Environ. Health, 1989; 15:130-135.
Hammerschlag R, Brady S. Axonal transport and the neuronal
cytockeleton. In: Siegal GJ, Agranoff BW, Albers RW, Molinoff PB,
eds. Basic Neurochemistry. New York: Raven Press, 1989:457-478.
Hanninen H. Methods in behavioral toxicology: current test batteries
and need for development. In: Russell RW, Flattau PE, Pope AM, eds.
Behavioral Measures of Neurotoxicity. Washington, DC: National
Academy Press, 1990.
Hanninen H, Mantere P, Hernberg S, Seppalainen AM, Kock B.
Subjective symptoms in low-level exposure to lead. Neurotoxicology
1979; 1:333-347.
Hattis D, Shapiro K. Analysis of dose/time/response relationships
for chronic toxic effects: the case of acrylamide. Neurotoxicology
1990; 11:219-236.
HAZDAT. ATSDR Database. Agency for Toxic Substances and Disease
Registry, Atlanta, GA, 1992.
Heikkila RE, Hess A, Duvoisin RC. Dopaminergic neurotoxicity of 1-
methyl-4-phenyl-1,2,5,6-tetrahydropyridine in mice. Science 1984;
224:1451-1453.
Heise GA. Behavioral methods for measuring effects of drugs on
learning and memory in animals. Med Res Rev 1984; 4:535-558.
Hill RM, Tennyson LM. Maternal drug therapy: effect on fetal and
neonatal growth and neurobehavior. Neurotoxicology 1986; 7:121-140.
Hjelm, EW, Hagberg, M, Iregren, A, Lof, A. Exposure to methyl
isobutyl ketone: toxicants and occurrence of irritative and CNS
symptoms in man. Int. Arch. Occup. Environ. Health, 1990; 62:19-26.
Hooisma J, Emmen HH, Kulig BM, Muijser H, Poortvliet D, Letz R.
Factor analysis of tests from the neurobehavioral evaluation system
and the WHO neurobehavioral core test battery. In: Johnson B, Anger
WK, Durao A, Xintaras C, eds. Advances in Neurobehavioral
Toxicology: Applications in Environmental and Occupational Health.
Chelsea: Lewis Publishers, 1990.
Horvath M, Frantik E. Quantitative interpretation of experimental
toxicological data: the use of reference substances. In: Horvath M,
ed. Adverse Effects of Environmental Chemicals and Psychotropic
Drugs. Amsterdam: Elsevier, 1973:11-21.
Hruska RE, Kennedy S, Silbergeld EK. Quantitative aspects of normal
locomotion in rats. Life Sci 1979; 25:171-179.
Hutchings DE, Fico TA, Dow-Edwards DL. Prenatal cocaine: maternal
toxicity, fetal effects and locomotor activity of rat offspring.
Neurotoxicol Teratol 1989; 11:65-69.
Iregren A, Gamberale F, Kjellberg A. A microcomputer-based
behavioral testing system. In: Neurobehavioral Methods in
Occupational and Environmental Health. Copenhagen: World Health
Organization, 1985.
Joffe JM, Soyka LF. Effects of drug exposure on male reproductive
processes and progeny. Period Biol 1981; 83:351-362.
Johnson MK. Organophosphates and delayed neuropathy--Is NTE alive
and well? Toxicol Appl Pharmacol 1990; 102:385-399.
Johnson BL. Prevention of neurotoxic illness in working populations.
New York: Wiley, 1987.
Johnson BL, Boyd J, Burg JR, Lee ST, Xintaras C, Albright BE.
Effects on the peripheral nervous system of workers exposed to
carbon disulfide. Neurotoxicology 1983; 4:53-66.
Jones KL, Smith DW. Recognition of the fetal alcohol syndrome in
early infancy. Lancet 1973; 2:999-1001.
Katzman R. Blood-brain-CSF barriers. In: Siegal GJ, ed. Basic
Neurochemistry. Boston: Little, Brown and Company, 1976:414-428.
Kimmel CA. Current status of behavioral teratology: science and
regulation. CRC Critical Reviews in Toxicology 1988; 19:1-10.
Kimmel CA. Quantitative approaches to human risk assessment for
noncancer health effects. Neurotoxicology 1990; 11:189-198.
Kimmel CA, Gaylor DW. Issues in qualitative and quantitative risk
analysis for developmental toxicology. Risk Analysis 1988; 8:15-20.
Kimmel CA, Rees DC, Francis EZ. Qualitative and quantitative
comparability of human and animal developmental neurotoxicology.
Teratology 1990; 12:175-292.
Klaassen CD. Absorption, distribution, and excretion of toxicants.
In: Doull J, Klaassen CD, Amdur M, eds. Toxicology: The Basic
Science of Poisonings. New York: MacMillan, 1980:28-55.
Kopin IJ, Markey SP. MPTP toxicity: implications for research in
Parkinson's disease. Ann Rev Neurosci 1988; 11:91-96.
Krinke GJ. Neuropathologic screening in rodent and other species. J
Am Coll Toxicol 1989; 8:141-155.
Kurata H. Mycotoxins and mycotoxicoses: overview. In: Pohland AE,
Dowell VR Jr, Richards JL, eds. Microbial Toxins in Foods and Feeds,
Cellular and Molecular Modes of Action. New York: Plenum Press,
1990:249-259.
Langston JW, Ballard P, Tetrud JW, Irwin I. Chronic parkinsonism in
humans due to a product of meperidine-analog synthesis. Science
1983; 219:979-980.
Last JM. Epidemiology and health information. In: Last JM, ed.
Maxcy-Rosenau Public Health and Preventive Medicine. New York:
Appleton-Century-Crofts, 1986.
Letz R. The neurobehavioral evaluation system: an international
effort. In: Johnson B, Anger WK, Durao A, Xintaras C, eds. Advances
in Neurobehavioral Toxicology: Applications in Environmental and
Occupational Health. Chelsea: Lewis Publishers, 1990.
Levine MS, Fox NL, Thompson B, Taylor W, Darlington AC, Van Der
Hoeden J, Emmett EA, Rutten W. Inhibition of esterase activity and
an undercounting of circulating monocytes in a population of
production workers. J Occup Med 1986; 28:207-211.
Liang Y-X, Chen Z-Q, Sun R-K, Fan Y-F, Yu J-H. Application of the
WHO neurobehavioral core test battery and other neurobehavioral
screening methods. In: Johnson B, Anger WK, Durao A, Xintaras C,
eds. Advances in Neurobehavioral Toxicology: Applications in
Environmental and Occupational Health. Chelsea: Lewis Publishers,
1990.
Lim DJ. Effects of noise and ototoxic drugs at the cellular level in
the cochlea: a review. Am J Otolaryngol 1986; 7:73-99.
Little RE, Anderson KW, Ervin CH, Worthington-Roberts B, Clarren SK.
Maternal alcohol use during breast-feeding and infant mental and
motor development at one year. N Engl J Med 1989; 321:425-430.
Lowndes HE, Baker T. Toxic site of action in distal axonopathies.
In: Spencer PS, Schaumburg HH, eds. Experimental and Clinical
Neurotoxicology. Baltimore: Williams and Wilkins, 1980:193-205.
MacPhail RC. Effects of pesticides on schedule-controlled behavior.
In: Seiden LS, Balster RL, eds. Behavioral Pharmacology: The Current
Status. New York: Alan R. Liss, 1985:519-535.
MacPhail RC. Environmental modulation of neurotoxicity. In: Russell
RW, Flattau PE, Pope AM, eds. Behavioral Measures of Neurotoxicity.
Washington, DC: National Academy Press, 1990.
MacPhail RC, Crofton KM, Reiter LW. Use of environmental challenges
in behavioral toxicology. Fed Proc 1983; 42:3196-3200.
MacPhail RC, Peele DB, Crofton KM. Motor activity and screening for
neurotoxicity. J. Am Coll Toxicol 1989; 8:117-125.
Mailman RB. Mechanisms of CNS injury in behavioral dysfunction.
Neurotoxicol Teratol 1987; 9:417-426.
Matthews HB, Dixon D, Herr DW, Tilson HA. Subchronic toxicity
studies indicate that tris(2-chloroethyl)phosphate administration
results in lesions in the rat hippocampus. Toxicol Indust Health
1990; 6:1-15.
Mattsson JL, Albee RR. Sensory evoked potentials in neurotoxicology.
Neurotoxicol Teratol 1988; 10:435-443.
Maurissen JP. Quantitative sensory assessment in toxicology and
occupational medicine: applications, theory and critical appraisal.
Toxicol Lett 1988; 43:321-343.
Maurissen JP, Weiss B, Davis HT. Somatosensory thresholds in monkeys
exposed to acrylamide. Toxicol Appl Pharmacol 1983; 71:266-279.
Mausner JS, Kramer S. Epidemiology--An Introductory Text (2nd ed).
Philadelphia: W.B. Saunders Co., 1985.
McMillan DE. Risk assessment for neurobehavioral toxicity. Environ
Health Perspect 1987; 76:155-161.
Merigan WH. Effects of toxicants on visual systems. Neurobehav
Toxicol 1979; 1 (Suppl.):15-22.
Morell P, Mailman RB. Selective and nonselective effects of
organometals on brain neurochemistry. In: Tilson HA, Sparber SB,
eds. Neurotoxicants and Neurobiological Function: Effects of
Organoheavy Metals. New York: Wiley, 1987:201-230.
Moser VC. Screening approaches to neurotoxicity: a functional
observational battery. J Am Coll Toxicol 1989; 8:85-93.
Moser VC. Approaches for assessing the validity of a functional
observational battery. Neurotoxicol Teratol 1990; 12:483-488.
Moser VC, McDaniel KL, Phillips PM. Rat strain and stock comparisons
using a functional observational battery: baseline values and
effects of amitraz. Toxicol Appl Pharmacol 1991; 108:267-283.
National Institute for Occupational Safety and Health (NIOSH). NIOSH
Recommendations for Occupational Safety and Health: Compendium of
Policy Documents and Statement. DHHS Pub. No. 92-100, 1992.
National Research Council, Committee on Toxicology. Effects on
behavior. In: Principles for Evaluating Chemicals in the
Environment. Washington, DC: National Academy of Sciences, 1975.
National Research Council (NRC). Risk Assessment in the Federal
Government. Washington, DC: National Academy Press, 1983.
National Research Council (NRC). Toxicity Testing: Strategies to
Determine Needs and Priorities. Washington, DC: National Academy of
Sciences, 1984.
National Research Council (NRC). Environmental Neurotoxicology.
Washington, DC: National Academy Press, 1992.
Needleman HL. Introduction: biomarkers in neurodevelopmental
toxicology. Environ Health Perspect 1987; 74:149-152.
Needleman HL. Lessons from the history of childhood blumbism for
pediatric neurotoxicology. In: Johnson BL, Anger WK, Durano A,
Xintaras C, eds. Advances in Neurobehavioral Toxicology: Application
in Experimental and Occupational Health. Chelsea: Lewis Publishers,
1990:331-337.
Needleman HL, Gunnoe CE, Leviton A. Deficits in psychologic and
classroom performance of children with elevated blood lead levels. N
Engl J Med 1979; 300:689-695.
Nelson BK. Evidence for behavioral teratogenicity in humans. J Appl
Toxicol 1991a; 11:33-37.
Nelson BK. Selecting exposure parameters in developmental
neurotoxicity assessments. Neurotoxicol Teratol 1991b; 13:569-573.
Newland MC. Quantification of motor function in toxicology. Toxicol
Lett 1988; 43:295-319.
O'Callaghan JP. Neurotypic and gliotypic proteins as biochemical
markers of neurotoxicity. Neurotoxicol Teratol 1988; 10:445-452.
O'Donoghue JL. Screening for neurotoxicity using a neurologically
based examination and neuropathology. J Am Coll Toxicol 1989; 8:97-
116.
Office of Science and Technology Policy (OST). Chemical carcinogens:
a review of the science and its associated principles. Federal
Register 1985:1-85.
Office of Technology Assessment (OTA). Neurotoxicity: identifying
and controlling poisons of the nervous system. U.S. Congress. Office
of Technology Assessment (OTA-BA-436), Washington, DC: U.S.
Government Printing Office, 1990.
Otto DA, Hudnell HK. Electrophysiological systems for neurotoxicity
field testing: PEARL II and alternatives. In: Johnson B, Anger WK,
Durao A, Xintaras C, eds. Advances in Neurobehavioral Toxicology:
Applications in Environmental and Occupational Health. Chelsea:
Lewis Publishers, 1990.
Otto DA, Hudnell HK. Problems in studying low level solvent
mixtures. Arbete och Halsa 1991; 35:35-38.
Overstreet DH. Pharmacological approaches to habituation of the
acoustic startle response in rats. Physiol Psychol 1977; 5:230-238.
Pardridge WM. Recent advances in blood-barrier transport. Ann Rev
Pharmacol Toxicol 1988; 28:25-39.
Paule MG, Forrester TM, Maher MA, Cranmer JM, Allen RR. Monkey
versus human performance in the NCTR operant test battery.
Neurotoxicol Teratol 1990; 12:503-507.
Paule MG, McMillan DE. Incremental repeated acquisition in the rat:
acute effects of drugs. Pharmacol Biochem Behav 1984; 21:431-439.
Pearson DT, Dietrich KN. The behavioral toxicology and teratology of
childhood: models, methods, and implications for intervention.
Neurotoxicology 1985; 6:165-182.
Peele DB, Vincent A. Strategies for assessing learning and memory,
1978-1987: a comparison of behavioral toxicology, psychopharmacology
and neurobiology. Neurosci Biobehav Rev 1989; 13:33-38.
Perl TM, Bedard L, Kosatsky T, Hockin JC, Todd ECD, Remis RS. An
outbreak of toxic encephalopathy caused by eating mussels
contaminanted with domoic acid. N Engl J Med 1990; 322:1775-1780.
Peters, A, Palay, SL, Webster, H. def. The Fine Structure of the
Nervous System. Neurons and Their Supporting Cells, 3rd Ed. New
York: Oxford University Press, 1991.
Piikivi L, Tolonen U. EEG findings in chloralkali workers subjected
to low long term exposure to mercury vapor. Br J Ind Med 1989;
46:30-35.
Politis MJ, Schaumburg HH, Spencer PS. Neurotoxicity of selected
chemicals. In: Spencer PS, Schaumburg HH, eds. Experimental and
Clinical Neurotoxicology. Baltimore: Williams and Wilkins, 1980:
613-630.
Pryor G, Dickinson J, Howd RA, Rebert CS. Transient cognitive
deficits and high- frequency hearing loss in weanling rats exposed
to toluene. Neurobehav Toxicol Teratol 1983; 5:53-57.
Putz VR, Johnson BL, Setzer JV. A comparative study of the effects
of carbon monoxide and methylene chloride on human performance. J
Environ Path Toxicol 1979; 2:97-112.
Raine CS. Neurocellular anatomy. In: Siegal GJ, Agranoff BW, Albers
RW, Molinoff PB, eds. Basic Neurochemistry. New York: Raven Press,
1989:3-34.
Rao BL, Husain A. Presence of cyclopiazonic acid in Kodo millet
(Paspalum scrobiculatum) causing 'Kodua poisoning' in man and its
production by associated fungi. Mycopathologia 1985; 89:177-180.
Rebert CS. Multisensory evoked potentials in experimental and
applied neurotoxicology. Neurobehav Toxicol Teratol 1983; 5:659-671.
Reiter LW. Neurotoxicology in regulatory and risk assessment.
Develop Pharmacol Therap 1987; 10:354-368.
Ricaurte GA, Delanney LE, Weiner SG, Irwin I, Langston JW. 5-
Hydroxyindoleacetic acid in cerebrospinal fluid reflects
serotonergic damage induced by 3,4- methylenedioxymethamphetamine in
CNS of nonhuman primates. Brain Res 1986; 474:359-363.
Rice DC. Quantification of operant behavior. Toxicol Lett 1988;
43:361-379.
Riley AL, Tuck DL. Conditioned taste aversions: a behavioral index
of toxicity. Ann NY Acad Sci 1985; 443:272-292.
Riley EP, Vorhees CV, eds. Handbook of Behavioral Teratology. New
York: Plenum Press, 1986.
Rodier PM. Time of exposure and time of testing in developmental
neurotoxicology. Neurotoxicology 1986; 7:69-76.
Rodier P. Developmental neurotoxicology. Toxicologic Pathol 1990;
18:89-95.
Russell RW. Essential roles for animal models in understanding human
toxicities. Neurosci Biobehav Rev 1991; 15:7-11.
Ryan CM, Morrow LA, Bromet EJ, Parkinson DK. Assessment of
neuropsychological dysfunction in the workplace: normative data from
the Pittsburgh Occupational Exposures Test Battery. J Clin Exp
Neuropsychol 1987; 9:665-679.
Salzman C. Antianxiety agents. In: Crook T, Cohen G, eds.
Physicians' Handbook on Psychotherapeutic Drug Use in the Aged. New
Canaan, NY: Mark Powley Associates, 1981.
Schrot J, Thomas JR, Robertson RF. Temporal changes in repeated
acquisition behavior after carbon monoxide exposure. Neurobehav
Toxicol Teratol 1984; 6:23-28.
Schwartz J, Landrigan PF, Feldman RG, Silbergeld EK, Baker EL, Von
Lindern IH. Threshold effect in lead-induced peripheral neuropathy.
J Pediatr 1988; 11:212-217.
Seppalainen AM, Haltia M. Carbon disulfide. In: Spencer PS,
Schaumburg HH, eds. Experimental and Clinical Neurotoxicology.
Baltimore: Williams and Wilkins, 1980:356-373.
Seppalainen AM, Harkonen H. Neurophysiological findings among
workers occupationally exposed to styrene. Scand J Work Environ
Health 1976; 2:140.
Sette WF, MacPhail RC. Qualitative and quantitative issues in
assessment of neurotoxic effects. In: Tilson H, Mitchell C, eds.
Neurotoxicology. New York: Raven Press, 1992:345-361.
Sheehan DM, Young JF, Slikker W Jr, Gaylor DW, Mattison DR. Workshop
on risk assessment in reproductive and developmental toxicology:
addressing the assumptions and identifying the research needs.
Regulat Toxicol Pharmacol 1989; 10:110-122.
Silbergeld EK. Maternally mediated exposure of the fetus: in utero
exposure to lead and other toxins. Neurotoxicology 1986; 7:557-568.
Silbergeld EK. Developing formal risk assessment methods for
neurotoxicants: an evaluation of the state of the art. In: Johnson
BL, Anger WK, Durao A, Xintaras C, eds. Advances in Neurobehavioral
Toxicology: Applications in Environmental and Occupational Health.
Chelsea: Lewis, 1990:133-148.
Sjogren B, Gustavsson P, Hogstedt C. Neuropsychiatric symptoms among
welders exposed to neurotoxic metals. Brit J Indust Med 1990;
47:704-707.
Slikker W Jr. Biomarkers of neurotoxicity: an overview. Recent
advances on biomarker research. Biomed Environ Sci 1991; 4:192-196.
Slikker W Jr, Gaylor DW. Biologically based dose-response model for
neurotoxicity risk assessment. Korean J Toxicol 1990; 6:204-213.
Slikker W Jr, Holson RR, Ali SF, Kolta MG, Paule MG, Scallet AC,
McMillan DE, Bailey JR, Hong JS, Scalzo FM. Behavioral and
neurochemical effects of orally administered MDMA in the rodent and
nonhuman primate. Neurotoxicology 1989; 10:529-542.
Smith MA, Grant LD, Sors AI, eds. Lead Exposure and Child
Development: An International Assessment. Dordrecht, Netherlands:
Kluwer Academic Publishers, 1989.
Solomon PR, Pendlebury WW. A model systems approach to age-related
memory disorders. Neurotoxicology 1988; 9:443-462.
Spencer PS, Schaumburg HH, eds. Experimental and Clinical
Neurotoxicology. Baltimore: Williams and Wilkins Co, 1980.
Spencer PS, Nunn PB, Hugon J, Ludolph AC, Ross SM, Roy DN, Robertson
RC. Guam amyotrophic lateral sclerosis-Parkinsonism-dementia linked
to a plant excitant neurotoxin. Science 1987; 237:517-522.
Squibb, RE, Tilson, HA, Meyer, OA, Lamartiniere, CA. Neonatal
exposure to monosodium glutamate alter the neurobehavioral
performance of adult rats. Neurotoxicology, 1991; 2:471-484.
Stanton ME, Spear LP. Comparability of endpoints across species in
developmental neurotoxicity. Neurotoxicol Teratol 1990; 12:261-268.
Stebbins WC, Coombs C. Behavioral assessment of ototoxicity in
nonhuman primates. In: Weiss B, Laties VG, eds. Behavioral
Toxicology. New York: Plenum Press, 1975.
Sterman AB, Schaumburg HH. Neurotoxicity of selected drugs. In:
Spencer PS, Schaumburg HH, eds. Experimental and Clinical
Neurotoxicology. Baltimore: Williams and Wilkins, 1980:593-612.
Stewart RD, Fisher TN, Hosko R. Experimental human exposure to
methylene chloride. Arch Environ Health 1972; 25:342-348.
Strong MJ, Garruto RM. Potentiation in the neurotoxic induction of
experimental chronic neurodegenerative disorders: n-butyl
benzenesulfonamide and aluminum chloride. Neurotoxicology 1991;
12:415-426.
Suzuki K. Special vulnerabilities of the developing nervous system
to toxic substances. In: Spencer PS, Schaumburg HH, eds.
Experimental and Clinical Neurotoxicology. Baltimore: Williams and
Wilkins, 1980:48-61.
Terrace HS. Errorless discrimination learning in the pigeon: Effects
of chlorpromazine and imipramine. Science 1963; 140:318-319.
Thompson FN Jr, Porter JK. Tall fescue toxicosis in cattle: could
there be a public health problem. Vet Human Toxicol 1990; 32:51-57.
Tilson HA, Cabe PA. Strategy for the assessment of neurobehavioral
consequences of environmental factors. Environ Health Perspect 1978;
26:287-299.
Tilson HA. Practical considerations in establishing valid and
sensitive neurobehavioral test methods. Zbl Bakt Hyg B 1987; 185:10-
15.
Tilson HA. Screening for neurotoxicity: principles and practices. J
Am Coll Toxicol 1989; 8:13-17.
Tilson HA. Behavioral indices of neurotoxicity. Toxicologic
Pathology 1990a; 18:96-104.
Tilson HA. Neurotoxicology in the 1990s. Neurotoxicol Teratol 1990b;
12:293-300.
Tilson HA, Mitchell CL. Neurotoxicants and adaptive responses of the
nervous system. Fed Proc 1983; 42:3189-3190.
Tilson HA, Mitchell CL. Neurobehavioral techniques to assess the
effects of chemicals on the nervous system. Ann Rev Pharmacol
Toxicol 1984; 24:25-50.
Tilson HA, Moser VC. Comparison of screening approaches.
Neurotoxicology 1992; 13:1-14.
United States Environmental Protection Agency (U.S. EPA).
Neurotoxicity Testing Guidelines. Springfield, VA: National
Technical Information Service, 1991.
Valciukas JA. Foundations of Environmental and Occupational
Neurotoxicology. New York: Van Nostrand Reinhold, 1991.
Veronesi B. The use of cell culture for evaluating neurotoxicity.
In: Tilson HA, Mitchell CL, eds. New York: Raven Press, 1991:21-50.
Veronesi B, Jones K, Pope C. The neurotoxicity of subchronic
acetylcholinesterase (AChE) inhibition in rat hippocampus. Toxicol
Appl Pharmacol 1990; 104:440-456.
Verity MA, Sarafian TS, Guerra W, Ettinger A, Sharp J. Ionic
modulation of triethyllead neurotoxicity in cerebellar granule cell
culture. Neurotoxicology 1990; 11:415-426.
Vorhees CV. Reliability, sensitivity and validity of indices of
neurotoxicity. Neurotoxicol Teratol 1987; 9:445-464.
Waddell, WJ. The science of toxicology and its relevance to MC5.
Reg. Toxicol. Pharmacol. 1993; 18:13-22.
Walker CH, Faustman WO, Fowler SC, Kazar DB. A multivariate analysis
of some operant variables used in behavioral pharmacology.
Psychopharmacology 1981; 74:182-186.
Weiss B. Risk assessment: The insidious nature of neurotoxicity and
the aging basis. Neurotoxicology 1990; 11:305-313.
Williamson AM. The influence of subject characteristics on the
identification of acute effects of solvent exposure. Arbete och
Halsa 1991; 35:14-18.
Willis WD Jr, Grossman RG. Neurotransmission. In: Medical
Neurobiology. St. Louis: C.V. Mosby, 1973:457.
World Health Organization (WHO). Principles and Methods for the
Assessment of Neurotoxicity Associated with Exposure to Chemicals.
Environmental Health Criteria Document 60. Geneva: World Health
Organization, 1986.
Wyllie TD, Morehouse LC. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses,
An Encyclopedic Handbook. New York: Marcel Dekker, Inc., 1978, Vols.
1-3.
Wyzga R. Towards quantitative risk assessment for neurotoxicity.
Neurotoxicology 1990; 11:199-208.
Yokel RA. Repeated systemic aluminum exposure effects on classical
conditioning of the rabbit. Neurobehav Toxicol Teratol 1983; 5:41-
46.
Yokel RA, Provan SD, Meyer JJ, Campbell SR. Aluminum intoxication
and the victim of Alzheimer's disease: similarities and differences.
Neurotoxicology 1988; 9:429-442.
Zenick H. Use of pharmacological challenge to disclose
neurobehavioral deficits. Fed Proc 1983; 42:3191-3195

Prepared by

Working Party on Neurotoxicology
Subcommittee on Risk Assessment
Federal Coordinating Council on Science, Engineering, and Technology

Lawrence W. Reiter, USEPA, Chair
Hugh A. Tilson, USEPA, Executive Secretary
John Dougherty, NIOSH
G. Jean Harry, NIEHS
Carol J. Jones, OSHA
Suzanne McMaster, USEPA
William Slikker, NCTR/FDA
Thomas J. Sobotka, FDA

Ad Hoc Interagency Committee on Neurotoxicology

William Boyes, U.S. Environmental Protection Agency
Joy Cavagnaro, Food and Drug Administration
Selene Chou, Agency for Toxic Substances and Disease Registry
Murray Cohn, U.S. Consumer Product Safety Commission
Joseph F. Contrera, Food and Drug Administration
Miriam Davis, National Institute of Environmental Health Sciences,
National Institutes of Health
Joseph DeGeorge, Food and Drug Administration
Robert Dick, National Institute for Occupational Safety and Health
John Dougherty, National Institute for Occupational Safety and
Health
Lynda Erinoff, National Institute on Drug Abuse
Joseph P. Hanig, Food and Drug Administration
G. Jean Harry, National Institute of Environmental Health Sciences
David G. Hattan, Food and Drug Administration
Norman A. Krasnegor, National Institutes of Health
Robert C. MacPhail, U.S. Environmental Protection Agency
Suzanne McMaster, U.S. Environmental Protection Agency
Lakshmi C. Mishra, U.S. Consumer Product Safety Commission
Andres Negro-Vilar, National Institute of Environmental Health
Sciences
James K. Porter, U.S. Department of Agriculture/Agricultural
Research Service
Lawrence W. Reiter, U.S. Environmental Protection Agency
Jane Robens, U.S. Department of Agriculture/Agricultural Research
Service
Barry Rosloff, Food and Drug Administration
Harry Salem, U.S. Army Chemical Research, Development, and
Engineering Center
Bernard A. Schwetz, National Institute of Environmental Health
Sciences
William F. Sette, U.S. Environmental Protection Agency
William Slikker, Jr., National Center for Toxicological Research
D. Stephen Snyder, National Institute on Aging
Thomas J. Sobotka, Food and Drug Administration
Hugh A. Tilson, U.S. Environmental Protection Agency
Mildred Williams-Johnson, Agency for Toxic Substances and Disease
Registry

[FR Doc. 94-20033 Filed 8-16-94; 8:45 am]
BILLING CODE 6560-50-P}}}