# Guidelines for Neurotoxicity Risk Assessment

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URL: https://www.frixlaw.com/law-library/documents/fr%3A98-12303

## Record

- **Collection:** Federal Register
- **Document type:** Notice
- **Published:** May 14, 1998
- **Citation:** 63 FR 26926

## Text

SUMMARY: The U.S. Environmental Protection Agency (EPA) is today
publishing in final form a document entitled Guidelines for
Neurotoxicity Risk Assessment (hereafter ``Guidelines''). These
Guidelines were developed as part of an interoffice guidelines
development program by a Technical Panel of the Risk Assessment Forum.
The Panel was composed of scientists from throughout the Agency, and
selected drafts were peer-reviewed internally and by experts from
universities, environmental groups, industry, and other governmental
agencies. The Guidelines are based, in part, on recommendations derived
from various scientific meetings and workshops on neurotoxicology, from
public comments, and from recommendations of the Science Advisory
Board. An earlier draft underwent external peer review in a workshop
held on June 2-3, 1992, and received internal review by the Risk
Assessment Forum. The Risk Assessment Subcommittee of the Committee on
the Environment and Natural Resources of Office of Science and
Technology Policy reviewed the proposed Guidelines during a meeting
held on August 15, 1995. The Guidelines were revised and proposed for
public comment on October 4, 1995 (60 FR 52032-52056). The proposed
Guidelines were reviewed by the Science Advisory Board on July 18,
1996. EPA appreciates the efforts of all participants in the process,
and has tried to address their recommendations in these Guidelines.
This notice describes the scientific basis for concern about
exposure to agents that cause neurotoxicity, outlines the general
process for assessing potential risk to humans because of environmental
contaminants, and addresses Science Advisory Board and public comments
on the 1995 Proposed Guidelines for Neurotoxicity Risk Assessment (60
FR:52032-52056). These Guidelines are intended to guide Agency
evaluation of agents that are suspected to cause neurotoxicity, in line
with the policies and procedures established in the statutes
administered by the Agency.

DATES: The Guidelines will be effective on April 30, 1998.

ADDRESSES: The Guidelines will be made available in several ways:
(1) The electronic version will be accessible from EPA's National
Center for Environmental Assessment home page on the Internet at http:/
/www.epa.gov/ncea.
(2) 3\1/2\'' high-density computer diskettes in WordPerfect format
will be available from ORD Publications, Technology Transfer and
Support Division, National Risk Management Research Laboratory,
Cincinnati, OH; Tel: 513-569-7562; Fax: 513-569-7566. Please provide
the EPA No.: EPA/630/R-95/001Fa when ordering.
(3) This notice contains the full document. Copies of the
Guidelines will be available for inspection at EPA headquarters and
regional libraries, through the U.S. Government Depository Library
program, and for purchase from the National Technical Information
Service (NTIS), Springfield, VA; telephone: 703-487-4650, fax: 703-321-
8547. Please provide the NTIS PB No. (PB98-117831) when ordering.

FOR FURTHER INFORMATION CONTACT: Dr. Hugh A. Tilson, Neurotoxicology
Division, National Health and Environmental Effects Research
Laboratory, U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711, Tel: 919-541-2671; Fax: 919-541-4849; E-mail:
[email protected].

SUPPLEMENTARY INFORMATION: In its 1983 book Risk Assessment in the
Federal Government: Managing the Process, the National Academy of
Sciences recommended that Federal regulatory agencies establish
``inference guidelines'' to promote consistency and technical quality
in risk assessment, and to ensure that the risk assessment process is
maintained as a scientific effort separate from risk management. A task
force within EPA accepted that recommendation and requested that Agency
scientists begin to develop such guidelines. In 1984, EPA scientists
began work on risk assessment guidelines for carcinogenicity,
mutagenicity, suspect developmental toxicants, chemical mixtures, and
exposure assessment. Following extensive scientific and public review,
these first five guidelines were issued on September 24, 1986 (51 FR
33992-34054). Since 1986, additional risk assessment guidelines have
been proposed, revised, reproposed, and finalized. These guidelines
continue the process initiated in 1984. As with other EPA guidelines
(e.g., developmental toxicity, 56 FR 63798-63826; exposure assessment,
57 FR 22888-22938; and carcinogenicity, 61 FR 17960-18011), EPA will
revisit these guidelines as experience and scientific consensus evolve.
These Guidelines set forth principles and procedures to guide EPA
scientists in the conduct of Agency risk assessments and to inform
Agency decision makers and the public about these procedures. Policies
in this document are intended as internal guidance for EPA. Risk
assessors and risk managers at EPA are the primary audience, although
these Guidelines may be useful to others outside the Agency. In
particular, the Guidelines emphasize that risk assessments will be
conducted on a case-by-case basis, giving full consideration to all
relevant scientific information. This approach means that Agency
experts study scientific information on each chemical under review and
use the most scientifically appropriate interpretation to assess risk.
The Guidelines also stress that this information will be fully
presented in Agency risk assessment documents, and that Agency
scientists will identify the strengths and weaknesses of each
assessment by describing uncertainties, assumptions, and limitations,
as well as the scientific basis and rationale for each assessment. The
Guidelines are formulated in part to bridge gaps in risk assessment
methodology and data. By identifying these gaps and the importance of
the missing information to the risk assessment process, EPA wishes to
encourage research and analysis that will lead to new risk assessment
methods and data.

Dated: April 30, 1998.
Carol M. Browner,
Administrator.

Contents

Part A: Guidelines for Neurotoxicity Risk Assessment

List of Tables
1. Introduction
1.1. Organization of These Guidelines
1.2. The Role of Environmental Agents in Neurotoxicity
1.3. Neurotoxicity Risk Assessment
1.4. Assumptions
2. Definitions and Critical Concepts
3. Hazard Characterization
3.1. Neurotoxicological Studies: Endpoints and Their
Interpretation
3.1.1. Human Studies
3.1.1.1. Clinical Evaluations
3.1.1.2. Case Reports
3.1.1.3. Epidemiologic Studies
3.1.1.4. Human Laboratory Exposure Studies

[[Page 26927]]

3.1.2. Animal Studies
3.1.2.1. Structural Endpoints of Neurotoxicity
3.1.2.2. Neurophysiological Endpoints of Neurotoxicity
3.1.2.3. Neurochemical Endpoints of Neurotoxicity
3.1.2.4. Behavioral Endpoints of Neurotoxicity
3.1.3. Other Considerations
3.1.3.1. Pharmacokinetics
3.1.3.2. Comparisons of Molecular Structure
3.1.3.3. Statistical Considerations
3.1.3.4. In Vitro Data in Neurotoxicology
3.1.3.5. Neuroendocrine Effects
3.2. Dose-Response Evaluation
3.3. Characterization of the Health-Related Database
4. Quantitative Dose-Response Analysis
4.1. LOAEL/NOAEL and BMD Determination
4.2. Determination of the Reference Dose or Reference
Concentration
5. Exposure Assessment
6. Risk Characterization
6.1. Overview
6.2. Integration of Hazard Characterization, Dose-Response
Analysis, and Exposure Assessment
6.3. Quality of the Database and Degree of Confidence in the
Assessment
6.4. Descriptors of Neurotoxicity Risk
6.4.1. Estimation of the Number of Individuals
6.4.2. Presentation of Specific Scenarios
6.4.3. Risk Characterization for Highly Exposed Individuals
6.4.4. Risk Characterization for Highly Sensitive or Susceptible
Individuals
6.5.5. Other Risk Descriptors
6.5. Communicating Results
6.6. Summary and Research Needs
References

Part B: Response to Science Advisory Board and Public Comments

1. Introduction
2. Response to Science Advisory Board Comments
3. Response to Public Comments

List of Tables

Table 1. Examples of possible indicators of a neurotoxic effect
Table 2. Neurotoxicants and disorders with specific neurological
targets
Table 3. Examples of neurophysiological measures of neurotoxicity
Table 4. Examples of neurotoxicants with known neurochemical
mechanisms
Table 5. Examples of measures in a representative functional
observational battery
Table 6. Examples of specialized behavioral tests to measure
neurotoxicity
Table 7. Examples of compounds or treatments producing developmental
neurotoxicity
Table 8. Characterization of the health-related database

Part A: Guidelines for Neurotoxicity Risk Assessment

1. Introduction

These Guidelines describe the principles, concepts, and procedures
that the U.S. Environmental Protection Agency (EPA) will follow in
evaluating data on potential neurotoxicity associated with exposure to
environmental toxicants. The Agency's authority to regulate substances
that have the potential to interfere with human health is derived from
a number of statutes that are implemented through multiple offices
within EPA. The procedures outlined here are intended to help develop a
sound scientific basis for neurotoxicity risk assessment, promote
consistency in the Agency's assessment of toxic effects on the nervous
system, and inform others of the approaches used by the Agency in those
assessments. This document is not a regulation and is not intended for
EPA regulations. The Guidelines set forth current scientific thinking
and approaches for conducting and evaluating neurotoxic risk
assessments. They are not intended, nor can they be relied upon, to
create any rights enforceable by any party in litigation with the
United States.
1.1. Organization of These Guidelines
This introduction (section 1) summarizes the purpose of these
Guidelines within the overall framework of risk assessment at EPA. It
also outlines the organization of the guidance and describes several
default assumptions to be used in the risk assessment process, as
discussed in the recent National Research Council report ``Science and
Judgment in Risk Assessment'' (NRC, 1994).
Section 2 sets forth definitions of particular terms widely used in
the field of neurotoxicology. These include ``neurotoxicity'' and
``behavioral alterations.'' Also included in this section are
discussions concerning reversible and irreversible effects and direct
versus indirect effects.
Risk assessment is the process by which scientific judgments are
made concerning the potential for toxicity in humans. The National
Research Council (NRC, 1983) has defined risk assessment as including
some or all of the following components (paradigm): hazard
identification, dose-response assessment, exposure assessment, and risk
characterization. In its 1994 report ``Science and Judgment in Risk
Assessment'' the NRC extended its view of the paradigm to include
characterization of each component (NRC, 1994). In addition, it noted
the importance of an approach that is less fragmented and more
holistic, less linear and more interactive, and that deals with
recurring conceptual issues that cut across all stages of risk
assessment. These Guidelines describe a more interactive approach by
organizing the process around the qualitative evaluation of the
toxicity data (hazard characterization), the quantitative dose-response
analysis, the exposure assessment, and the risk characterization. In
these Guidelines, hazard characterization includes deciding whether a
chemical has an effect by means of qualitative consideration of dose-
response relationships, route, and duration of exposure. Determining a
hazard often depends on whether a dose-response relationship is present
(Kimmel et al., 1990). This approach combines the information important
in comparing the toxicity of a chemical with potential human exposure
scenarios (section 3). In addition, it avoids the potential for
labeling chemicals as ``neurotoxicants'' on a purely qualitative basis.
This organization of the risk assessment process is similar to that
discussed in the Guidelines for Developmental Toxicity Risk Assessment
(56 FR 63798), the main difference being that the quantitative dose-
response analysis is discussed under a separate section in these
Guidelines.
Hazard characterization involves examining all available
experimental animal and human data and the associated doses, routes,
timing, and durations of exposure to determine qualitatively if an
agent causes neurotoxicity in that species and under what conditions.
From the hazard characterization and criteria provided in these
Guidelines, the health-related database can be characterized as
sufficient or insufficient for use in risk assessment (section 3.3).
Combining hazard identification and some aspects of dose-response
evaluation into hazard characterization does not preclude the
evaluation and use of data for other purposes when quantitative
information for setting reference doses (RfDs) and reference
concentrations (RfCs) is not available.
The next step in the dose-response analysis (section 4) is the
quantitative analysis, which includes determining the no-observed-
adverse-effect-level (NOAEL) and/or the lowest-observed-adverse-effect-
level (LOAEL) for each study and type of effect. Because of the
limitations associated with the use of the NOAEL, the Agency is
beginning to use an additional approach, the benchmark dose approach
(BMD) (Crump, 1984; U.S. EPA, 1995a), for

[[Page 26928]]

more quantitative dose-response evaluation when sufficient data are
available. The benchmark dose approach takes into account the
variability in the data and the slope of the dose-response curve, and
provides a more consistent basis for calculation of the RfD or RfC. If
data are considered sufficient for risk assessment, and if
neurotoxicity is the effect occurring at the lowest dose level (i.e.,
the critical effect), an oral or dermal RfD or an inhalation RfC, based
on neurotoxic effects, is then derived. This RfD or RfC is derived
using the NOAEL or benchmark dose divided by uncertainty factors to
account for interspecies differences in response, intraspecies
variability, and other factors of study design or the database. A
statement of the potential for human risk and the consequences of
exposure can come only from integrating the hazard characterization and
dose-response analysis with the human exposure estimates in the final
risk characterization.
The section on exposure assessment (section 5) identifies human
populations exposed or potentially exposed to an agent, describes their
composition and size, and presents the types, magnitudes, frequencies,
and durations of exposure to the agent. The exposure assessment
provides an estimate of human exposure levels for particular
populations from all potential sources.
In risk characterization (section 6), the hazard characterization,
dose-response analysis, and exposure assessment for given populations
are combined to estimate some measure of the risk for neurotoxicity. As
part of risk characterization, a summary of the strengths and
weaknesses of each component of the risk assessment is given, along
with major assumptions, scientific judgments and, to the extent
possible, qualitative and quantitative estimates of the uncertainties.
This characterization of the health-related database is always
presented in conjunction with information on the dose, route, duration,
and timing of exposure as well as the dose-response analysis including
the RfD or RfC. If human exposure estimates are available, the exposure
basis used for the risk assessment is clearly described, e.g., highly
exposed individuals or highly sensitive or susceptible individuals. The
NOAEL may be compared to the various estimates of human exposure to
calculate the margin(s) of exposure (MOE). The considerations for
judging the acceptability of the MOE are similar to those for
determining the appropriate size of the uncertainty factor for
calculating the RfD or RfC.
The Agency recently issued a policy statement and associated
guidance for risk characterization (U.S. EPA, 1995b, 1995c), which is
currently being implemented throughout EPA. This statement is designed
to ensure that critical information from each stage of a risk
assessment is used in forming conclusions about risk and that this
information is communicated from risk assessors to risk managers
(policy makers), from middle to upper management, and from the Agency
to the public. Additionally, the policy provides a basis for greater
clarity, transparency, reasonableness, and consistency in risk
assessments across Agency programs.
Final neurotoxicity risk assessment guidelines may reflect
additional changes in risk characterization practices resulting from
implementation activities. Risk assessment is just one component of the
regulatory process and defines the potential adverse health
consequences of exposure to a toxic agent. The other component, risk
management, combines risk assessment with statutory directives
regarding socioeconomic, technical, political, and other considerations
in order to decide whether to control future exposure to the suspected
toxic agent and, if so, the nature and level of control. One major
objective of these Guidelines is to help the risk assessor determine
whether the experimental animal or human data indicate the potential
for a neurotoxic effect. Such information can then be used to
categorize evidence that will identify and characterize neurotoxic
hazards, as described in section 3.3, Characterization of the Health-
Related Database, and Table 8 of these Guidelines. Risk management is
not dealt with directly in these Guidelines because the basis for
decision making goes beyond scientific considerations alone, but the
use of scientific information in this process is discussed. For
example, the acceptability of the MOE is a risk management decision,
but the scientific bases for establishing this value are discussed
here.
1.2. The Role of Environmental Agents in Neurotoxicity
Chemicals are an integral part of life, with the capacity to
improve as well as endanger health. The general population is exposed
to chemicals in air, water, foods, cosmetics, household products, and
drugs used therapeutically or illicitly. During daily life, a person
experiences a multitude of exposures to potentially neuroactive
substances, singly and in combination, both synthetic and natural.
Levels of exposure vary and may or may not pose a hazard, depending on
dose, route, and duration of exposure.
A link between human exposure to some chemical substances and
neurotoxicity has been firmly established (Anger, 1986; OTA, 1990).
Because many natural and synthetic chemicals are present in today's
environment, there is growing scientific and regulatory interest in the
potential for risks to humans from exposure to neurotoxic agents. If
sufficient exposure occurs, the effects resulting from such exposures
can have a significant adverse impact on human health. It is not known
how many chemicals may be neurotoxic in humans (Reiter, 1987). EPA's
TSCA inventory of chemical substances manufactured, imported, or
processed in the United States includes more than 65,000 substances and
is increasing yearly. An overwhelming majority of the materials in
commercial use have not been tested for neurotoxic potential (NRC,
1984).
Estimates of the number of chemicals with neurotoxic properties
have been made for subsets of substances. For instance, a large
percentage of the more than 500 registered active pesticide ingredients
affect the nervous system of the target species to varying degrees. Of
588 chemicals listed by the American Conference of Governmental
Industrial Hygienists, 167 affected the nervous system or behavior at
some exposure level (Anger, 1984). Anger (1990) 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 if sufficient exposure occurs. Anger (1984) also
recognized neurotoxic effects as one of the 10 leading workplace
disorders. A number of therapeutic substances, including some
anticancer and antiviral agents and abused drugs, can cause adverse or
neurotoxicological side effects at therapeutic levels (OTA, 1990). The
number of chemicals with neurotoxic potential has been estimated to
range from 3% to 28% of all chemicals (OTA, 1990). Thus, estimating the
risks of exposure to chemicals with neurotoxic potential is of concern
with regard to their overall impact on human health.
1.3. Neurotoxicity Risk Assessment
In addition to its primary role in psychological functions, the
nervous system controls most, if not all, other bodily processes. It is
sensitive to perturbation from various sources and has limited ability
to regenerate. There is evidence that even small anatomical,
biochemical, or physiological insults to the nervous system may result
in

[[Page 26929]]

adverse effects on human health. Therefore, there is a need for
consistent guidance on how to evaluate data on neurotoxic substances
and assess their potential to cause transient or persistent and direct
or indirect effects on human health.
These Guidelines develop principles and concepts in several areas.
They outline the scientific basis for evaluating effects due to
exposure to neurotoxicants and discuss principles and methods for
evaluating data from human and animal studies on behavior,
neurochemistry, neurophysiology, and neuropathology. They also discuss
adverse effects on neurological development and function in infants and
children following prenatal and perinatal exposure to chemical agents.
They outline the methods for calculating reference doses or reference
concentrations when neurotoxicity is the critical effect, discuss the
availability of alternative mathematical approaches to dose-response
analyses, characterize the health-related database for neurotoxicity
risk assessment, and discuss the integration of exposure information
with results of the dose-response assessment to characterize risks.
These Guidelines do not advocate developing reference doses specific
for neurotoxicity, but rather support the use of neurotoxicity as one
possible endpoint to develop reference doses. EPA offices have
published guidelines for neurotoxicity testing in animals (U.S. EPA,
1986, 1987, 1988a, 1991a). The testing guidelines address the
development of new data for use in risk assessment.
These neurotoxicity risk assessment guidelines provide the Agency's
first comprehensive guidance on the use and interpretation of
neurotoxicity data, and are part of the Agency's risk assessment
guidelines development process, which was initiated in 1984. As part of
its neurotoxicity guidelines development program, EPA has sponsored or
participated in several conferences on relevant issues (Tilson, 1990);
these and other sources (see references) provide the scientific basis
for these Guidelines.
This guidance is intended for use by Agency risk assessors and is
separate and distinct from the recently published document on
principles of neurotoxicity risk assessment (U.S. EPA, 1994). The
document on principles was prepared under the auspices of the
Subcommittee on Risk Assessment of the Federal Coordinating Council for
Science, Engineering, and Technology and was not intended to provide
specific directives for how neurotoxicity risk assessment should be
performed. It is expected that, like other EPA risk assessment
guidelines for noncancer endpoints (U.S. EPA, 1991b), this document
will encourage research and analysis leading to new risk assessment
methods and data, which in turn would be used to revise and improve the
Guidelines and better guide Agency risk assessors.
1.4. Assumptions
There are a number of unknowns in the extrapolation of data from
animal studies to humans. Therefore, a number of default assumptions
are made that are generally applied in the absence of data on the
relevance of effects to potential human risk. Default assumptions
should not be applied indiscriminately. First, all available
mechanistic and pharmacokinetic data should be considered. If these
data indicate that an alternative assumption is appropriate or if they
obviate the need for applying an assumption, such information should be
used in risk assessment. For example, research in rats may determine
that the neurotoxicity of a chemical is caused by a metabolite. If
subsequent research finds that the chemical is metabolized to a lesser
degree or not at all in humans, then this information should be used in
formulating the default assumptions. The following default assumptions
form the basis of the approaches taken in these Guidelines:
(1) It is assumed that an agent that produces detectable adverse
neurotoxic effects in experimental animal studies will pose a potential
hazard to humans. This assumption is based on the comparisons of data
for known human neurotoxicants (Anger, 1990; Kimmel et al., 1990;
Spencer and Schaumburg, 1980), which indicate that experimental animal
data are frequently predictive of a neurotoxic effect in humans.
(2) It is assumed that behavioral, neurophysiological,
neurochemical, and neuroanatomical manifestations are of concern. In
the past, the tendency has been to consider only neuropathological
changes as endpoints of concern. Based on data on agents that are known
human neurotoxicants (Anger, 1990; Kimmel et al., 1990; Spencer and
Schaumberg, 1980), there is usually at least one experimental species
that mimics the types of effects seen in humans, but in other species
tested, the neurotoxic effect may be different or absent. For example,
certain organophosphate compounds produce a delayed-onset neuropathy in
hens similar to that seen in humans, whereas rodents are
characteristically insensitive to these compounds. A biologically
significant increase in any of the manifestations is considered
indicative of an agent's potential for disrupting the structure or
function of the human nervous system.
(3) It is assumed that the neurotoxic effects seen in animal
studies may not always be the same as those produced in humans.
Therefore, it may be difficult to determine the most appropriate
species in terms of predicting specific effects in humans. The fact
that every species may not react in the same way is probably due to
species-specific differences in maturation of the nervous system,
differences in timing of exposure, metabolism, or mechanisms of action.
(4) It is also assumed that, in the absence of data to the
contrary, the most sensitive species is used to estimate human risk.
This is based on the assumption that humans are as sensitive as the
most sensitive animal species tested. This provides a conservative
estimate of sensitivity for added protection to the public. As with
other noncancer endpoints, it is assumed that there is a nonlinear
dose-response relationship for neurotoxicants. Although there may be a
threshold for neurotoxic effects, these are often difficult to
determine empirically. Therefore, a nonlinear relationship is assumed
to exist for neurotoxicants.
These assumptions are ``plausibly conservative'' (NRC, 1994) in
that they are protective of public health and are also well founded in
scientific knowledge about the effects of concern.

2. Definitions and Critical Concepts

This section defines the key terms and concepts that EPA will use
in the identification and evaluation of neurotoxicity. The various
health effects that fall within the broad classification of
neurotoxicity are described and examples are provided. Adverse effects
include alterations from baseline or normal conditions that diminish an
organism's ability to survive, reproduce, or adapt to the environment.
Neurotoxicity is an adverse change in the structure or function of the
central and/or peripheral nervous system following exposure to a
chemical, physical, or biological agent (Tilson, 1990). Functional
neurotoxic effects include adverse changes in somatic/autonomic,
sensory, motor, and/or cognitive function. Structural neurotoxic
effects are defined as neuroanatomical changes occurring at any level
of nervous system organization; functional changes are defined as
neurochemical, neurophysiological, or behavioral effects. Chemicals can
also be categorized into four classes: Those that act on the central
nervous system, the peripheral nerve fibers, the peripheral

[[Page 26930]]

nerve endings, or muscles or other tissues (Albert, 1973). Changes in
function can result from toxicity to other specific organ systems, and
these indirect changes may be considered adverse. For example, exposure
to a high dose of a chemical may cause damage to the liver, resulting
in general sickness and a decrease in a functional endpoint such as
motor activity. In this case, the change in motor activity could be
considered as adverse, but not necessarily neurotoxic. A discussion
concerning problems associated with risk assessment of high doses of
chemicals in the context of drinking water and health was published by
the National Research Council (1986).
The risk assessor should also know that there are different levels
of concern based on the magnitude of effect, duration of exposure, and
reversibility of some neurotoxic effects. Neurotoxic effects may be
irreversible (the organism cannot return to the state prior to
exposure, resulting in a permanent change) or reversible (the organism
can return to the pre-exposure condition). Clear or demonstrable
irreversible change in either the structure or function of the nervous
system causes greater concern than do reversible changes. If neurotoxic
effects are observed at some time during the lifespan of the organism
but are slowly reversible, the concern is also high. There is lesser
concern for effects that are rapidly reversible or ``transient,'' i.e.,
measured in minutes, hours, or days, and that appear to be associated
with the pharmacokinetics of the causal agent and its presence in the
body. Reversible changes that occur in the occupational setting or
environment, however, may be of high concern if, for example, exposure
to a short-acting solvent interferes with operation of heavy equipment
in an industrial plant. The context of the exposure should be
considered in evaluating reversible effects. Setting of exposure limits
is not always associated with the determination of a reference dose,
which is based on chronic dosing. Data from acute or subacute dosing
can be used for health advisories or in studies involving developmental
exposures.
It should also be noted that the nervous system is known for its
reserve capacity (Tilson and Mitchell, 1983). That is, repeated insult
to the nervous system could lead to an adaptation. There are, however,
limits to this capacity, and when these limits are exceeded, further
exposure could lead to frank manifestations of neurotoxicity at the
structural or functional level. The risk assessor should be aware that
once damaged, neurons, particularly in the central nervous system, have
a limited capacity for regeneration. Reversibility of effects resulting
from cell death or from the destruction of cell processes may represent
an activation of repair capacity, decreasing future potential
adaptability. Therefore, even reversible neurotoxic changes should be
of concern. Evidence of progressive effects (those that continue to
worsen even after the causal agent has been removed), delayed-onset
effects (those that occur at a time distant from the last contact with
the causal agent), residual effects (those that persist beyond a
recovery period), or latent effects (those that become evident only
after an environmental challenge or aging) have a high level of
concern.
Environmental challenges can include stress, increased physical or
cognitive workload, pharmacological manipulations, and nutritional
deficiency or excess. Evidence for reversibility may depend on the
region of the nervous system affected, the chemical involved, and
organismic factors such as the age of the exposed population. Some
regions of the nervous system, such as peripheral nerves, have a high
capacity for regeneration, while regions in the brain such as the
hippocampus are known for their ability to compensate or adapt to
neurotoxic insult. For example, compensation is likely to be seen with
solvents (e.g., n-hexane) that produce peripheral neuropathy because of
the repair capacity of the peripheral nerve. In addition, tolerance to
some cholinergic effects of cholinesterase-inhibiting compounds may be
due to compensatory down-regulation of muscarinic receptors. Younger
individuals may have more capacity to adapt than older individuals,
suggesting that the aged may be at greater risk to neurotoxic exposure.
Neurotoxic effects can be observed at various levels of
organization of the nervous system, including neurochemical,
anatomical, physiological, or behavioral. At the neurochemical level,
for example, an agent that causes neurotoxicity might inhibit
macromolecule or transmitter synthesis, alter the flow of ions across
cellular membranes, or prevent release of neurotransmitter from the
nerve terminals. Anatomical changes may include alterations of the cell
body, the axon, or the myelin sheath. At the physiological level, a
chemical might change the thresholds for neural activation or reduce
the speed of neurotransmission. Behavioral alterations can include
significant changes in sensations of sight, hearing, or touch;
alterations in simple or complex reflexes and motor functions;
alterations in cognitive functions such as learning, memory, or
attention; and changes in mood, such as fear or rage, disorientation as
to person, time, or place, or distortions of thinking and feeling, such
as delusions and hallucinations. At present, relatively few neurotoxic
syndromes have been thoroughly characterized in terms of the initial
neurochemical change, structural alterations, physiological
consequence, and behavioral effects. Knowledge of exact mechanisms of
action is not, however, necessary to conclude that a chemically induced
change is a neurotoxic effect.
Neurotoxic effects can be produced by chemicals that do not require
metabolism prior to interacting with their sites in the nervous system
(primary neurotoxic agents) or those that require metabolism prior to
interacting with their sites (secondary neurotoxic agents). Chemically
induced neurotoxic effects can be direct (due to an agent or its
metabolites acting directly on sites in the nervous system) or indirect
(due to agents or metabolites that produce their effects primarily by
interacting with sites outside the nervous system). For example,
excitatory amino acids such as domoic acid damage specific neurons
directly by activating excitatory amino acid receptors in the nervous
system, whereas carbon monoxide decreases oxygen availability, which
can indirectly kill neurons. Other examples of indirect effects include
cadmium-induced spasms in blood vessels supplying the nervous system,
dichloroacetate-induced perturbation of metabolic pathways, and
chemically induced alterations in skeletomuscular function or structure
and effects on the endocrine system. Professional judgment may be
required in making determinations about direct versus indirect effects.
The interpretation of data as indicative of a potential neurotoxic
effect involves the evaluation of the validity of the database. This
approach and these terms have been adapted from the literature on human
psychological testing (Sette, 1987; Sette and MacPhail, 1992), where
they have long been used to evaluate the level of confidence in
different measures of intelligence or other abilities, aptitudes, or
feelings. There are four principal questions that should be addressed:
whether the effects result from exposure (content validity); whether
the effects are adverse or toxicologically significant (construct
validity); whether there are correlative measures among behavioral,
physiological, neurochemical, and

[[Page 26931]]

morphological endpoints (concurrent validity); and whether the effects
are predictive of what will happen under various conditions (predictive
validity). Addressing these issues can provide a useful framework for
evaluating either human or animal studies or the weight of evidence for
a chemical (Sette, 1987; Sette and MacPhail, 1992). The next sections
indicate the extent to which chemically induced changes can be
interpreted as providing evidence of neurotoxicity.

3. Hazard Characterization

3.1. Neurotoxicological Studies: Endpoints and Their Interpretation
The qualitative characterization of neurotoxic hazard can be based
on either human or animal data (Anger, 1984; Reiter, 1987; U.S. EPA,
1994). Such data can result from accidental, inappropriate, or
controlled experimental exposures. This section describes many of the
general and some of the specific characteristics of human studies and
reports of neurotoxicity. It then describes some features of animal
studies of neuroanatomical, neurochemical, neurophysiological, and
behavioral effects relevant to risk assessment. The process of
characterizing the sufficiency or insufficiency of neurotoxic effects
for risk assessment is described in section 3.3. Additional sources of
information relevant to hazard characterization, such as comparisons of
molecular structure among compounds and in vitro screening methods, are
also discussed.
The hazard characterization should:
a. Identify strengths and limitations of the database:
Epidemiological studies (case reports, cross-sectional,
case-control, cohort, or human laboratory exposure studies);
Animal studies (including structural or neuropathological,
neurochemical, neurophysiological, behavioral or neurological, or
developmental endpoints).
b. Evaluate the validity of the database:
Content validity (effects result from exposure);
Construct validity (effects are adverse or toxicologically
significant);
Concurrent validity (correlative measures among
behavioral, physiological, neurochemical, or morphological endpoints);
Predictive validity (effects are predictive of what will
happen under various conditions).
c. Identify and describe key toxicological studies.
d. Describe the type of effects:
Structural (neuroanatomical alternations);
Functional (neurochemical, neurophysiological, behavioral
alterations).
e. Describe the nature of the effects (irreversible, reversible,
transient, progressive, delayed, residual, or latent).
f. Describe how much is known about how (through what biological
mechanism) the chemical produces adverse effects.
g. Discuss other health endpoints of concern.
h. Comment on any nonpositive data in humans or animals.
I. Discuss the dose-response data (epidemiological or animal)
available for further dose-response analysis.
j. Discuss the route, level, timing, and duration of exposure in
studies demonstrating neurotoxicity as compared to expected human
exposures.
k. Summarize the hazard characterization:
Confidence in conclusions;
Alternative conclusions also supported by the data;
Significant data gaps; and
Highlights of major assumptions.
3.1.1. Human Studies
It is well established that information from the evaluation of
human exposure can identify neurotoxic hazards (Anger and Johnson,
1985; Anger, 1990). Prominent among historical episodes of
neurotoxicity in human populations are the outbreaks of methylmercury
poisoning in Japan and Iraq and the neurotoxicity seen in miners of
metals, including mercury, manganese, and lead (Carson et al., 1987;
Silbergeld and Percival, 1987; OTA, 1990). In the past decade, lead
poisoning in children has been a prominent issue of concern (Silbergeld
and Percival, 1987). Neurotoxicity in humans has been studied and
reviewed for many pesticides (Hayes, 1982; NRDC, 1989; Ecobichon and
Joy, 1982; Ecobichon et al., 1990). Organochlorines, organophosphates,
carbamates, pyrethroids, certain fungicides, and some fumigants are all
known neurotoxicants. They may pose occupational risks to manufacturing
and formulation workers, pesticide applicators and farm workers, and
consumers through home application or consumption of residues in foods.
Families of workers may also be exposed by transport into the home from
workers' clothing. Data on humans can come from a number of sources,
including clinical evaluations, case reports, epidemiologic studies,
and human laboratory exposure studies. A more extensive description of
issues concerning human neurotoxicology and risk assessment has been
published elsewhere (U.S. EPA, 1993). A review of the types of tests
used to assess cognitive and neurological function in children, in
addition to a discussion of methodological issues in the design of
prospective, longitudinal studies of developmental neurotoxicity in
humans, has recently been published (Jacobson and Jacobson, 1996).
Stanton and Spear (1990) reviewed assessment measures used in
developmental neurotoxicology for their comparability in humans and
laboratory animals and their ability to detect comparable adverse
effects across species. At the level of the various functional
assessments for sensory, motivational, cognitive and motor function,
and social behavior, there was good agreement across species among the
neurotoxic agents reviewed.
3.1.1.1. Clinical Evaluations
Clinical methods are used extensively in neurology and
neuropsychology to evaluate patients suspected of having neurotoxicity.
An 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 neurophysiological studies and medical history to
derive a working diagnosis. Brain functional imaging techniques based
on magnetic resonance imaging or emission tomography may also be useful
in helping diagnose neurodegenerative disorders following chemical
exposures in humans (Omerand et al., 1994; Callender et al., 1994).
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
chemical-or drug-induced changes in nervous system processes.
Neurological and neuropsychological methods have long been employed
to identify the adverse health effects of environmental workplace
exposures (Sterman and Schaumburg, 1980).

[[Page 26932]]

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 these clinical methods. It is
very important to make external/internal dose measurements in humans to
determine the actual dose(s) that can cause unwanted effects.
Aspects of the neurological examination approach limit its
usefulness for neurotoxicological risk assessment. Information obtained
from the neurological 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. For example, the magnitude and
symmetry of muscle strength are often judged by having the patient push
against the resistance of the examiner's hands. The endpoints are
therefore the absolute and relative amount of muscle load sensed by the
examiner in his or her arms.
Compared with other methods, the neurological 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 neurological evaluation of persons with documented
neurobehavioral impairment would be helpful for identifying nonchemical
causes of neurotoxicity, 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 neurological exams
share two disadvantages with respect to neurotoxicity risk assessment.
First, neurological 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 is
generally thought to be limited, decreasing the usefulness of clinical
assessment approaches for epidemiologic risk assessment.
Second, neurological exams and neuropsychological test batteries
were 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 validated against
these neurological disease states. With a few notable exceptions,
chemicals are not believed to produce impairment similar to that from
trauma or lesions; neurotoxic effects are more similar to the effects
of degenerative disease. There has been insufficient research to
demonstrate which tests designed to assess functional expression of
neurologic disease are useful in characterizing the modes of central
nervous system impairment produced by chemical agents and drugs.
It should be noted that alternative approaches are available that
avoid many of the limitations of clinical and neurological and
traditional neuropsychological methods. Computerized behavioral
assessment systems designed for field testing of populations exposed to
chemicals in the community or workplace have been developed during the
past decade. The most widely used system is the Neurobehavioral
Evaluation System (NES) developed by Baker et al. (1985). Advantages of
computerized tests include (1) standardized administration to eliminate
intertester variability and minimize subject-experimenter interaction;
(2) automated data collection and scoring, which is faster, easier, and
less error-prone than traditional methods; and (3) test administration
requires minimal training and experience. NES tests have proven
sensitive to a variety of solvents, metals, and pesticides (Otto,
1992). Computerized systems available for human neurotoxicity testing
are critically reviewed in Anger et al. (1996).
3.1.1.2. Case Reports
The first type of human data available is often 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. However, case reports involving a single neurotoxic
agent, although informative, are rare in the literature; for example,
farmers are likely to be 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 can be obtained more quickly than more complex
studies. Case reports of acute high-level exposure to a toxicant can be
useful for identifying signs and symptoms that may also apply to lower
exposure. Case reports can also be useful when corroborating
epidemiological data are available.
3.1.1.3. 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 (Friedlander and Hearn, 1980).
Epidemiologic studies are a useful means of evaluating the effects of
neurotoxic substances on human populations, particularly if effects of
exposure are cumulative or exposures are repeated. Such studies are
less useful in cases of acute exposure, where the effects are short-
term. Frequently, determining the precise dose or exposure
concentration in epidemiological studies can be difficult.
3.1.1.3.1. 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 are
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

[[Page 26933]]

required to complete compared with case reports and more complex
analytical studies, but should be augmented with additional data.
3.1.1.3.2. 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 neurotoxicants
in the environment provides a great deal of useful information for the
risk assessor. In his textbook, 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.
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.
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 nonexposed population (or that the prevalence of
the characteristic under study is the same in the control group as in
the general population). Failure to satisfy these assumptions may
result in selection bias that may invalidate 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 should 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
factors requires a larger study.
The definition of exposure is critical in epidemiologic studies. In
occupational settings, exposure assessment often 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. Positive
results from a properly controlled retrospective study should weigh
heavily in the risk assessment process.
3.1.1.3.3. Cohort (Prospective, Follow-Up) Studies.
In a prospective study design, a healthy group of people is
assembled and followed forward in time and observed for the development
of dysfunction. Such studies are invaluable for determining the time
course for development of dysfunction (e.g., follow-up 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 toxic incidence rates in the
cohort can be determined. Prospective study designs also allow 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. More credence should be
given to those studies in which both observer and subject bias are
carefully controlled (e.g., double-blind studies).
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. Positive or negative results from a
properly controlled prospective study should weigh heavily in the risk
assessment process.
3.1.1.4. Human Laboratory Exposure Studies
Neurotoxicity assessment has an advantage not afforded to 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. This makes it 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.
Human exposure studies sometimes offer advantages over
epidemiologic field studies. Combined with appropriate sampling of
biological fluids (urine or blood), it is possible to calculate body
concentrations, examine toxicokinetics, and identify metabolites.
Bioavailability, elimination, dose-related changes in metabolic
pathways, individual variability, time course of effects, interactions
between chemicals, and interactions between chemical and environmental/
biobehavioral processes (stressors, workload/respiratory rate) are
factors that are generally easier to collect under controlled
conditions.
Other goals of laboratory studies include the in-depth
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

[[Page 26934]]

study of environmental and biobehavioral variables that affect the
action of chemical agents. The effects of ambient temperature, task
difficulty, rate of ongoing behavior, conditioning variables,
tolerance/sensitization, sleep deprivation, motivation, and so forth
are sometimes studied.
From a methodological 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. There should be a sufficient
number of test sessions in the pre-exposure phase to allow performance
on all tests to achieve a relatively stable baseline level.
Participants in laboratory exposure studies may have been recruited
from populations of persons already exposed to the chemical/drug or
from chemical-naive populations. Although the use of exposed volunteers
has ethical advantages, can mitigate against novelty effects, and
allows evaluation of tolerance/sensitization, finding an accessible
exposed population in reasonable proximity to the laboratory can be
difficult. Chemical-naive participants are more easily recruited but
may differ significantly in important characteristics from a
representative sample of exposed persons. Chemical-naive volunteers are
often younger, healthier, and better educated than the populations
exposed environmentally, in the workplace, or pharmacotherapeutically.
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, whereas 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. In all cases, the potential for participant bias should
be as carefully controlled for as possible. Even the consent form can
lead to participant bias, as toxic effects have been reported in some
individuals who were warned of such effects in an informed consent
form. In addition, double-blind studies have been shown to provide some
control for observer bias that may occur in single-blind studies. More
credence should be given to those studies in which both observer and
subject bias are carefully controlled (Benignus, 1993).
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 limited number
of functions in multiple ways may be more useful for elucidating
mechanisms or validating specific effects.
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 another
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.
3.1.2. Animal Studies
This section provides an overview of the major types of endpoints
that may be evaluated in animal neurotoxicity studies, describes the
kinds of effects that may be observed and some of the tests used to
detect and quantify these effects, and provides guidance for
interpreting data. Compared with human studies, animal studies are more
often available for specific chemicals, provide more precise exposure
information, and control environmental factors better (Anger, 1984).
For these reasons, risk assessments tend to rely heavily on animal
studies.
Many tests that can measure some aspect of neurotoxicity have been
used in the field of neurobiology in the past 50 years. The Office of
Prevention, Pesticides and Toxic Substances (OPPTS) has published
animal testing guidelines that were developed in cooperation with the
Office of Research and Development (U.S. EPA, 1991a). While the test
endpoints included in the 1991 document serve as a convenient focus for
this section, there are many other endpoints for which there are no
current EPA guidelines. The goal of the current document is to provide
a framework for interpreting data collected in tests frequently used by
neurotoxicologists.
Five categories of endpoints will be described: structural or
neuropathological, neurophysiological, neurochemical, behavioral, and
developmental. Table 1 lists a number of endpoints in each of these
categories. It is imperative for the risk assessor to understand that
the interpretation of the indicators listed in Table 1 as neurotoxic
effects is dependent on the dose at which such changes occur and the
possibility that damage to other organ systems may contribute to or
cause such changes indirectly.

Table 1.--Examples of Possible Indicators of a Neurotoxic Effect
------------------------------------------------------------------------

-------------------------------------------------------------------------
Structural or neuropathological endpoints:
Gross changes in morphology, including brain weight.
Histologic changes in neurons or glia (neuronopathy, axonopathy,
myelinopathy).
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.
Inhibition and aging of neuropathy enzyme.
Increases in glial fibrillary acidic protein in adults.
Neurophysiological endpoints:
Change in velocity, amplitude, or refractory period of nerve
conduction.

[[Page 26935]]

Change in latency or amplitude of sensory-evoked potential.
Change in electroencephalographic pattern.
Behavioral and neurological endpoints:
Increases or decreases in motor activity.
Changes in touch, sight, sound, taste, or smell sensations.
Changes in motor coordination, weakness, paralysis, abnormal
movement or posture, tremor, ongoing performance.
Absence or decreased occurrence, magnitude, or latency of
sensorimotor reflex.
Altered magnitude of neurological measurement, including grip
strength, hindlimb splay.
Seizures.
Changes in rate or temporal patterning of schedule-controlled
behavior.
Changes in learning, memory, and attention.
Developmental endpoints:
Chemically induced changes in the time of appearance of behaviors
during development.
Chemically induced changes in the growth or organization of
structural or neurochemical elements.
------------------------------------------------------------------------

3.1.2.1. Structural Endpoints of Neurotoxicity
Structural endpoints are typically defined as neuropathological
changes evident by gross observation or light microscopy, although most
neurotoxic changes will be detectable only at the light microscopic
level. Gross changes in morphology can include discrete or widespread
lesions in nerve tissue. A change in brain weight is considered to be a
biologically significant effect. This is true regardless of changes in
body weight, because brain weight is generally protected during
undernutrition or weight loss, unlike many other organs or tissues. It
is inappropriate to express brain weight changes as a ratio of body
weight and thereby dismiss changes in absolute brain weight. Changes in
brain weight are a more reliable indicator of alteration in brain
structure than are measurements of length or width in fresh brain,
because there is little historical data in the toxicology literature.
Neurons are composed of a neuronal body, axon, and dendritic
processes. Various types of neuropathological lesions may be classified
according to the site where they occur (Spencer and Schaumburg, 1980;
WHO, 1986; Krinke, 1989; Griffin, 1990). Neurotoxicant-induced lesions
in the central or peripheral nervous system may be classified as a
neuronopathy (changes in the neuronal cell body), axonopathy (changes
in the axons), myelinopathy (changes in the myelin sheaths), or nerve
terminal degeneration. Nerve terminal degeneration represents a very
subtle change that may not be detected by routine histopathology, but
requires detection by special procedures such as silver staining or
neurotransmitter-specific immunohistochemistry. For axonopathies, a
more precise location of the changes may also be described (i.e.,
proximal, central, or distal axonopathy). In the case of some
developmental exposures, a neurotoxic chemical might delay or
accelerate the differentiation or proliferation of cells or cell types.
Alteration in the axonal termination site might also occur with
exposure. In an aged population, exposure to some neurotoxicants might
accelerate the normal loss of neurons associated with aging (Reuhl,
1991). In rare cases, neurotoxic agents have been reported to produce
neuropathic conditions resembling neurodegenerative disorders, such as
Parkinson's disease, in humans (WHO, 1986). Table 2 lists examples of
such neurotoxic chemicals, their putative site of action, the type of
neuropathology produced, and the disorder or condition that each
typifies. Inclusion of any chemical in any of the following tables is
for illustrative purposes, i.e., it has been reported that the chemical
will produce a neurotoxic effect at some dose; any individual chemical
listed may also adversely affect other organs at lower doses. It is
important that the severity of each structural union be graded
objectively and the grading criteria reported.

Table 2.--Neurotoxicants and Disorders With Specific Neurological Targets
----------------------------------------------------------------------------------------------------------------
Corresponding
Site of action Neurotoxic change Neurotoxic chemical neurodegenerative
disorder
----------------------------------------------------------------------------------------------------------------
Neuron cell body.................... Neuronopathy........... Methylmercury.......... Minamata disease.
Quinolinic acid........ Huntington's disease.
3-Acetylpyridine....... Cerebellar ataxia.
Nerve terminal...................... Terminal destruction... 1-Methyl-4-phenyl 1,2,. Parkinson's disease.
3,6-tetrahydro-........
pyridine (MPTP)
(dopaminergic).
Schwann cell myelin................. Myelinopathy........... Hexachlorophene........ Congenital
hypomyelinogenesis.
Centra-peripheral distal axon....... Distal axonopathy...... Acrylamide, carbon Peripheral neuropathy.
disulfide, n-hexane.
Central axons....................... Central axonopathy..... Clioquinol............. Subacute
myeloopticoneuro-pathy.
Proximal axon....................... Proximal axonopathy.... B,B'- Motor neuron disease.
Iminodipropionitrile.
----------------------------------------------------------------------------------------------------------------

Alterations in the structure of the nervous system (i.e.,
neuronopathy, axonopathy, myelinopathy, terminal degeneration) are
regarded as evidence of a neurotoxic effect. The risk assessor should
note that pathological changes in many cases require time for the
perturbation to become observable, especially with evaluation at the
light microscopic level. Neuropathological studies should control for
potential differences in the area(s) and section(s) of the nervous
system sampled; in the age, sex, and body weight of the subject; and in
fixation artifacts (WHO, 1986). Concern for the structural integrity of
nervous system tissues derives from

[[Page 26936]]

their functional specialization and lack of regenerative capacity.
Within general class of nervous system structural alteration, there
are various histological changes that can result after exposure to
neurotoxicants. For example, specific changes in nerve cell bodies
include chromatolysis, vacuolization, and cell death. Axons can undergo
swelling, degeneration, and atrophy, while myelin sheath changes
include folding, edematous splitting, and demyelination. Although
terminal degeneration does occur, it is not readily detectable by light
microscopy. Many of these changes are a result of complex effects at
specific subcellular organelles, such as the axonal swelling that
occurs as a result of neurofilament accumulation in acrylamide
toxicity. Other changes may be associated with regenerative or adaptive
processes that occur after neurotoxicant exposure.
3.1.2.2. Neurophysiological Endpoints of Neurotoxicity
Neurophysiological studies measure the electrical activity of the
nervous system. The term ``neurophysiology'' is often used synonymously
with ``electrophysiology'' (Dyer, 1987). Neurophysiological techniques
provide information on the integrity of defined portions of the nervous
system. Several neurophysiological procedures are available for
application to neurotoxicological studies. Examples are listed in Table
3. They 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. Microelectrode
procedures typically are used to study mechanisms of action and are
frequently performed in vitro. Macroelectrode procedures are generally
used in studies to detect or characterize the potential neurotoxic
effects of agents of interest because of potential environmental
exposure. The present discussion concentrates on macroelectrode
neurophysiological procedures because it is more likely that they will
be the focus of decisions regarding critical effects in risk
assessment. All of the procedures described below for use in animals
also have been used in humans to determine chemically induced
alterations in neurophysiological function.

Table 3.--Examples of Neurophysiological Measures of Neurotoxicity
------------------------------------------------------------------------
Representative
System/function Procedure agents
------------------------------------------------------------------------
Retina.......................... Electroretinograph Developmental
y (ERG). lead.
Visual pathway.................. Flash-evoked Carbon disulfide.
potential (FEP).
Visual function................. Pattern-evoked Carbon disulfide.
potential (PEP)
(pattern size and
contrast).
Auditory pathway................ Brain stem Aminoglycoside,
auditory evoked antibiotics,
potential (BAER) toluene, styrene.
(clicks).
Auditory function............... BAER (tones)...... Aminoglycoside,
antibiotics,
toluene, styrene.
Somatosensory pathway........... Somatosensory Acrylamide, n-
provoked. hexane.
Somatosensory function.......... Sensory-evoked Acrylamide, n-
potential (SEP) hexane.
(tactile).
Spinocerebellar pathway......... SEP recorded from Acrylamide, n-
cerebellum. hexane.
Mixed nerve..................... Peripheral nerve Triethyltin.
compound action
potential (PNAP).
Motor axons..................... PNAP isolate motor Triethyltin.
components.
Sensory axons................... PNAP isolate Triethyltin.
sensory
components.
Neuromuscular................... Electromyography Dithiobiuret.
(EMG).
General central nervous system/ Electroencephalogr Toluene.
level of arousal. aphy (EEG).
------------------------------------------------------------------------

3.1.2.2.1. Nerve Conduction Studies. Nerve conduction studies,
generally performed on peripheral nerves, can be useful in
investigations of possible peripheral neuropathy. Most peripheral
nerves contain mixtures of 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 sensory nerves or by measuring
the muscle response evoked by nerve stimulation to measure motor
effects. While a number of endpoints can be recorded, the most critical
variables are nerve conduction velocity, response amplitude, and
refractory period. It is important to recognize that damage to nerve
fibers may not be reflected in changes in these endpoints if the damage
is not sufficiently extensive. Thus, the interpretation of data from
such studies may be enhanced if evaluations such as nerve pathology
and/or other structural measures are also included.
Nerve conduction measurements are influenced by a number of
factors, the most important of which is temperature. An adequate nerve
conduction study will either measure the temperature of the limb under
study and mathematically adjust the results according to well-
established temperature factors or will control limb temperature within
narrow limits. Studies that measure peripheral nerve function without
regard for temperature are not adequate for risk assessment.
In well-controlled studies, statistically significant decreases in
nerve conduction velocity are indicative of a neurotoxic effect. While
a decrease in nerve conduction velocity is indicative of demyelination,
it frequently occurs later 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 rule out peripheral axonal degeneration if
other signs of peripheral nerve dysfunction are present.
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 is
conduction velocity. Measurements of response amplitude, however, can
be more variable and require careful application of experimental
techniques, a larger sample size, and greater statistical power than
measurements of velocity to detect changes. The refractory period
refers to the time required after stimulation before a nerve can fire
again and reflects the functional status of nerve membrane ion
channels. Chemically induced changes in

[[Page 26937]]

refractory periods in a well-controlled study indicate a neurotoxic
effect.
In summary, alterations in peripheral nerve response amplitude and
refractory period in studies that are well controlled for temperature
are indicative of a neurotoxic effect. Alterations in peripheral nerve
function are frequently associated with clinical signs such as
numbness, tingling, or burning sensations or with motor impairments
such as weakness. Examples of compounds that alter peripheral nerve
function in humans or experimental animals include acrylamide, carbon
disulfide, n-hexane, lead, and some organophosphates.
3.1.2.2.2. Sensory, Motor, and Other Evoked Potentials. Evoked
potential studies are electrophysiological procedures that measure the
response elicited from a defined stimulus such as a tone, a light, or a
brief electrical pulse. Evoked potentials reflect the function of the
system under study, including visual, auditory, or somatosensory;
motor, involving motor nerves and innervated muscles; or other neural
pathways in the central or peripheral nervous system (Rebert, 1983;
Dyer, 1985; Mattsson and Albee, 1988; Mattsson et al., 1992; Boyes,
1992, 1993). Evoked potential studies should be interpreted with
respect to the known or presumed neural generators of the responses,
and their likely relationships with behavioral outcomes, when such
information is available. Such correlative information strengthens the
confidence in electrophysiological outcomes. In the absence of such
supportive information, the extent to which evoked potential studies
provide convincing evidence of neurotoxicity is a matter of
professional judgment on a case-by-case basis. Judgments should
consider the nature, magnitude, and duration of such effects, along
with other factors discussed elsewhere in this document.
Data are in the form of a voltage record collected over time and
can be quantified in several ways. Commonly, the latency (time from
stimulus onset) and amplitude (voltage) of the positive and negative
voltage peaks are identified and measured. Alternative measurement
schemes may involve substitution of spectral phase or template shifts
for peak latency and spectral power, spectral amplitude, root-mean-
square, or integrated area under the curve for peak amplitude. Latency
measurements are dependent on both the velocity of nerve conduction and
the time of synaptic transmission. Both of these factors depend on
temperature, as discussed in regard to nerve conduction, and similar
caveats apply for sensory evoked potential studies. In studies that are
well controlled for temperature, increases in latencies or related
measures can reflect deficits in nerve conduction, including
demyelination or delayed synaptic transmission, and are indicators of a
neurotoxic effect.
Decreases in peak latencies, like increases in nerve conduction
velocity, are unusual, but the neural systems under study in sensory
evoked potentials are complex, and situations that might cause a peak
measurement to occur earlier are conceivable. Two such situations are a
reduced threshold for spatial or temporal summation of afferent neural
transmission and a selective loss of cells responding late in the peak,
thus making the measured peak occur earlier. Decreases in peak latency
should not be dismissed outright as experimental or statistical error,
but should be examined carefully and perhaps replicated to assess
possible neurotoxicity. A decrease in latency is not conclusive
evidence of a neurotoxic effect.
Changes in peak amplitudes or equivalent measures reflect changes
in the magnitude of the neural population responsive to stimulation.
Both increases and decreases in amplitude are possible following
exposure to chemicals. Whether excitatory or inhibitory neural activity
is translated into a positive or negative deflection in the sensory
evoked potential is dependent on the physical orientation of the
electrode with respect to the tissue generating the response, which is
frequently unknown. Comparisons should be based on the absolute change
in amplitude. Therefore, either increases or decreases in amplitude may
be indicative of a neurotoxic effect.
Within any given sensory system, the neural circuits that generate
various evoked potential peaks differ as a function of peak latency. In
general, early latency peaks reflect the transmission of afferent
sensory information. Changes in either the latency or amplitude of
these peaks are considered convincing evidence of a neurotoxic effect
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 (Dyer, 1987). Thus, changes in later-latency evoked
potential peaks should be interpreted in light of the behavioral status
of the subject and would generally be considered evidence of a
neurotoxic effect.
3.1.2.2.3. Seizures/Convulsions. Some neurotoxicants (e.g.,
lindane, pyrethroids, trimethyltin, dichlorodiphenyltrichloroethane
[DDT]) produce observable convulsions. When convulsionlike behaviors
are observed, as described in the behavioral section on convulsions,
neurophysiological recordings can provide additional information to
help interpret the results. Recordings of brain electrical activity
that demonstrate seizurelike activity are indicative of a neurotoxic
effect.
In addition to producing seizures directly, chemicals may also
alter the frequency, severity, duration, or threshold for eliciting
seizures through other means by a phenomenon known as ``kindling.''
Such alterations can occur after acute exposure or after repeated
exposure to dose levels below the acute threshold. In experiments
demonstrating changes in sensitivity following repeated exposures to
the test compound, information regarding possible changes in the
pharmacokinetic distribution of the compound is required before the
seizure susceptibility changes can be interpreted as evidence of
neurotoxicity. Increases in susceptibility to seizures are considered
adverse.
3.1.2.2.4. 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 using EEG in
either setting is the relationship between specific patterns of EEG
waveforms and specific behavioral states. Because states of alertness
and 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 after exposure to some chemicals. Changes in the pattern of the
EEG can be elicited by anesthetic drugs and stimuli producing arousal
(e.g., lights, sounds). In studies with toxicants, changes in EEG
pattern can sometimes precede alterations in other objective signs of
neurotoxicity (Dyer, 1987).
EEG studies should be done under highly controlled conditions, and
the data should be considered on a case-by-case basis. Chemically
induced seizure activity detected in the EEG pattern is evidence of a
neurotoxic effect.

[[Page 26938]]

3.1.2.3. Neurochemical Endpoints of Neurotoxicity
Many different neurochemical endpoints have been measured in
neurotoxicological studies, and some have proven useful in advancing
the understanding of mechanisms of action of neurotoxic chemicals
(Bondy, 1986; Mailman, 1987; Morell and Mailman, 1987; Costa, 1988;
Silbergeld, 1993). Normal functioning of the nervous system depends on
the synthesis and release of specific neurotransmitters and activation
of their receptors at specific presynaptic and postsynaptic sites.
Chemicals can interfere with the ionic balance of a neuron, act as a
cytotoxicant after transport into a nerve terminal, block reuptake of
neurotransmitters and their precursors, act as a metabolic poison,
overstimulate receptors, block transmitter release, and inhibit
transmitter synthetic or catabolic enzymes. Table 4 lists several
chemicals that produce neurotoxic effects at the neurochemical level
(Bondy, 1986; Mailman, 1987; Morell and Mailman, 1987; Costa, 1988).

Table 4.--Examples of Neurotoxicants With Known Neurochemical Mechanisms
------------------------------------------------------------------------
Site of action Examples
------------------------------------------------------------------------
Neurotoxicants acting on ionic balance:
Inhibit sodium entry................. Tetrodotoxin.
Block closing of sodium channel...... p,p'-DDT, pyrethroids.
Increase permeability to sodium...... Batrachotoxin.
Increase intracellular calcium....... Chlorodecone.
Synaptic neurotoxicants................ MPTP.
Uptake blockers........................ Hemicholinium.
Metabolic poisons...................... Cyanide.
Hyperactivation of receptors........... Domoic acid.
Blocks transmitter release............. Botulinum toxin.
Inhibition of transmitter degradation.. Pesticides of the
organophosphate and carbamate
classes.
Blocks axonal transport................ Acrylamide.
------------------------------------------------------------------------

As stated previously, any neurochemical change is potentially
neurotoxic. Persistent or irreversible chemically induced neurochemical
changes are indicative of neurotoxicity. Because the ultimate
functional significance of some biochemical changes is not known at
this time, neurochemical studies should be interpreted with reference
to the presumed neurotoxic consequence(s) of the neurochemical changes.
For example, many neuroactive agents can increase or decrease
neurotransmitter levels, but such changes are not indicative of a
neurotoxic effect. If, however, these neurochemical changes may be
expected to have neurophysiological, neuropathological, or
neurobehavioral correlates, then the neurochemical changes could be
classified as neurotoxic effects.
Some neurotoxicants, such as the organophosphate and carbamate
pesticides, are known to inhibit the activity of a specific enzyme,
acetylcholinesterase (for a review see Costa, 1988), which hydrolyzes
the neurotransmitter acetylcholine. Inhibition of the enzyme in either
the central or peripheral nervous system prolongs the action of the
acetylcholine at the neuron's synaptic receptors and is thought to be
responsible for the range of effects these chemicals produce, although
it is possible that these compounds have other modes of action
(Eldefrawi et al., 1992; Greenfield et al., 1984; Small, 1990).
There is agreement that objective clinical measures of cholinergic
overstimulation (e.g., salivation, sweating, muscle weakness, tremor,
blurred vision) can be used to evaluate dose-response and dose-effect
relationships and define the presence and absence of effects. A given
depression in peripheral and central cholinesterase activity may or may
not be accompanied by clinical manifestations. A depression in RBC and/
or plasma cholinesterase activity may or may not be accompanied by
clinical manifestations. It should be noted, however, that reduction in
cholinesterase activity, even if the anticholinesterase exposure is not
severe enough to precipitate clinical signs or symptoms, may impair the
organism's ability to adapt to additional exposures to
anticholinesterase compounds. Inhibition of RBC and/or plasma
cholinesterase activity is a biomarker of exposure, as well as a
reflection of cholinesterase inhibition in other peripheral tissues
(e.g., neuromuscular junction, peripheral nerve, or ganglia) (Maxwell
et al., 1987; Nagymajtenyi et al., 1988; Padilla et al., 1994), thereby
contributing to the overall hazard identification of cholinesterase-
inhibiting compounds.
The risk assessor should also be aware that tolerance to the
cholinergic overstimulation may be observed following repeated exposure
to cholinesterase-inhibiting chemicals. It has been reported, however,
that although tolerance can develop to some effects of cholinesterase
inhibition, the cellular mechanisms responsible for the development of
tolerance may also lead to the development of other effects, i.e.,
cognitive dysfunction, not present at the time of initial exposure
(Bushnell et al., 1991). These adaptive biochemical changes in the
tolerant animal may render it supersensitive to subsequent exposure to
cholinergically active compounds (Pope et al., 1992).
In general, the risk assessor should understand that assessment of
cholinesterase-inhibiting chemicals should be done on a case-by-case
basis using a weight-of evidence approach in which all of the available
data (e.g., brain, blood, and other tissue cholinesterase activity, as
well as the presence or absence of clinical signs) is considered in the
evaluation. Generally, the toxic effects of anticholinesterase
compounds are viewed as reversible, but there is human and experimental
animal evidence indicating that there may be residual, if not
permanent, effects of exposure to these compounds (Steenland et al.,
1994; Tandon et al., 1994; Stephens et al., 1995).
A subset of organophosphate agents also produces organophosphate-
induced delayed neuropathy (OPIDN) after acute or repeated exposure.
Inhibition and aging of neurotoxic esterase (or neuropathy enzymes) are
associated with agents that produce OPIDN (Johnson, 1990; Richardson,
1995). The conclusion that a chemical may produce OPIDN should be based
on at least two of three factors: (1) Evidence of a clinical syndrome,
(2) pathological lesions, and (3) neurotoxic esterase (NTE) inhibition.
NTE inhibition is necessary, but not sufficient, evidence

[[Page 26939]]

of the potential to produce OPIDN when there is at least 55%-70%
inhibition after acute exposure (Ehrich et al., 1995) and at least 45%
inhibition following repeated exposure.
Chemically induced injury to the central nervous system may be
accompanied by hypertrophy of astrocytes. In some cases, these
astrocytic changes can be seen light microscopically with
immunohistochemical stains for glial fibrillary acidic protein (GFAP),
the major intermediate filament protein in astrocytes. In addition,
GFAP can be quantified by an immunoassay, which has been proposed as a
marker of astrocyte reactivity (O'Callaghan, 1988). Immunohistochemical
stains have the advantage of better localization of GFAP increases,
whereas immunoassay evaluations are superior at detecting and
quantifying changes in GFAP levels and establishing dose-response
relationships. The ability to detect and quantify changes in GFAP by
immunoassay is improved by dissecting and analyzing multiple brain
regions. The interpretation of a chemical-induced change in GFAP is
facilitated by corroborative data from the neuropathology or
neuroanatomy evaluation. A number of chemicals known to injure the
central nervous system, including trimethyltin, methylmercury, cadmium,
3-acetylpyridine, and methylphenyltetrahydropyridine (MPTP), have been
shown to increase levels of GFAP. Measures of GFAP are now included as
an optional test in the Neurotoxicity Screening Battery (U.S. EPA,
1991a).
Increases in GFAP above control levels may be seen at dosages below
those necessary to produce damage seen by standard microscopic or
histopathological techniques. Because increases in GFAP reflect an
astrocyte response in adults, treatment-related increases in GFAP are
considered to be evidence that a neurotoxic effect has occurred. There
is less agreement as to how to interpret decreases in GFAP relative to
an appropriate control group. The absence of a change in GFAP following
exposure does not mean that the chemical is devoid of neurotoxic
potential. Known neurotoxicants such as cholinesterase-inhibiting
pesticides, for example, would not be expected to increase brain levels
of GFAP. Interpretation of GFAP changes prior to weaning may be
confounded by the possibility that chemically induced increases in GFAP
could be masked by changes in the concentration of this protein
associated with maturation of the central nervous system, and these
data may be difficult to interpret.
3.1.2.4. Behavioral Endpoints of Neurotoxicity
Behavior reflects the integration of the various functional
components of the nervous system. Changes in behavior can arise from a
direct effect of a toxicant on the nervous system, or indirectly from
its effects on other physiological systems. Understanding the
interrelationship between systemic toxicity and behavioral changes
(e.g., the relationship between liver damage and motor activity) is
extremely important. The presence of systemic toxicity may complicate,
but does not preclude, interpretation of behavioral changes as evidence
of neurotoxicity. In addition, a number of behaviors (e.g., schedule-
controlled behavior) may require a motivational component for
successful completion of the task. In such cases, experimental
paradigms designed to assess the motivation of an animal during
behavior might be necessary to interpret the meaning of some chemical-
induced changes in behavior.
EPA's testing guidelines developed for the Toxic Substances Control
Act and the Federal Insecticide, Fungicide and Rodenticide Act describe
the use of functional observational batteries (FOB), motor activity,
and schedule-controlled behavior for assessing neurotoxic potential
(U.S. EPA, 1991a). Examples of measures obtained in a typical FOB are
presented in Table 5. There are many other measures of behavior,
including specialized tests of motor and sensory function and of
learning and memory (Tilson, 1987; Anger, 1984).

Table 5.--Examples of Measures in a Representative Functional
Observational Battery
------------------------------------------------------------------------
Home cage and open field Manipulative Physiological
------------------------------------------------------------------------
Arousal......................... Approach response. Body temperature.
Autonomic signs................. Click response.... Body weight.
Convulsions, tremors............ Foot splay........
Gait............................ Grip strength..... ..................
Mobility........................ Righting reflex... ..................
Posture......................... Tail pinch ..................
response.
Rearing.
Stereotypy.
Touch response. .................. ..................
------------------------------------------------------------------------

Table 6.--Examples of Specialized Behavioral Tests To Measure
Neurotoxicity
------------------------------------------------------------------------
Representative
Function Procedure agents
------------------------------------------------------------------------
Motor Function
------------------------------------------------------------------------
Weakness........................ Grip strength, n-Hexane, methyl.
swimming n-Butylketone,
endurance, carbaryl.
suspension rod,
discriminative
motor function.
Incoordination.................. Rotorod, gait 3-Acetylpyridine,
assessments, ethanol.
righting reflex.
Tremor.......................... Rating scale, Chlordecone, Type
spectral analysis. I.
pyrethroids, DDT.
Myoclonic spasms................ Rating scale...... DDT, Type II
pyrethroids.
------------------------------------------------------------------------
Sensory Function
------------------------------------------------------------------------
Auditory........................ Discrimination Toluene,
conditioning. trimethyltin.
Reflex ..................
modification.
Visual.......................... Discrimination Methylmercury.
conditioning.
Somatosensory................... Discrimination Acrylamide.
conditioning.
Pain sensitivity................ Discrimination Parathion.
conditioning.
Olfactory....................... Discrimination 3-Methylindole,
conditioning. methylbromide.
------------------------------------------------------------------------
Cognitive Function
------------------------------------------------------------------------
Habituation..................... Startle reflex.... Diisopropylfluorop
hosphate.
Pre/neonatal
methylmercury.

[[Page 26940]]

Classical conditioning.......... Nictitating Aluminum.
membrane.
Conditioned flavor Carbaryl.
aversion.......... Trimethyltin,
IDPN.
Passive avoidance. Neonatal
trimethyltin.
Olfactory ..................
conditioning.
Instrumental conditioning....... One-way avoidance. Chlordecone.
Two-way avoidance. Pre/neonatal lead.
Y-maze avoidance.. Hypervitaminosis
A.
Biel water maze... Styrene.
Morris water maze. DFP.
Radial arm maze... Trimethyltin.
Delayed matching DFP.
to sample.
Repeated Carbaryl.
acquisition.
------------------------------------------------------------------------

At the present time, there is no clear consensus concerning the use
of specific behavioral tests to assess chemical-induced sensory, motor,
or cognitive dysfunction in animal models. The risk assessor should
also know that the literature is clear that a number of other behaviors
besides those listed in Tables 1, 5, and 6 could be affected by
chemical exposure. For example, alterations in food and water intake,
reproduction, sleep, temperature regulation, and circadian rhythmicity
are controlled by specific regions of the brain, and chemical-induced
alterations in these behaviors could be indicative of neurotoxicity. It
is reasonable to assume that an NOAEL or LOAEL could be based on one or
more of these endpoints.
The following sections describe, in general, behavioral tests and
their uses and offer guidance on interpreting data.
3.1.2.4.1. Functional Observational Battery (FOB). An FOB is
designed to detect and quantify major overt behavioral, physiological,
and neurological signs (Gad, 1982; O'Donoghue, 1989; Moser, 1989). A
number of batteries have been developed, each consisting of tests
generally intended to evaluate various aspects of sensorimotor function
(Tilson and Moser, 1992). Many FOB tests are essentially clinical
neurological examinations that rate the presence or absence, and in
many cases the severity, of specific neurological signs. Some FOBs in
animals are similar to clinical neurological examinations used with
human patients. Most FOBs have several components or tests. A typical
FOB is summarized in Table 5 and 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.
The relevance of statistically significant test results from an FOB
is judged according to the number of signs affected, the dose(s) at
which effects are observed, and the nature, severity, and persistence
of the effects and their incidence in relation to control animals. In
general, if only a few unrelated measures in the FOB are affected, or
the effects are unrelated to dose, the results may not be considered
evidence of a neurotoxic effect. If several neurological signs are
affected, but only at the high dose and in conjunction with other overt
signs of toxicity, including systemic toxicity, large decreases in body
weight, decreases in body temperature, or debilitation, there is less
persuasive evidence of a direct neurotoxic effect. In cases where
several related measures in a battery of tests are affected and the
effects appear to be dose dependent, the data are considered to be
evidence of a neurotoxic effect, especially in the absence of systemic
toxicity. The risk assessor should be aware of the potential for a
number of false positive statistical findings in these studies because
of the large number of endpoints customarily included in the FOB.
FOB data can be grouped into one or more of several neurobiological
domains, including neuromuscular (i.e., weakness, incoordination,
abnormal movements, gait), sensory (i.e., auditory, visual,
somatosensory), and autonomic functions (Tilson and Moser, 1992). This
statistical technique may be useful when separating changes that occur
on the basis of chance or in conjunction with systemic toxicity from
those treatment-related changes indicative of neurotoxic effects. In
the case of the developing organism, chemicals may alter the maturation
or appearance of sensorimotor reflexes. Significant alterations in or
delay of such reflexes is evidence of a neurotoxic effect.
Examples of chemicals that affect neuromuscular function are 3-
acetylpyridine, acrylamide, and triethyltin. Organophosphate and
carbamate insecticides produce autonomic dysfunction, while
organochlorine and pyrethroid insecticides increase sensorimotor
sensitivity, produce tremors and, in some cases, cause seizures and
convulsions (Spencer and Schaumburg, 1980).
3.1.2.4.2. Motor Activity. Motor activity represents a broad class
of behaviors involving coordinated participation of sensory, motor, and
integrative processes. Assessment of motor activity is noninvasive and
has been used to evaluate the effects of acute and repeated exposure to
neurotoxicants (MacPhail et al., 1989). An organism's level of activity
can, however, be affected by many different types of environmental
agents, including non-neurotoxic agents. Motor activity measurements
also have been used in humans to evaluate disease states, including
disorders of the nervous system (Goldstein and Stein, 1985).
Motor activity is usually quantified as the frequency of movements
over a period of time. The total counts generated during a test period
will depend on the recording mechanism and the size and configuration
of the testing apparatus. Effects of agents on motor activity can be
expressed as absolute activity counts or as a percentage of control
values. In some cases, a transformation (e.g., square root) may be used
to achieve a normal distribution of the data. In these cases, the
transformed data and not raw data should be used for risk assessment
purposes. The frequency of motor activity within a session usually
decreases and is reported as the average number of counts occurring in
each successive block of time. The EPA's

[[Page 26941]]

Office of Prevention, Pesticides and Toxic Substances guidelines (U.S.
EPA, 1991a), for example, call for test sessions of sufficient duration
to allow motor activity to approach steady-state levels during the last
20 percent of the session for control animals. A sum of the counts in
each epoch will add up to the total number of counts per session.
Motor activity can be altered by a number of experimental factors,
including neurotoxic chemicals. Decreases in activity could occur
following high doses of non-neurotoxic agents (Kotsonis and Klaassen,
1977; Landauer et al., 1984). Examples of neurotoxic agents that
decrease motor activity include many pesticides (e.g., carbamates,
chlorinated hydrocarbons, organophosphates, and pyrethroids), heavy
metals (lead, tin, and mercury), and other agents (3-acetylpyridine,
acrylamide, and 2,4-dithiobiuret). Some neurotoxicants (e.g., toluene,
xylene, triadimefon) produce transient increases in activity by
presumably stimulating neurotransmitter release, while others (e.g.,
trimethyltin) produce persistent increases in motor activity by
destroying specific regions of the brain (e.g., hippocampus).
Following developmental exposures, neurotoxic effects are often
observed as a change in the ontogenetic profile or maturation of motor
activity patterns. Frequently, developmental exposure to neurotoxic
agents will produce an increase in motor activity that persists into
adulthood or that results in changes in other behaviors. This is
evidence of a neurotoxic effect. Like other organ systems, the nervous
system may be differentially sensitive to toxicants in groups such as
the young. For example, toxicants introduced to the developing nervous
system may kill stem cells and thus cause profound effects on adult
structure and function. Moreover, toxicants may have greater access to
the developing nervous system before the blood-brain barrier is
completely formed or before metabolic detoxifying systems are
functional.
Motor activity measurements are typically used with other tests
(e.g., FOB) to help detect neurotoxic effects. Agent-induced changes in
motor activity associated with other 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, are related to structural or other functional changes in the
nervous system, or occur in the absence of life-threatening toxicity.
13.1.2.4.3. Schedule-Controlled Operant Behavior. Schedule-
controlled operant behavior (SCOB) involves the maintenance of behavior
(e.g., performance of a lever-press or key-peck response) by
reinforcement. Different rates and patterns of responding are
controlled by the relationship between response and subsequent
reinforcement. SCOB provides a measure of performance of a learned
behavior (e.g., lever press or key peck) and involves training and
motivational variables that should be considered in evaluating the
data. Agents may interact with sensory processing, motor output,
motivational variables (i.e., related to reinforcement), training
history, and baseline characteristics (Rice, 1988; Cory-Slechta, 1989).
Qualitatively, rates and patterns of SCOB display cross-species
generality, but the quantitative measures of rate and pattern of
performance can vary within and between species.
In laboratory animals, SCOB has been used to study a wide range of
neurotoxicants, including methylmercury, many pesticides, organic and
inorganic lead, triethyltin, and trimethyltin (MacPhail, 1985; Tilson,
1987; Rice, 1988). The primary SCOB endpoints for evaluation are
response rate and the temporal pattern of responding. These endpoints
may vary as a function of the contingency between responding and
reinforcement presentation (i.e., schedule of reinforcement). Schedules
of reinforcement that have been used in toxicology studies include
fixed ratio and fixed interval schedules. Fixed ratio schedules
engender high rates of responding and a characteristic pause after
delivery of each reinforcement. Fixed interval schedules engender a
relatively low rate of responding during the initial portion of the
interval and progressively higher rates near the end of the interval.
For some schedules of reinforcement, the temporal pattern of responding
may play a more important role in defining the performance
characteristics than the rate of responding. For other schedules, the
reverse may be true. For example, the temporal pattern of responding
may be more important than rate of responding for defining performance
on a fixed interval schedule. For a fixed ratio schedule, more
importance might be placed on the rate of responding than on the post-
reinforcement pause.
The overall qualitative patterns are important properties of the
behavior. Substantial qualitative changes in operant performance, such
as elimination of characteristic response patterns, can be evidence of
an adverse effect. Most chemicals, however, can disrupt operant
behavior at some dose, and such adverse effects may be due either to
neurotoxic or non-neurotoxic mechanisms. Unlike large qualitative
changes in operant performance, small quantitative changes are not
adverse. Some changes may actually represent an improvement, e.g., an
increase in the index of curvature with a decrease in fixed interval
rate of responding. Assessing the toxicological importance of these
effects requires considerable professional judgment and evaluation of
converging evidence from other types of toxicological endpoints. While
most chemicals decrease the efficiency of responding at some dose, some
agents may increase response efficiency on schedules requiring high
response rates because of a stimulant effect or an increase in central
nervous system excitability. Agent-induced changes in responding
between reinforcements (i.e., the temporal pattern of responding) may
occur independently of changes in the overall rate of responding.
Chemicals may also affect the reaction time to respond following
presentation of a stimulus. Agent-induced changes in response rate or
temporal patterning associated with other overt signs of toxicity
(e.g., body weight loss, systemic toxicity, or occurring in a 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.
3.1.2.4.4. Convulsions. Observable convulsions in animals are
indicative of an adverse effect. These events can reflect central
nervous system activity comparable to that of epilepsy in humans and
could be defined as neurotoxicity. Occasionally, other toxic actions
of compounds, such as direct effects on muscle, might mimic some
convulsionlike behaviors. In some cases, convulsions or
convulsionlike behaviors may be observed in animals that are
otherwise severely compromised, moribund, or near death. In such
cases, convulsions might reflect an indirect effect of systemic
toxicity and are less clearly indicative of neurotoxicity. As
discussed in the section on neurophysiological measures, electrical
recordings of brain activity could be used to determine specificity
of effects on the nervous system.
3.1.2.4.5. Specialized Tests for Neurotoxicity. Several procedures
have been developed to measure agent-induced changes in specific
neurobehavioral functions such as motor, sensory, or cognitive function
(Tilson, 1987; Cory-Slechta, 1989). Table 6 lists several behavioral
tests, the neurobehavioral functions they were designed to assess, and
agents known to affect the response. Many of these tests in animals
have been designed to assess neural functions in humans using similar
testing procedures.
A statistically or biologically significant chemically induced
change

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in any measure in Table 6 may be evidence of an adverse effect.
However, judgments of neurotoxicity may involve not only the analysis
of changes seen but the structure and class of the chemical and other
available neurochemical, neurophysiological, and neuropathological
evidence. In general, behavioral changes seen across broader dose
ranges indicate more specific actions on the systems underlying those
changes, i.e., the nervous system. Changes that are not dose dependent
or that are confounded with body weight changes and/or other systemic
toxicity may be more difficult to interpret as neurotoxic effects.
3.1.2.4.5.1. Motor Function. Neurotoxicants commonly affect motor
function. These effects can be categorized generally into (1) weakness
or decreased strength, (2) tremor, (3) incoordination, and (4) spasms,
myoclonia, or abnormal motor movements (Tilson, 1987; Cory-Slechta,
1989). Specialized tests used to assess strength include measures of
grip strength, swimming endurance, suspension from a hanging rod, and
discriminative motor function. Rotorod and gait assessments are used to
measure coordination, while rating scales and spectral analysis
techniques can be used to quantify tremor and other abnormal movements.
3.1.2.4.5.2. Sensory Function. Gross perturbations of sensory
function can be observed in simple neurological assessments such as the
hot plate or tail flick test. However, these tests may not be
sufficiently sensitive to detect subtle sensory changes. Psychophysical
procedures that study the relationship between a physical dimension
(e.g., intensity, frequency) of a stimulus and behavior may be
necessary to quantify agent-induced alterations in sensory function.
Examples of psychophysical procedures include discriminated
conditioning and startle reflex modification.
3.1.2.4.5.3. Cognitive Function. Alterations in learning and memory
in experimental animals should be inferred from changes in behavior
following exposure when compared with that seen prior to exposure or
with a nonexposed control group. Learning is defined as a relatively
lasting change in behavior due to experience, and memory is defined as
the persistence of a learned behavior over time. Table 6 lists several
examples of learning and memory tests and representative neurotoxicants
known to affect these tests. Measurement of changes in learning and
memory should be separated from other changes in behavior that do not
involve cognitive or associative processes (i.e., motor function,
sensory capabilities, motivational factors). In addition, any apparent
toxicant-induced change in learning or memory should ideally be
demonstrated over a range of stimulus and response conditions and
testing conditions. In developmental exposures, it should be shown that
the animals have matured enough to perform the specified task.
Developmental neurotoxicants can accelerate or delay the ability to
learn a response or may interfere with cognitive function at the time
of testing. Older animals frequently perform poorly on some types of
tests, and it should be demonstrated that control animals in this
population are capable of performing the procedure. Neurotoxicants
might accelerate age-related dysfunction or alter motivational
variables that are important for learning to occur. Further, it is not
the case that a decrease in responding on a learning task is adverse
while an increase in performance on a learning task is not. It is well
known that lesions in certain regions of the brain can facilitate the
acquisition of certain types of behaviors by removing preexisting
response tendencies (e.g., inhibitory responses due to stress) that
moderate the rate of learning under normal circumstances.
Apparent improvement in performance is not either adverse or
beneficial until demonstrated to be so by converging evidence with a
variety of experimental methods. Examples of procedures to assess
cognitive function include simple habituation, classical conditioning,
and operant (or instrumental) conditioning, including tests for spatial
learning and memory.
3.1.2.4.5.4. Developmental Neurotoxicity. Although the previous
discussion of various neurotoxicity endpoints and tests applies to
studies in which developmental exposures are used, there are particular
issues of importance in the evaluation of developmental neurotoxicity
studies. This section underscores the importance of detecting
neurotoxic effects following developmental exposure because an NRC
(1993) report has indicated that infants and children may be
differentially sensitive to environmental chemicals such as pesticides.
Exposure to chemicals during development can result in a spectrum of
effects, including death, structural abnormalities, altered growth, and
functional deficits (U.S. EPA, 1991b). A number of agents have been
shown to cause developmental neurotoxicity when exposure occurred
during the period between conception and sexual maturity (e.g., Riley
and Vorhees, 1986; Vorhees, 1987).
Table 7 lists several examples of agents known to produce
developmental neurotoxicity in experimental animals. Animal models of
developmental neurotoxicity have been shown to be sensitive to several
environmental agents known to produce developmental neurotoxicity in
humans, including lead, ethanol, x-irradiation, methylmercury, and
polychlorinated biphenyls (PCBs) (Kimmel et al., 1990; Needleman, 1990;
Jacobson et al., 1985; Needleman, 1986). In many of these cases,
functional deficits are observed at dose levels below those at which
other indicators of developmental toxicity are evident or at minimally
toxic doses in adults. Such effects may be transient, but generally are
considered adverse. Developmental exposure to a chemical could result
in transient or reversible effects observed during early development
that could reemerge as the individual ages (Barone et al., 1995).

Table 7.--Examples of Compounds or Treatments Producing Developmental
Neurotoxicity
------------------------------------------------------------------------

------------------------------------------------------------------------
Alcohols.................................. Methanol, ethanol.
Antimitotics.............................. X-radiation, azacytidine.
Insecticides.............................. DDT, chlordecone.
Metals.................................... Lead, methylmercury,
cadmium.
Polyhalogenated hydrocarbons.............. PCBs, PBBs.
------------------------------------------------------------------------

Testing for developmental neurotoxicity has not been required
routinely by regulatory agencies in the United States, but is required
by EPA when other information indicates the potential for developmental
neurotoxicity (U.S. EPA, 1986, 1988a, 1988b, 1989, 1991a, 1991b).
Useful data for decision making may be derived from well-conducted
adult neurotoxicity studies, standard developmental toxicity studies,
and multigeneration studies, although the dose levels used in the
latter may be lower than those in studies with shorter term exposure.
Important design issues to be evaluated for developmental
neurotoxicity studies are similar to those for standard developmental
toxicity studies (e.g., a dose-response approach with the highest dose
producing minimal overt maternal or perinatal toxicity, with number of
litters large enough for adequate statistical power, with randomization
of animals to dose groups and test groups, with litter generally
considered as the statistical unit). In addition, the use of a
replicate study design provides added confidence in the interpretation
of data. A pharmacological/physiological challenge may also be valuable
in

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evaluating neurological function and ``unmasking'' effects not
otherwise detectable. For example, a challenge with a psychomotor
stimulant such as d-amphetamine may unmask latent developmental
neurotoxicity (Hughes and Sparber, 1978; Adams and Buelke-Sam, 1981;
Buelke-Sam et al., 1985).
Direct extrapolation of developmental neurotoxicity to humans is
limited in the same way as for other endpoints of toxicity, i.e., by
the lack of knowledge about underlying toxicological mechanisms and
their significance (U.S. EPA, 1991b). However, comparisons of human and
animal data for several agents known to cause developmental
neurotoxicity in humans showed many similarities in effects (Kimmel et
al., 1990). As evidenced primarily by observations in laboratory
animals, comparisons at the level of functional category (sensory,
motivational, cognitive, motor function, and social behavior) showed
close agreement across species for the agents evaluated, even though
the specific endpoints used to assess these functions varied
considerably across species (Stanton and Spear, 1990). Thus, it can be
assumed that developmental neurotoxicity effects in animal studies
indicate the potential for altered neurobehavioral development in
humans, although the specific types of developmental effects seen in
experimental animal studies will not be the same as those that may be
produced in humans. Therefore, when data suggesting adverse effects in
developmental neuro

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Source: Frix Law Library, https://www.frixlaw.com/law-library/documents/fr%3A98-12303. Public record. Not legal advice.