Proposed Guidelines for Neurotoxicity Risk Assessment

Note: EPA no longer updates this information, but it may be useful as a reference or resource.

[Federal Register: October 4, 1995 (Volume 60, Number 192)]
[Notices]
[Page 52031-52056]
>From the Federal Register Online via GPO Access [wais.access.gpo.gov]

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ENVIRONMENTAL PROTECTION AGENCY
[FRL-5306-2]

AGENCY: Environmental Protection Agency (EPA).
ACTION: Proposed guidelines for Neurotoxicity Risk Assessment and
request for comments.

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SUMMARY: The U.S. Environmental Protection Agency (EPA; Agency) is
today issuing proposed guidelines for assessing the risks for
neurotoxicity from exposure to environmental agents. As background
information for this guidance, this notice describes the scientific
basis for concern about exposure to agents that cause neurotoxicity and
outlines the general process for assessing potential risk to humans
because of environmental contaminants.
These proposed Guidelines for Neurotoxicity Risk Assessment
(hereafter ``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
EPA. The Guidelines were developed as part of an interoffice guidelines
development program under the auspices of the Risk Assessment Forum,
within EPA's Office of Research and Development. Draft Guidelines were
developed by an Agency work group 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. A subsequent draft has
undergone peer review in a workshop held on June 2-3, 1992, and has
received internal review by the Concordance and Oversight Subcommittees
of the Risk Assessment Forum. Most recently, the Committee on the
Environment and Natural Resources of the Office of Science and
Technology Policy reviewed the guidelines at a meeting held on August
15, 1995. The proposed Guidelines are based, in part, on
recommendations derived from these reviews and on those made at various
scientific meetings and workshops on neurotoxicology.
The public is invited to comment, and public comments will be
considered in EPA decisions in formulating the final Guidelines.
Commenters are asked to focus on several special issues, particularly,
(1) the issue of compensation and recovery of function in
neurotoxicological studies and how to account for compensation in
neurotoxicology risk assessment; (2) the use of blood and/or brain
acetylcholinesterase activity as an indication of neurotoxicity for
risk assessment; (3) endpoints indicative of neurotoxicity that may not
be covered by these guidelines, i.e., endocrine disruption or
neuroendocrine-mediated neurotoxicity; and (4) the possibility of no
threshold for some neurotoxic agents.
The EPA Science Advisory Board (SAB) also will review these
proposed Guidelines at a meeting to be announced in a future Federal
Register. Agency staff will prepare summaries of the public and SAB
comments, analyses of major issues presented by commenters, and Agency
responses to those comments. Appropriate comments will be incorporated,
and the revised Guidelines will be submitted to the Risk Assessment
Forum for review. The Agency will consider comments from the public,
the SAB, and the Risk Assessment Forum in its recommendations to the
EPA Administrator.

DATES: The Proposed Guidelines are being made available for a 120-day
public review and comment period. Comments must be in writing and must
be postmarked by February 1, 1996. Please submit one unbound original
with pages consecutively numbered, and three copies. If there are
attachments, include an index numbered consecutively with comments, and
three copies.

FOR FURTHER INFORMATION CONTACT: Dr. Hugh A. Tilson, Tel: 919-541-2671;
Fax: 919-541-4849.

ADDRESSES: Comments on the proposed Guidelines may be mailed or
delivered to: Dr. Hugh A. Tilson, Neurotoxicology Division (MD-74B),
National Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, NC 27711.
Please note that all comments received in response to this notice will
be placed in a public record. Commenters should not send any item of
personal information, such as medical information or home address, if
they do not wish it to be part of the public record.

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'' (1) to promote consistency and technical
quality in risk assessment, and (2) 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 for male and female
reproductive risk (53 FR 24834-847; 53 FR 24850-869), and two of the
1986 guidelines, suspect developmental toxicants (56 FR 63798-826) and
exposure assessment (57 FR 22888-938), have been revised, reproposed,
and finalized.
The Guidelines proposed today continue the guidelines development
process initiated in 1984. 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. 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 case-by-case
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: September 25, 1995.
Carol M. Browner,
Administrator.

Proposed Guidelines for Neurotoxicity Risk Assessment Contents

I. Introduction
A. Organization of These Guidelines
B. The Role of Environmental Agents in Neurotoxicity

[[Page 52033]]

C. Neurotoxicity Risk Assessment
D. Assumptions
II. Definitions and Critical Concepts
III. Hazard Characterization
A. Neurotoxicological Studies: End Points and Their
Interpretation

Human Studies
a. Clinical Evaluations
b. Case Reports
c. Epidemiologic Studies
(1) Cross-sectional studies
(2) Case-control (retrospective) studies
(3) Cohort (prospective, follow-up) studies
d. Human Laboratory Exposure Studies
Animal Studies
a. Structural End Points of Neurotoxicity
b. Neurophysiological End Points of Neurotoxicity
(1) Nerve conduction studies
(2) Sensory, motor, and other evoked potentials
(3) Seizures/convulsions
(4) Electroencephalography (EEG)
c. Neurochemical End Points of Neurotoxicity
d. Behavioral End Points of Neurotoxicity
(1) Functional observational battery
(2) Motor activity
(3) Schedule-controlled operant behavior
(4) Convulsions
(5) Specialized tests for neurotoxicity
(a) Motor function
(b) Sensory function
(c) Cognitive function
e. Developmental Neurotoxicity
Other Considerations
a. Pharmacokinetics
b. Comparisons of Molecular Structure
c. Statistical Considerations
d. In Vitro Data in Neurotoxicology
B. Dose-Response Evaluation
C. Characterization of the Health-Related Data Base
IV. Dose-Response Analysis
A. LOAEL/NOAEL and Benchmark Dose (BMD) Determination
B. Determination of the Reference Dose or Reference
Concentration
V. Exposure Assessment
VI. Risk Characterization
A. Overview
B. Integration of Hazard Characterization, Dose-Response
Analysis, and Exposure Assessment
C. Quality of the Data Base and Degree of Confidence in the
Assessment
D. Descriptors of Neurotoxicity Risk
Estimation of the Number of Individuals
Presentation of Specific Scenarios
Risk Characterization for Highly Exposed Individuals
Risk Characterization for Highly Sensitive or Susceptible
Individuals
Other Risk Descriptors
E. Communicating Results
F. Summary and Research Needs
VII. References

List of Tables

Table 1. Examples of possible indicators of a neurotoxic effect
Table 2. Neurotoxicants and diseases with specific neuronal targets
Table 3. Examples of neurophysiological measures of neurotoxicity
Table 4. Examples of neurotoxicants with known neurochemical
mechanisms
Table 5. Summary of measures in a representative functional
observational battery, and the type of data produced by each
Table 6. Examples of specialized behavioral tests to measure
neurotoxicity
Table 7. Examples of developmental neurotoxicants
Table 8. Characterization of the Health-Related Database

I. Introduction

These proposed Guidelines describe the principles, concepts, and
procedures that the U.S. Environmental Protection Agency (EPA; Agency)
would 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 the 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.

A. Organization of These Guidelines

This Introduction (section I) summarizes the purpose of these
proposed Guidelines within the overall framework of risk assessment at
the 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 II 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 to occur 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 one that deals with
recurring conceptual issues that cut across all stages of risk
assessment. These Guidelines propose a more interactive approach by
organizing the process around components that focus on evaluation of
the toxicity data (hazard characterization), the quantitative doseresponse
analysis, the exposure assessment, and the risk
characterization. This is done because, in practice, hazard
identification for neurotoxicity and other noncancer health effects is
usually done in conjunction with an evaluation of dose-response
relationships in the studies used to identify the hazard. Determining a
hazard often depends on whether a dose-response relationship is present
(Kimmel et al., 1990). Thus, the hazard characterization provides an
evaluation of a hazard within the context of the dose, route, duration,
and timing of exposure. This approach combines the information
important in comparing the toxicity of a chemical to potential human
exposure scenarios (Section V). Secondly, 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 doseresponse
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 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 data base can be characterized as sufficient or
insufficient for use in risk assessment (section III.C). Combining
hazard identification and some aspects of dose-response evaluation into
hazard characterization does not preclude the evaluation and use of
data when quantitative information for setting reference doses (RfDs)
and reference concentrations (RfCs) are not available.
The next step, the dose-response analysis (section IV) is the
quantitative analysis, and includes determining the no-observedadverse
-effect-level (NOAEL) and/or the lowest-observed-adverse-effectlevel
(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, i.e., the

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benchmark dose approach (Crump, 1984; U.S. EPA, 1995a), for 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 data base. 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 V) 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 VI), the hazard characterization,
dose-response analysis, and the 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 data base 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 policy 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, 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 risk
assessment 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 subsequently to
categorize evidence to identify and characterize neurotoxic hazards as
described in section III.3.C, Characterization of the Health-Related
Data Base, 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.

B. 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 with neurotoxic properties in air, water, foods,
cosmetics, household products, and drugs used therapeutically or
illicitly. Naturally occurring neurotoxins, such as animal and plant
toxins, present additional hazards. During daily life, a person
experiences a multitude of exposures, both voluntary and unintentional,
to neuroactive substances, singly and in combination. 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). The
EPA's inventory of toxic chemicals is greater than 65,000 and
increasing yearly. An overwhelming majority of the materials in
commercial use have not been tested for their 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 are neurotoxic 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 one 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). Thus, estimating the risks of exposure
to chemicals with neurotoxic potential is of concern with regard to the
overall impact of these exposures on human health.

C. Neurotoxicity Risk Assessment

In addition to its primary role in cognitive 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 52035]]

adverse effects on human health. Therefore, there is a need for
consistent guidance on how to evaluate data on neurotoxic substances
and assess to what degree, if any, they have the potential to cause
transient or persistent, direct or indirect effects on human health.
To help address these needs, these Guidelines develop principles
and concepts in several areas. First, these Guidelines 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. This guidance document also
discusses adverse effects on neurological development and function in
infants and children following prenatal and perinatal exposure to
chemical agents. Other sections of these Guidelines outline the method
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 data base for neurotoxicity risk
assessment, and discuss integration of exposure information with the
results of the dose-response assessment to characterize risks of
exposures of concern. These Guidelines do not advocate developing
reference doses specific for neurotoxicity, but rather the use of
neurotoxicity as one possible end point 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
proposed neurotoxicity risk assessment Guidelines provide the Agency's
first comprehensive guidance on the use and interpretation of
neurotoxicity data. These proposed Guidelines are part of the Agency's
risk assessment guidelines development process, which was initiated in
1984. As part of its neurotoxicity guidelines development program, the
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 proposed risk assessment 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, 1993). 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
(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.

D. 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 indiscriminantly. First, all available
mechanistic and pharmacokinetic data should be considered. If these
data indicate that an alternative assumption is appropriate or obviate
the need for applying an assumption, such information should be used in
the risk assessment of that agent. The following default assumptions
form the basis of the approaches taken in these Guidelines.
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.
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 end
points of concern. Based on the 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 type of neurotoxic effect may be different or absent. Thus,
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.
It is assumed that the types of neurotoxic effects seen in animal
studies may not always be the same as those produced in humans.
Therefore, it may be difficult to determine which will be the most
appropriate species in terms of predicting the specific types of
effects seen 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.
It is assumed that the most appropriate species will be used when
data are available to estimate human risk. In the absence of such data,
the most sensitive species is used, based on the fact that for the
majority of known human neurotoxicants, humans are as sensitive or more
so than the most sensitive animal species tested.
In general, a threshold is assumed for the dose-response curve for
most neurotoxicants. This is based on the known capacity of the nervous
system to compensate for or to repair a certain amount of damage at the
cellular, tissue, or organ level. In addition, because of the
multiplicity of cells in the nervous system, multiple insults at the
molecular or cellular level may be required to produce an effect on the
whole organism.
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.

II. Definitions and Critical Concepts

This section defines the key terms and concepts that the 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 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). Neurotoxic
effects include changes in somatic/autonomic, sensory, motor, and/or
cognitive function. Structural effects are defined as neuroanatomical
changes occurring at any level of nervous system organization;
functional changes are defined as neurochemical, neurophysiological, or
behavioral alterations. Changes in function can also result from
toxicity to other specific organ systems, and these indirect changes
may be considered adverse but not necessarily neurotoxic.

[[Page 52036]]

The risk assessor also should know that there are different levels
of concern based on the magnitude of effect and reversibility of some
neurotoxic effects. Neurotoxic effects may be irreversible, i.e.,
cannot return to the state prior to exposure, resulting in a permanent
change in the organism, or reversible, i.e., can return to the preexposure
condition, allowing the organism to return to its state prior
to exposure. 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 life span 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 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. The risk assessor should note 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, i.e., those that continue
to worsen even after the causal agent has been removed; or delayed
effects, i.e., those that occur at a time distant from the last contact
with the causal agent; or residual effects, i.e., those that persist
beyond a recovery period; or latent effects, i.e., 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.
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 target sites in the nervous
system, i.e., primary neurotoxic agents, or those that require
metabolism prior to interacting with their target sites in the nervous
system, i.e., secondary neurotoxic agents. Chemically induced
neurotoxic effects can be direct, i.e., due to an agent or its
metabolites acting directly on target sites in the nervous system, or
indirect, i.e., due to agents or metabolites that produce their effects
primarily by interacting with target sites outside the nervous system,
which subsequently affect target sites in the nervous system.
Excitatory amino acids such as domoic acid damage specific neurons
directly by activating excitatory amino acid receptors in the nervous
system, while carbon monoxide decreases oxygen availability, which
indirectly kills neurons. Other examples of indirect effects of
chemicals that could lead to altered structure and/or function of the
nervous system 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 data base. 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 morphological end points (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.

III. Hazard Characterization

A. Neurotoxicological Studies: End Points and Their Interpretation

Identification and characterization of neurotoxic hazard can be
based on either human or animal data (Anger, 1984; Reiter, 1987; U.S.
EPA, 1993). 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 III.C. 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 end points).

[[Page 52037]]

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 end points);
--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 effects).
f. Describe how much is known about how (through what biological
mechanism) the chemical produces adverse effects.
g. Discuss other health end points of concern.
h. Comment on any non-positive 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
--Highlight of major assumptions.

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 last 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, and epidemiologic
studies. A more extensive description of issues concerning human
neurotoxicology and risk assessment has been published elsewhere (U.S.
EPA, 1993).
a. Clinical Evaluations. Clinical methods are used extensively in
neurology and neuropsychology to evaluate patients suspected of having
neurotoxicity. An extensive array of examiner-administered and paperand
-pencil tasks are used to assess sensory, motor, cognitive, and
affective functions and personality states/traits. Neurobehavioral data
are synthesized with information from neurophysiologic studies and
medical history to derive a working diagnosis. Brain imaging techniques
based on magnetic resonance imaging or emission tomography may also be
useful in helping diagnose neurodegenerative disorders following
chemical exposures in humans (Omerod 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
chemically or drug-induced changes in nervous system processes.
Neurologic and neuropsychologic methods have long been employed to
identify the adverse health effects of environmental workplace
exposures (Sterman and Schaumburg, 1980). 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 neurologic examination approach limit its usefulness
for neurotoxicologic risk assessment. Information obtained from the
neurologic exam is mostly qualitative and descriptive rather than
quantitative. Estimates of the severity of functional impairment can be
reliably placed into only three or four categories (for example, mild,
moderate, severe). Much of the assessment depends on the subjective
judgment of the examiner. 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 end points are therefore the
absolute and relative amount of muscle load sensed by the examiner in
his or her arms.
Compared with other methods, the neurologic exam may be less
sensitive in detecting early neurotoxicity in peripheral sensory and
motor nerves. While clinicians' judgments are equal in sensitivity to
quantitative methods in assessing the amplitude of tremor, tremor
frequency is poorly quantified by clinicians. Thus, important aspects
of the clinical neurologic exam may be insufficiently quantified and
lack sufficient sensitivity for detecting early neurobehavioral
toxicity produced by environmental or workplace exposure conditions.
However, a neurologic evaluation of persons with documented
neurobehavioral impairment would be helpful for identifying nonchemical
causes 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 neurologic exams share
two disadvantages with respect to neurotoxicity risk assessment. First,

[[Page 52038]]

neurologic exams and neuropsychological test batteries are designed to
confirm and classify functional problems in individuals selected on the
basis of signs and symptoms identified by the patient, family, or other
health professionals. Their usefulness in detecting low base-rate
impairment in workers or the general population is generally thought to
be limited, decreasing the usefulness of clinical assessment approaches
for epidemiologic risk assessment.
Second, neurologic exams and neuropsychologic 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 neurologic 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.
b. 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 where
exposure involved 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 are inexpensive compared with epidemiologic studies
and can be obtained more quickly than more complex studies. However,
they provide little information about disease frequency or population
at risk, but their importance has been clearly demonstrated,
particularly in accidental poisoning or acute exposure to high levels
of toxicant. They remain an important source of index cases of new
diseases and for surveillance.
c. 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 means of evaluating the effects of
neurotoxic substances on human populations, but such studies are
limited because they must be performed shortly after exposure if the
effect is acute. Most often these effects are suspected to be a result
of occupational exposures due to the increased opportunity for exposure
to industrial and other chemicals. Frequently, determining the precise
dose or exposure concentration can be difficult in epidemiological
studies.
(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 required to complete compared with case reports and more
complex analytical studies but should be augmented with additional
data.
(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 casecontrol
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 of the
study. Analysis of the case-comparison study design assumes that the
selected cases are representative persons with the disease--either all
cases with the disease or a representative sample of them have been
ascertained. It further assumes that the control or comparison group is
representative of the nondiseased population (or that the prevalence of
the characteristic under study is the same in the control group as in
the general population). Failure to satisfy these assumptions may
result in selection bias, but violation of assumptions does not
necessarily invalidate the study results.
An additional source of bias in case-control studies is the
presence of confounding variables, i.e., factors known to be associated
with the exposure and causally related to the disease under study.
These must be controlled either in the design of the study by matching
cases to controls on the basis of the confounding factor or in the
analysis of the data by using statistical techniques such as
stratification or regression. Matching requires time to identify an
adequate number of potential controls to distinguish those with the
proper characteristics, while statistical control of confounding
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

[[Page 52039]]

study should weigh heavily in the risk assessment process.
(3) Cohort (prospective, followup) studies. In a prospective study
design, a healthy group of people is assembled and followed forward in
time and observed for the development of disease. Such studies are
invaluable for determining the time course for development of disease
(e.g., followup studies performed in various cities on the effects of
lead on child development). This approach allows the direct estimate of
risks attributed to a particular exposure since disease incidence rates
in the cohort 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.
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 results from a properly
controlled prospective study should weigh heavily in the risk
assessment process.
d. Human Laboratory Exposure Studies. Neurotoxicity assessment has
an advantage not afforded the evaluation of other toxic end points,
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 sometime offer advantages over epidemiologic
field studies. Combined with appropriate sampling of biologic 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 indepth
characterization of effects, the development of new assessment methods,
and the examination of the sensitivity, specificity, and reliability of
neurobehavioral assessment methods across chemical classes. The
laboratory is the most appropriate setting for the study of
environmental and biobehavioral variables that affect the action of
chemical agents. The effects of ambient temperature, task difficulty,
rate of ongoing behavior, conditioning variables, tolerance/
sensitization, sleep deprivation, motivation, and so forth are
sometimes studied.
From a methodologic standpoint, human laboratory studies can be
divided into two categories--between-subjects and within-subjects
designs. In the former, the neurobehavioral performance of exposed
volunteers is compared with that of nonexposed participants. In the
latter, preexposure performance is compared with neurobehavioral
function under the influence of the chemical or drug. Within-subjects
designs have the advantage of requiring fewer participants, eliminating
individual differences as a source of variability, and controlling for
chronic mediating variables, such as caffeine use and educational
achievement. A disadvantage of the within-subjects design is that
neurobehavioral tests must be administered more than once. Practice on
many neurobehavioral tests often leads to improved performance that may
confound the effect of the chemical/drug. There should be a sufficient
number of test sessions in the pre-exposure phase of the study 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 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 is difficult.
Naive participants are more easily recruited but may differ
significantly in important characteristics from a representative sample
of exposed persons. Naive volunteers are often younger, healthier, and
better educated than the populations exposed environmentally, in the
workplace, or pharmacotherapeutically.
Compared with workplace and environmental exposures, laboratory
exposure conditions can be controlled more precisely, but exposure
periods are much shorter. Generally only one or two relatively pure
chemicals are studied for several hours while the population of
interest may be exposed to multiple chemicals containing impurities for
months or years. Laboratory studies are therefore better at identifying
and characterizing effects with acute onset and the selective effects
of pure agents.
Neurobehavioral test methods may have been selected according to
several strategies. A test battery that examines multiple
neurobehavioral functions may be more useful for screening and the
initial characterization of acute effects. Selected neurobehavioral
tests that measure a more limited number of functions in multiple ways
may be more useful for elucidating mechanisms or validating specific
effects.
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
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.
2. Animal Studies
This section provides an overview of the major types of end points
that may be evaluated in animal neurotoxicity

[[Page 52040]]

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 last 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
end points included serve as a convenient focus for this section, there
are many other end points for which there are no current EPA
guidelines. The goal of this document is to provide a framework for
interpreting data collected with tests frequently used by
neurotoxicologists.
Five categories of end points will be described: Structural or
neuropathological, neurophysiological, neurochemical, behavioral, and
developmental end points. Table 1 lists a number of end points in each
of these categories.

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

I. Structural or Neuropathological End Points

Gross changes in morphology, including brain weight
Hemorrhage in nerve tissue
Breakdown of neurons, glial cells
Accumulation, proliferation, or rearrangement of structural
elements
Glial fibrillary acidic protein increases (in adults)
II. Neurochemical End Points
Alterations in synthesis, release, uptake, degradation of
neurotransmitters
Alterations in second messenger associated signal
transduction
Alterations in membrane-bound enzymes regulating neuronal
activity
Inhibition of neuropathy target enzyme (<gr-thn-eq>40)
III. Neurophysiological End Points
Change in velocity, amplitude, or refractory period of nerve
conduction
Change in latency or amplitude of sensory-evoked potential
Change in electroencephalographic pattern
IV. Behavioral and Neurological End Points
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, intelligence, attention
V. Developmental End Points
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.
a. Structural End Points of Neurotoxicity. Structural end points
are typically defined as neuropathological changes measured through
gross observation or with the aid of a microscope. Gross changes in
morphology can include discrete or widespread lesions in nerve tissue.
Changes in brain size (weight, width, or length) are considered to be
indicative of neurotoxic events. This is true regardless of changes in
body weight, because brain size 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. The risk
assessor should be aware that a unit of measurement that is
biologically meaningful should be used for analysis. Brain length
measurements, for example, expressed to 1 or 10 micron units is
biologically meaningless. The same is true for brain width.
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 (WHO, 1986; Krinke, 1989;
Griffin, 1990). Neurodegenerative 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 terminal degeneration. 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 in humans
such as Parkinson's disease (WHO, 1986). Table 2 lists examples of such
neurotoxic chemicals, their putative site of action, the type of
neuropathology produced, and the disease or condition that each
typifies.

Table 2.--Neurotoxicants and Diseases With Specific Neuronal Targets

Corresponding
Site of action Neuropathology Neurotoxicant neurodegenerative disease
or condition

Neuron cell body............ Neuronopathy................ Methylmercury Quinolinic Minamata disease,
acid 3-Acetylpyridine. Huntington's disease,
Cerebellar ataxia.
Nerve terminal.............. Terminal destruction........ 1-Methyl-4-phenyl- Parkinson's disease.
1,2,3,6-
tetrahydropyridine
(dopaminergic).
Schwann cell Myelin......... Myelinopathy................ Hexachlorophene......... Congenital
hypomyelinogenesis.
Central-peripheral distal Distal axonopathy........... Acrylamide Carbon Peripheral neuropathy.
axon. disulfide n-Hexane.
Central axons............... Central axonopathy.......... Clioquinol.............. Subacute
myeloopticoneuropathy.
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

[[Page 52041]]

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 its functional specialization and
the lack of regenerative capacity in the central nervous system.
In general, chemical effects can lead to two types of structural
alteration at the cellular level: the breakdown of cells, in whole or
in part, or the accumulation, proliferation, or rearrangement of
structural elements (e.g., intermediate filaments, microtubules) or
organelles (e.g., mitochondria). Some changes may be associated with
regenerative processes that reflect adaptive changes associated with
exposure to a toxicant.
Chemically induced injury to the central nervous system may be
associated with astrocytic hypertrophy. Such changes may be seen using
immunocytochemical techniques visualized by light microscopy or
quantified more precisely by radioimmunoassay (RIA) procedures. Assays
of glial fibrillary acidic protein (GFAP), the major intermediate
filament protein of astrocytes, have been proposed as a biomarker of
this response (O'Callaghan, 1988). The interpretation of a chemicalinduced
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 in the Neurotoxicity
Test Battery testing guidelines (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.
Decreases in GFAP are not clearly interpretable as indicative of
neurotoxicity. The absence of a change in GFAP following exposure does
not necessarily 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 is confounded
by the possibility that chemically induced increases in GFAP may 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.
b. Neurophysiological End Points of Neurotoxicity.
Neurophysiological studies are those that 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 of neurophysiological measures of neurotoxicity 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

System/function Procedure Representative agents

Retina................. Electroretinography Developmental lead.
(ERG).
Visual pathway......... Flash evoked potential Carbon disulfide.
(FEP).
Visual function........ Pattern evoked Carbon disulfide.
potential (PEP)
pattern size and
contrast).
Auditory pathway....... Brain stem auditory Aminoglycoside,
evoked potential Antibiotics, Toluene,
(BAER) (clicks). styrene.
Auditory function...... BAER (tones)........... Aminoglycoside,
Antibiotics, Toluene,
styrene.
Somatosensory pathway.. Somatosensory evoked Acrylamide, n-Hexane.
potential (SEP)
(shocks).
Somatosensory function. SEP (tactile).......... Acrylamide n-Hexane.
Spinocerebellar pathway SEP recorded from Acrylamide n-Hexane.
cerebellum.
Mixed nerve............ Peripheral nerve Triethyltin.
compound action
potential (PNAP).
Motor axons............ PNAP isolate motor Triethyltin.
components.
Sensory axons.......... PNAP isolate sensory Triethyltin.
components.
Neuromuscular.......... Electromyography (EMG), Dithiobiuret.
H-reflex, M-response.
General central nervous Electroencephalography Anesthetics.
system/level of (EEG).
arousal.

(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 purely sensory

[[Page 52042]]

nerves such as the sural nerve or by measuring the muscle response
evoked by nerve stimulation to measure motor effects. While a number of
end points can be recorded, the most critical variables are (1) nerve
conduction velocity, (2) response amplitude, and (3) refractory period.
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 wellestablished
temperature factors or 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
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 provides a measure reflecting the functional status of nerve
membrane ion channels. Chemically induced changes in 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.
(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-meansquare,
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 must be interpreted in light of the behavioral status
of the subject and would generally be considered evidence of a
neurotoxic effect.
(3) Seizures/convulsions. Neurophysiological recordings of brain
electrical activity that demonstrate seizure-like activity are
indicative of a neurotoxic effect. Occasionally, behaviors resembling
convulsions might follow actions outside the nervous system, such as
direct effects on muscle. When convulsion-like behaviors are observed,
as described in the behavioral section, neurophysiological recordings
can determine if these behaviors

[[Page 52043]]
originate from seizure activity in the brain.
In addition to producing seizures directly, neurotoxicants also may
alter the frequency, severity, duration, or threshold for eliciting
seizures produced through other means. Such changes can occur after
acute exposure or after repeated exposure to dose levels below the
acute threshold and are considered to be neurotoxic effects. Examples
of agents that produce convulsions include lindane, DDT (dichlorodiphenyl
-trichloroethane), pyrethroids, and trimethyltin.
(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 stimuli
producing arousal (e.g., lights, sounds) and anesthetic drugs. In
studies with toxicants, changes in EEG pattern can sometimes precede
alterations in other objective signs of neurotoxicity (Dyer, 1987).
EEG studies must be done under highly controlled conditions, and
the data must be considered on a case-by-case basis. Chemically induced
seizure activity detected in the EEG pattern is evidence of a
neurotoxic effect.
c. Neurochemical End Points of Neurotoxicity. Many different
neurochemical end points 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). 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:
A. Inhibit sodium entry........ Tetrodotoxin.
B. Block closing of sodium p,p'-DDT, pyrethroids.
channel.
C. Increase permeability to Batrachotoxin.
sodium.
D. Increase intracellular Chlordecone.
calcium.
Cytotoxicants--Depend on uptake MPTP.
into nerve terminal.
Uptake blockers................. Hemicholinium.
Metabolic poisons............... Cyanide.
Hyperactivation of receptors.... Domoic acid.
Blocks transmitter release Botulinum toxin.
(Acetylcholine [ACh]).
Inhibition of transmitter Pesticides of the organophosphate
degradation (ACh). and carbamate classes.
Blocks axonal transport......... Acrylamide.

As stated previously, any neurochemical change is potentially
neurotoxic, but each determination requires professional judgment.
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 necessarily
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 prolongs
the action of the acetylcholine at the neuron's synaptic receptors and
is responsible for the autonomic stimulation and death that these
agents cause.
Within EPA and elsewhere, questions have arisen as to whether
inhibition of cholinesterase activity constitutes an adverse effect for
defining hazard potential and evaluating risk. There is agreement among
scientists that statistically significant inhibition of cholinesterase
activity in multiple organs and tissues accompanied by clinical effects
constitutes a hazard. However, there is scientific uncertainty and
related controversy about the risk assessment implications of data
describing inhibition of cholinesterase enzyme activity in the absence
of observable clinical effects. While there is agreement that such
inhibition is a biomarker of exposure, there is continued disagreement
over whether cholinesterase inhibition, especially in blood,
constitutes an adverse effect.
At this point, it can be stated that there is general agreement
among scientists that objective clinical measures of dysfunction/
impairment can be overt manifestations of inhibition of cholinesterase
in the nervous system. On the basis of clinical manifestations, e.g.,
muscle weakness, tremor, blurred vision, one should be able to evaluate
dose-response and dose-effect relationships and define the presence and
absence of given effects. A relationship between the effect and
cholinesterase inhibition should be

[[Page 52044]]

confirmed by biochemical measures of reduced cholinesterase activity.
In addition, a reduction in brain cholinesterase activity may or
may not be accompanied by clinical manifestations. Most experts in the
field acknowledge that when significant reductions in brain
cholinesterase activity alone occur, reduced cholinesterase levels
either are themselves toxic or would lead to a neurotoxic effect if
exposure were to persist over time or increase in magnitude. Therefore,
statistically significant decreases in brain cholinesterase could be
considered to be a biologically significant effect.
A reduction in RBC and/or plasma cholinesterase activity also may
or may not be accompanied by clinical manifestations. At this time,
there is general agreement that the observation of inhibition of RBC
and/or plasma cholinesterase contributes to the overall hazard
identification of cholinesterase inhibiting agents by serving as
biomarkers. As such, these enzyme parameters can provide information
that will help scientists evaluate whether reported clinical effects
are associated with cholinesterase inhibition. There remains, however,
a lack of consensus as to whether RBC and/or plasma cholinesterase
represent biologically significant events. Discussions on this topic
are continuing within the Agency.
A subset of organophosphate agents also produces organophosphateinduced
delayed neuropathy (OPIDN) after acute or repeated exposure.
Prolonged inhibition (i.e., aging) of neurotoxic esterase (or
neuropathy target enzyme) has been associated with agents that produce
OPIDN (Johnson, 1990), a clear neurotoxic effect.
d. Behavioral End Points of Neurotoxicity. 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 schedulecontrolled
behavior for assessing neurotoxic potential (U.S. EPA,
1991a). There are many other measures of behavior, including
specialized tests of motor and sensory function and of learning and
memory (Tilson, 1987; Anger, 1984). Examples of behavioral end points
that have been used to detect neurotoxicity are included in Table 1.
The risk assessor should know that the literature is clear that a
number of other behaviors besides those listed in Tables 1 and 5 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 a NOAEL or LOAEL
could be based on one or more of these end points.

Table 5.--Summary of Measures in a Representative Functional Observational Battery, and the Type of Data

Produced by Each

Home cage and open field Manipulative Physiologic

Posture (D) Ease of removal (R) Body temperature (I).
Convulsions, tremors (D) Handling reactivity (R) Body weight (I).
Palpebral closure (R) Palpebral closure (R). .......................

Lacrimation (R) Approach response (R).
Piloerection (Q) Click response (R).
Salivation (R) Touch response (R).
Vocalizations (Q) Tail pinch response (R).
Rearing (C) Righting reflex (R).
Urination (C) Landing foot splay (I).
Defecation (C) Forelimb grip strength (I).
Gait (D, R) Hindlimb grip strength (I).
Arousal (R) Pupil response (Q).
Mobility (R).
Stereotypy (D).
Bizarre behavior (D)

D--descriptive data; R--rank order data; Q--quantal data; I--interval data; C--count data.

Behavior is an indication of the overall well-being of the
organism. 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 is extremely important (e.g.,
the relationship between liver damage and motor activity). The presence
of systemic toxicity may complicate, but does not necessarily 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.
The following sections describe in general behavioral tests and
their uses and offer guidance on interpreting data.
(1) Functional observational battery. A functional observational
battery 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. FOB data may be in the form of interval, ordinal,
or continuous measurements.
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

[[Page 52045]]

incidence in relation to control animals. If only a few unrelated
measures in the FOB are affected, or the effects are unrelated to dose,
the results are not 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 no conclusive 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. Recently, it
was proposed that data from FOB studies be grouped into 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 is 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 Schaumberg, 1980).
(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 nonneurotoxic 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 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. 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 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.
In the adult, neurotoxic agents generally decrease motor activity
(MacPhail et al., 1989). Examples 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 developmental 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 type of effect 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-doserelated
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) 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 must 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). Rates and patterns of
SCOB display remarkable species and experimental generality.
In laboratory animals, SCOB has been used to study a wide range of
neurotoxicants, including methylmercury, many pesticides, carbon
disulfide, organic and inorganic lead, and triethyl and trimethyltin
(MacPhail, 1985; Tilson, 1987; Rice, 1988). The primary SCOB end points
for evaluation are response rate and the temporal pattern of
responding. These end points may vary as a function of the contingency
between responding and reinforcement presentation (i.e., schedule of
reinforcement). While most chemicals decrease the efficiency of
responding at some dose, some agents may increase response efficiency
on schedules requiring high response rates due to 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.
(4) Convulsions. Observable convulsions in animals are indicative
of an adverse effect. These events can

[[Page 52046]]

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 convulsion-like behaviors. In some cases, convulsions or
convulsion-like 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.
(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 well-known
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.

Table 6.--Examples of Specialized Behavioral Tests to Measure
Neurotoxicity

Function Procedure Representative agents

Neuromuscular:
Weakness........... Grip strength; swimming n-Hexane, methl nendurance;
suspension butylketone,
rod; discriminative carbaryl.
motor function.
Incoordination..... Rotorod, gait 3-Acetylpyridine,
measurements; righting ethanol.
reflex.
Tremor............. Rating scale, spectral Chlordecone, Type I
analysis. pyrethroids, DDT.
Myoclonia spasms... Rating scale, spectral DDT, Type II
analysis. pyrethroids.
Sensory:
Auditory........... Discrimination Toluene, trimethyltin.
conditioning Reflex
modification.
Visual............. Discrimination Methylmercury.
conditioning.
Somatosensory...... Discrimination Acrylamide.
conditioning.
Pain sensitivity... Discrimination Parathion.
conditioning
(titration);
functional
observational battery.
Olfactory.......... Discrimination 3-Methylindole,
conditioning. methylbromide.
Learning/Memory:
Habituation........ Startle reflex......... Diisopropylfluorophosphate
(DFP)
Pre/neonatal
methylmercury.
Classical Nictitating membrane... Aluminum.
conditioning. Conditioned flavor Carbaryl.
aversion. Trimethyltin, IDPN.
Passive avoidance...... Neonatal trimethyltin.
Olfactory conditioning.
Operant One-way avoidance...... Chlordecone.
conditioning. Two-way avoidance...... Pre/neonatal lead.
Y-maze avoidance....... Hypervitaminois A.
Biel water maze........ Styrene.
Morris water maze...... DFP.
Radial arm maze........ Trimethyltin.
Delayed matching to DFP.
sample. Carbaryl.
Repeated acquisition... Lead.
Visual discrimination..

A statistically significant chemically induced change in any
measure in Table 6 is presumptive evidence of adverse effect. 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.
(a) 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 weakness include measures of
grip strength, swimming endurance, suspension from a hanging rod, and
discriminative motor function. Rotarod and gait assessments are used to
measure incoordination, while rating scales and spectral analysis
techniques can be used to quantify tremor and other abnormal movements.
(b) Sensory function: Gross perturbations of sensory function can
be observed in simple neurological assessments such as the FOB.
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.
(c) Cognitive function: Alterations in learning and memory in
experimental animals must be inferred from changes in behavior
following exposure when compared with that either 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 must 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

[[Page 52047]]

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 interfere with cognitive function at the time of
testing. Older animals frequently perform poorly on some types of
tests, and it must 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
necessarily 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.
Examples of learning and memory procedures include simple habituation,
classical conditioning, and operant (or instrumental) conditioning,
including tests for spatial learning and memory.
e. Developmental Neurotoxicity. Although the previous discussion of
various neurotoxicity end points and tests applies to studies in which
developmental exposures are used, there are particular issues of
importance in the evaluation of developmental neurotoxicity studies.
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). Children are often
differentially sensitive to chemical exposure. 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, xirradiation,
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 to
be adverse effects.

Table 7.--Examples of Developmental Neurotoxicants

Alcohols........................... Methanol, ethanol.
Antimitotics....................... X-radiation, azacytidine.
Insecticides....................... DDT, kepone.
Metals............................. Lead, methylmercury, cadmium.
Polyhalogenated hydrocarbons....... PCBs, PBBs.
Solvents........................... Carbon disulfide, toluene.

Testing for developmental neurotoxicity has not been required
routinely by regulatory agencies in the United States, but is required
by the 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 wellconducted
adult neurotoxicity studies, standard developmental toxicity
studies, and multigeneration studies, although the dose levels used in
the latter may be lower than that 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, number of
litters large enough for adequate statistical power, randomization of
animals to dose groups and test groups, 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
evaluating neurologic 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 end points 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). Comparisons at the level of functional category (sensory,
motivational, cognitive, and motor function and social behavior) showed
close agreement across species for the agents evaluated, even though
the specific end points 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 necessarily be the same as those
that may be produced in humans. Therefore, when data suggesting adverse
effects in developmental neurotoxicity studies are encountered for
particular agents, they should be considered in the risk assessment
process.
Functional tests with a moderate degree of background variability
(e.g., a coefficient of variability of 20 percent or less) may be more
sensitive to the effects of an agent on behavioral end points than are
tests with low variability that may be impossible to disrupt without
using life-threatening doses. A battery of functional tests, in
contrast to a single test, is usually needed to evaluate the full
complement of nervous system functions in an animal. Likewise, a series
of tests conducted in animals in several age groups may provide more
information about maturational changes and their persistence than tests
conducted at a single age.
It is a well-established principle that there are critical
developmental periods for the disruption of functional competence,
which include both the prenatal and postnatal periods to the time of
sexual maturation, and the effect of a toxicant is likely to vary
depending on the time and degree of exposure (Rodier, 1978, 1990). It
is also important to consider the data from studies in which postnatal
exposure is included, as there may be an interaction of the agent with
maternal behavior, milk composition, pup suckling behavior, as well as
possible direct exposure of pups via dosed food or water (Kimmel et
al., 1992).
Agents that produce developmental neurotoxicity at a dose that is
not toxic to the maternal animal are of special concern. However,
adverse developmental effects are often produced at doses that cause
maternal toxicity (e.g., <20 percent reduction in weight gain during
gestation and lactation). In these cases, the

[[Page 52048]]
developmental effects are still considered to represent neurotoxicity
and should not be discounted as being secondary to maternal toxicity.
At doses causing moderate maternal toxicity (i.e., <gr-thn-eq>20
percent reduction in weight gain during gestation and lactation),
interpretation of developmental effects may be confounded. Current
information is inadequate to assume that developmental effects at doses
causing minimal maternal toxicity result only from maternal toxicity;
rather, it may be that the mother and developing organism are equally
sensitive to that dose level. Moreover, whether developmental effects
are secondary to maternal toxicity or not, the maternal effects may be
reversible while the effects on the offspring may be permanent. These
are important considerations for agents to which humans may be exposed
at minimally toxic levels either voluntarily or involuntarily, because
several agents are known to produce adverse developmental effects at
minimally toxic doses in adult humans (e.g., alcohol) (Coles et al.,
1991).
Although interpretation of developmental neurotoxicity data may be
limited, it is clear that functional effects must be evaluated in light
of other toxicity data, including other forms of developmental toxicity
(e.g., structural abnormalities, perinatal death, and growth
retardation). For example, alterations in motor performance may be due
to a skeletal malformation rather than nervous system change. Changes
in learning tasks that require a visual cue might be influenced by
structural abnormalities in the eye. The level of confidence that an
agent produces an adverse effect may be as important as the type of
change seen, and confidence may be increased by such factors as
reproducibility of the effect either in another study of the same
function or by convergence of data from tests that purport to measure
similar functions. A dose-response relationship is an extremely
important measure of a chemical's effect; in the case of developmental
neurotoxicity both monotonic and biphasic dose-response curves are
likely, depending on the function being tested. The EPA Guidelines for
Developmental Toxicity Risk Assessment (U.S. EPA, 1991b) may be
consulted for more information on interpreting developmental toxicity
studies. The endpoints frequently used to assess developmental
neurotoxicity in exposed children was recently reviewed by Winneke
(1995).
3. Other Considerations
a. Pharmacokinetics. Extrapolation of test results between species
can be aided considerably by data on the pharmacokinetics of a
particular agent in the species tested and, if possible, in humans.
Information on a toxicant's half-life, metabolism, absorption,
excretion, and distribution to the peripheral and central nervous
system may be useful in predicting risk. Of particular importance for
the pharmacokinetics of neurotoxicants is the blood-brain barrier,
which ordinarily excludes ionic and nonlipid soluble chemicals from the
central nervous system. The brain contains circumventricular organs
whose purpose seems to be to sense the chemical composition of the
peripheral circulation and activate mechanisms to bring the composition
of the blood back to equilibrium if disturbed. These areas are
technically inside the brain, but they lie outside of the blood-brainbarrier.
Therefore, chemicals from the periphery can pass directly into
the brain at these sites. The majority of these structures are located
within or near the hypothalamus, an area that is crucial for
maintenance of neuroendocrine function. Pharmacokinetic data may be
helpful in defining the dose-response curve, developing a more accurate
basis for comparing species sensitivity (including that of humans),
determining dosimetry at target sites, and comparing pharmacokinetic
profiles for various dosing regimens or routes of administration. The
correlation of pharmacokinetic parameters and neurotoxicity data may be
useful in determining the contribution of specific pharmacokinetic
processes to the effects observed.
b. Comparisons of Molecular Structure. Comparisons of the chemical
or physical properties of an agent with those of known neurotoxicants
may provide some indication of the potential for neurotoxicity. Such
information may be helpful for evaluating potential toxicity when only
minimal data are available. The structure-activity relationships (SAR)
of some chemical classes have been studied, including hexacarbons,
organophosphates, carbamates, and pyrethroids. Therefore, class
relationships or SAR may help predict neurotoxicity or interpret data
from neurotoxicological studies. Under certain circumstances (e.g., in
the case of new chemicals), this procedure is one of the primary
methods used to evaluate the potential for toxicity when little or no
empirical toxicity data are available. It should be recognized,
however, that effects of chemicals in the same class can vary widely.
Moser (1994), for example, reported that the behavioral effects of
prototypic cholinesterase-inhibiting pesticides differed qualitatively
in a battery of behavioral tests.
c. Statistical Considerations. Properly designed studies on the
neurotoxic effects of compounds will include appropriate statistical
tests of significance. In general, the likelihood of obtaining a
significant effect will depend jointly on the magnitude of the effect
and the variability obtained in control and treated groups. A number of
texts are available on standard statistical tests (e.g., Siegel, 1956;
Winer, 1971; Sokal and Rohlf, 1969; Salsburg, 1986; Gad and Weil,
1988).
Neurotoxicity data present some unique features that must be
considered in selecting statistical tests for analysis. Data may
involve several different measurement scales, including categorical
(affected or not), rank (more or less affected), and interval and ratio
scales of measurement (affected by some percentage). For example,
convulsions are usually recorded as being present or absent
(categorical), whereas neuropathological changes are frequently
described in terms of the degree of damage (rank). Many tests of
neurotoxicity involve interval or ratio measurements (e.g., frequency
of photocell interruptions or amplitude of an evoked potential), which
are the most powerful and sensitive scales of measurement. In addition,
measurements are frequently made repeatedly in control and treated
subjects, especially in the case of behavioral and neurophysiological
end points. For example, OPPTS guidelines for FOB assessment call for
evaluations before exposure and at several times during exposure in a
subchronic study (U.S. EPA, 1991a).
Descriptive data (categorical) and rank order data can be analyzed
using standard nonparametric techniques (Siegel, 1956). In some cases,
if it is determined that the data fit the linear model, the categorical
modeling procedure can be used for weighted least-squares estimation of
parameters for a wide range of general linear models, including
repeated-measures analyses. The weighted least-squares approach to
categorical and rank data allows computation of statistics for testing
the significance of sources of variation as reflected by the model. In
the case of studies assessing effects in the same animals at several
time points, univariate analyses can be carried out at each time point
when the overall dose effect or the dose-by-time interaction is
significant.

[[Page 52049]]

Continuous data (e.g., magnitude, rate, amplitude), if found to be
normally distributed, can be analyzed with general linear models using
a grouping factor of dose and, if necessary, repeated measures across
time (Winer, 1971). Univariate analyses of dose, comparing dose groups
to the control group at each time point, are performed when there is a
significant overall dose effect or a dose-by-time interaction. Post hoc
comparisons between control and treatment groups can be made following
tests for overall significance. In the case of multiple end points
within a series of evaluations, some type of correction for multiple
observations is warranted (Winer, 1971).
d. In Vitro Data in Neurotoxicology. Methods and procedures that
fall under the general heading of short-term tests include an array of
in vitro tests that have been proposed as alternatives to whole-animal
tests (Goldberg and Frazier, 1989). In vitro approaches use animal or
human cells, tissues, or organs and maintain them in a nutritive
medium. Various types of in vitro techniques produce data for
evaluating potential and known neurotoxic substances, including primary
cell cultures, cell lines, and cloned cells. While such procedures are
important in studying the mechanism of action of toxic agents, their
use in hazard identification in human health risk assessment has not
been explored to any great extent.
Data from in vitro procedures are generally based on simplified
approaches that require less time to yield information than do many in
vivo techniques. However, in vitro methods generally do not take into
account the distribution of the toxicant in the body, the route of
administration, or the metabolism of the substance. It also is
difficult to extrapolate in vitro data to animal or human neurotoxicity
end points, which include behavioral changes, motor disorders, sensory
and perceptual disorders, lack of coordination, and learning deficits.
In addition, data from in vitro tests cannot duplicate the complex
neuronal circuitry characteristic of the intact animal.
Many in vitro systems are now being evaluated for their ability to
predict the neurotoxicity of various agents seen in intact animals.
This validation process requires considerations in study design,
including defined end points of toxicity and an understanding of how a
test agent would be handled in vitro as compared to the intact
organism. Demonstrated neurotoxicity in vitro in the absence of in vivo
data is suggestive but inadequate evidence of a neurotoxic effect. In
vivo data supported by in vitro data enhance the reliability of the in
vivo results.

B. Dose-Response Evaluation

Dose-response evaluation is a critical part of hazard
characterization and involves the description of the dose response
relationship in the available data. Human studies covering a range of
exposures are rarely available and therefore animal data are typically
used for estimating exposure levels likely to produce adverse effects
in humans. Evidence for a dose-response relationship is an important
criterion in establishing a neurotoxic effect, although this analysis
may be limited when based on standard studies using three dose groups
or fewer. The evaluation of dose-response relationships includes
identifying effective dose levels as well as doses associated with no
increase in incidence of adverse effects when compared with controls.
Much of the focus is on identifying the critical effect(s) observed at
the lowest-observed-adverse-effect-level and the no-observed-adverseeffect
-level associated with that effect. The NOAEL is defined as the
highest dose at which there is no statistically or biologically
significant increase in the frequency of an adverse neurotoxic effect
when compared with the appropriate control group in a data base
characterized as having sufficient evidence for use in a risk
assessment (see section C). Although a threshold is assumed for
neurotoxic effects, the existence of a NOAEL in an animal study does
not prove or disprove the existence or level of a biological threshold.
Alternatively, mathematical modeling of the dose-response relationship
may be performed to determine a quantitative estimate of responses in
the experimental range. This approach can be used to determine a BMD,
which may be used in place of the NOAEL (Crump, 1994) (see DoseResponse
Analysis, Section IV).
In addition to identifying the NOAEL/LOAEL or BMD, the doseresponse
evaluation defines the range of doses that are neurotoxic for
a given agent, species, route of exposure, and duration of exposure. In
addition to these considerations, pharmacokinetic factors and other
aspects that might influence comparisons with human exposure scenarios
should be taken into account. For example, dose-response curves may
exhibit not only monotonic but also U-shaped or inverted U-shaped
functions (Davis and Svendsgaard, 1990). Such curves are hypothesized
to reflect multiple mechanisms of action, the presence of homeostatic
mechanisms, and/or activation of compensatory or protective mechanisms.
In addition to considering the shape of the dose-response curve, it
should also be recognized that neurotoxic effects vary in terms of
nature and severity across dose or exposure level. At high levels of
exposure, frank lesions accompanied by severe functional impairment may
be observed. Such effects are widely accepted as adverse. At
progressively lower levels of exposure, however, the lesions may become
less severe and the impairments less obvious. At levels of exposure
near the NOAEL and LOAEL, the effects will often be mild, possibly
reversible, and inconsistently found. In addition, the end points
showing responses may be at levels of organization below the whole
organism (e.g., neurochemical or electrophysiological end points). The
adversity of such effects can be contentious (e.g., cholinesterase
inhibition), yet it is such effects that are likely to be the focus of
risk assessment decisions. To the extent possible, this document
provides guidance on determining the adversity of neurotoxic effects.
However, the identification of a critical adverse effect often requires
considerable professional judgment and should consider factors such as
the biological plausibility of the effect, the evidence of a doseeffect
continuum, and the likelihood for progression of the effect with
continued exposure.

C. Characterization of the Health-Related Data Base

This section describes a scheme for characterizing the sufficiency
of evidence for neurotoxic effects. This scheme defines two broad
categories: sufficient and insufficient (Table 8). Categorization is
aimed at providing certain criteria for the Agency to use to define the
minimum evidence necessary to define hazards and to conduct doseresponse
analyses. It does not address the issues related to
characterization of risk, which requires analysis of potential human
exposures and their relation to potential hazards to estimate the risks
of those hazards from anticipated or estimated exposures.

Table 8.--Characterization of the Health-Related Database

Sufficient Evidence

The sufficient evidence category includes data that collectively
provide enough information to judge whether or not a human
neurotoxic hazard could exist. This category may include both human
and experimental animal evidence.

Sufficient Human Evidence

This category includes agents for which there is sufficient
evidence from

[[Page 52050]]
epidemiologic studies, e.g., case control and cohort studies, to judge
that some neurotoxic effect is associated with exposure. A case
series in conjunction with other supporting evidence may also be
judged ``sufficient evidence.'' Epidemiologic and clinical case
studies should discuss whether the observed effects can be
considered biologically plausible in relation to chemical exposure.
(Historically, much often has been made of the notion of causality
in epidemiologic studies. Causality is a more stringent criterion
than association and has become a topic of scientific and
philosophical debate. See Susser [1986], for example, for a
discussion of inference in epidemiology.)

Sufficient Experimental Animal Evidence/Limited or No Human Data

This category includes agents for which there is sufficient
evidence from experimental animal studies and/or limited human data
to judge whether a potential neurotoxic hazard may exist. Generally,
agents that have been tested according to current test guidelines
would be included in this category. The minimum evidence necessary
to judge that a potential hazard exists would be data demonstrating
an effect in a single appropriate, well-executed study in a single
experimental animal species, whereas the minimum evidence needed to
judge that a potential hazard does not exist would include data from
appropriate, well-executed laboratory animal studies that evaluated
a variety of the potential manifestations of neuroxtoxicity and
showed no effects at doses that were at least minimally toxic.
Information on pharmacokinetics, mechanisms, or known properties of
the chemical class may also strengthen the evidence.

Insufficient Evidence

This category includes agents for which there is less than the
minimum evidence sufficient for identifying whether or not a
neurotoxic hazard exists, such as agents for which there are no data
on neurotoxicity or agents with data bases from studies in animals
or humans that are limited by study design or conduct (e.g.,
inadequate conduct or report of clinical signs). Many general
toxicity studies, for example, are considered insufficient in terms
of the conduct of clinical neurobehavioral observations or the
number of samples taken for histopathology of the nervous system.
Thus, a battery of negative toxicity studies with these shortcomings
would be regarded as providing insufficient evidence of the lack of
a neurotoxic effect of the test material. Further, most screening
studies based on simple observations involving autonomic and motor
function provide insufficient evaluation of many sensory or
cognitive functions. Data, which by itself would likely fall in this
category, would also include information on structure-activity
relationships or data from in vitro tests. While such information
would be insufficient by itself to proceed further in the assessment
it could be used to support the need for additional testing.
Data from all potentially relevant studies, whether indicative
of potential hazard or not, should be included in this
characterization. The primary sources of data are human studies and
case reports, experimental animal studies, other supporting data,
and in vitro and/or structure-activity relationship data. Because a
complex interrelationship exists among study design, statistical
analysis, and biological significance of the data, a great deal of
scientific judgment, based on experience with neurotoxicity data and
with the principles of study design and statistical analysis, is
required to adequately evaluate the data base on neurotoxicity. In
many cases, interaction with scientists in specific disciplines
either within or outside the field of neurotoxicology (e.g.,
epidemiology, statistics) may be appropriate.
The adverse nature of different neurotoxicity end points may be
a complex judgment. In general, most neuropathological and many
neurobehavioral changes are regarded as adverse. However, there are
adverse behavioral effects that may not reflect a direct action on
the nervous system. Neurochemical and electrophysiological changes
may be regarded as adverse as a function of their known or presumed
relation to neuropathological and/or neurobehavioral consequences.
In the absence of supportive information, a professional judgment
must be made regarding the adversity of such outcomes, considering
factors such as the nature, magnitude, and duration of the effects
reported. Thus, correlated measures of neurotoxicity strengthen the
evidence for a hazard. Correlations between functional and
morphological effects, such as the correlation between leg weakness
and paralysis and peripheral nerve damage from exposure to triortho
-cresyl phosphate, are the most common and striking example of
this form of validity. Correlations support a coherent and logical
link between behavioral effects and biochemical mechanisms.
Replication of a finding also strengthens the evidence for a hazard.
Some neurotoxicants cause similar effects across most species. Many
chemicals shown to produce neurotoxicity in laboratory animals have
similar effects in humans. Some neurologic effects may be considered
adverse even if they are small in magnitude, reversible, or the
result of indirect mechanisms.
Because of the inherent difficulty in ``proving any negative,''
it is more difficult to document a finding of no apparent adverse
effect than a finding of an adverse effect. Neurotoxic effects (and
most kinds of toxicity) can be observed at many different levels, so
that only a single end point needs to be found to demonstrate a
hazard, but many end points need to be examined to demonstrate no
effect. For example, to judge that a hazard for neurotoxicity could
exist for a given agent, the minimum evidence sufficient would be
data on a single adverse end point from a well-conducted study. In
contrast, to judge that an agent is unlikely to pose a hazard for
neurotoxicity, the minimum evidence would include data from a host
of end points that revealed no neurotoxic effects. This may include
human data from appropriate studies that could support a conclusion
of no evidence of a neurotoxic effect. With respect to clinical
signs and symptoms, human exposures can reveal far more about the
absence of effects than animal studies, which are confined to the
signs examined.
In some cases, it may be that no individual study is judged
sufficient to establish a hazard, but the total available data may
support such a conclusion. Pharmacokinetic data and structureactivity
considerations, data from other toxicity studies, as well
as other factors may affect the strength of the evidence in these
situations. For example, given that gamma diketones are known to
cause motor system neurotoxicity, a marginal data set on a candidate
gamma diketone, e.g., \1/10\ animals affected, might be more likely
to be judged sufficient than equivalent data from a member of a
chemical class about which nothing is known.
A judgment that the toxicology data base is sufficient to
indicate a potential neurotoxic hazard is not the end of analysis.
The circumstances of expression of hazard are essential to
describing human hazard potential. Thus, reporting should contain
the details of the circumstances under which effects have been
observed, e.g., ``long-term oral exposures of adult rodents to
compound X at levels of roughly 1 mg/kg have been associated with
ataxia and peripheral nerve damage.''

IV. Dose-Response Analysis

This section describes several approaches (including the LOAEL/
NOAEL and BMD) for determining the reference dose or reference
concentration. The NOAEL or BMD/uncertainty factor approach results in
a RfD or RfC, which is an estimate (with uncertainty spanning perhaps
an order of magnitude) of a daily exposure to the human population
(including sensitive subgroups) that is likely to be without an
appreciable risk of deleterious effects during a lifetime.
The dose-response analysis characterization should:
a. Describe how the RfD/RfC was calculated;
b. Discuss the confidence in the estimates;
c. Describe the assumptions or uncertainty factors used; and
d. Discuss the route and level of exposure observed, as compared to
expected human exposures. (Specifically, are the available data from
the same route of exposure as the expected human exposures? How many
orders of magnitude do you need to extrapolate from the observed data
to environmental exposures?)

A. LOAEL/NOAEL and Benchmark Dose (BMD) Determination

As indicated earlier, the LOAEL and NOAEL are determined for
endpoints that are seen at the lowest dose level (so-called critical
effect). Several limitations in the use of the NOAEL have been
identified and described (e.g., Barnes and Dourson, 1988; Crump, 1984).
For example, the NOAEL is derived from a single end point from a single
study (the critical study) and

[[Page 52051]]
ignores both the slope of the dose-response function and baseline
variability in the end point of concern. Because the baseline
variability is not taken into account, the NOAEL from a study using
small group sizes may be higher than the NOAEL from a similar study in
the same species that uses larger group sizes. The NOAEL is also
directly dependent on the dose spacing used in the study. Finally, and
perhaps most importantly, use of the NOAEL does not allow estimates of
risk or extrapolation of risk to lower dose levels.
Because of these and other limitations in the NOAEL approach,
mathematical curve-fitting techniques (Crump, 1984; Gaylor and Slikker,
1990; Glowa, 1991; U.S. EPA, 1995a) are beginning to be used with, or
as an alternative to, the NOAEL in calculating the RfD or RfC. The
Agency is in the process of implementing these newer techniques and
strongly encourages the calculation of BMDs for neurotoxicity and other
health effect end points. These techniques typically apply a
mathematical function that describes the dose-response relationship and
then interpolate to a level of exposure associated with a small
increase in effect over that occurring in the control group or under
baseline conditions. The BMD has been defined as a lower confidence
limit on the effective dose associated with some defined level of
effect, e.g., a 5 percent or 10 percent increase in response (i.e., a
BMD<INF>05 or BMD<INF>10 for a particular effect). Because the model is
only used to interpolate within the dose range of the study, no
assumptions about the existence (or nonexistence) of a threshold are
needed. Thus, any model that fits the data well is likely to provide a
reasonable estimate of the BMD.
Many neurotoxic end points provide continuous measures of response,
such as response speed, nerve conduction velocity, IQ score, degree of
enzyme inhibition, or the accuracy of task performance. Although it is
possible to impose a dichotomy on a continuous effects distribution and
to classify some level of response as ``affected'' and the remainder as
``unaffected,'' it may be very difficult and inappropriate to establish
such clear distinctions, because such a dichotomy would misrepresent
the true nature of the neurotoxic response. Alternatively, quantitative
models designed to analyze continuous effect variables may be
preferable. Other techniques that allow this approach, with
transformation of the information into estimates of the incidence or
frequency of affected individuals in a population, have been proposed
(Crump, 1984; Gaylor and Slikker, 1990). Categorical regression
analysis has been proposed since it can evaluate different types of
data and derive estimates for short-term exposures (Rees and Hattis,
1994). Decisions about the most appropriate approach require
professional judgment, taking into account the biological nature of the
continuous effect variable and its distribution in the population under
study.
Although dose-response functions in neurotoxicology are generally
linear or monotonic, curvilinear functions, especially U-shaped or
inverted U-shaped curves, have been reported as noted earlier (Section
III B). Dose-response analyses should consider the uncertainty that Ushaped
dose-response functions might contribute to the estimate of the
NOAEL/LOAEL or BMD. Typically, estimates of the NOAEL/LOAEL are taken
from the lowest part of the dose-response curve associated with
impaired function or adverse effect.

B. Determination of the Reference Dose or Reference Concentration

Since the availability of dose-response data in humans is limited,
extrapolation of data from animals to humans usually involves the
application of uncertainty factors to the NOAEL/LOAEL or BMD. The NOAEL
or BMD/uncertainty factor approach results in a RfD or RfC, which is an
estimate (with uncertainty spanning perhaps an order of magnitude) of a
daily exposure to the human population (including sensitive subgroups)
that is likely to be without an appreciable risk of deleterious effects
during a lifetime. The oral RfD and inhalation RfC are applicable to
chronic exposure situations and are based on an evaluation of all the
noncancer health effects, including neurotoxicity data. RfDs and RfCs
in the Integrated Risk Information System (IRIS-2) data base for
several agents are based on neurotoxicity end points and include a few
cases in which the RfD or RfC is calculated using the BMD approach
(e.g., methylmercury, carbon disulfide). The size of the final
uncertainty factor used will vary from agent to agent and will require
the exercise of scientific judgment, taking into account interspecies
differences, the shape of the dose-response curve, and the
neurotoxicity end points observed. Default uncertainty factors are
typically multiples of 10 and are used to compensate for human
variability in sensitivity, the need to extrapolate from animals to
humans, and the need to extrapolate from less than lifetime (e.g.,
subchronic) to lifetime exposures. An additional factor of up to 10 may
be included when only a LOAEL (and not a NOAEL) is available from a
study, or depending on the completeness of the data base, a modifying
factor of up to 10 may be applied, depending on the confidence one has
in the data base. Barnes and Dourson (1988) provide a more complete
description of the calculation, use, and significance of RfDs in
setting exposure limits to toxic agents by the oral route. Jarabek et
al. (1990) provide a more complete description of the calculation, use,
and significance of RfCs in setting exposure limits to toxic agents in
air. Neurotoxicity can result from acute, shorter term exposures, and
it may be appropriate in some cases, e.g., for air pollutants or water
contaminants, to set shorter term exposure limits for neurotoxicity as
well as for other noncancer health effects.

V. Exposure Assessment

Exposure assessment describes the magnitude, duration, frequency,
and routes of exposure to the agent of interest. This information may
come from hypothetical values, models, or actual experimental values,
including ambient environmental sampling results. Guidelines for
exposure assessment have been published separately (U.S. EPA, 1992) and
will, therefore, be discussed only briefly here.
The exposure assessment should include an exposure characterization
that:
a. Provides a statement of the purpose, scope, level of detail, and
approach used in the exposure assessment;
b. Presents the estimates of exposure and dose by pathway and route
for individuals, population segments, and populations in a manner
appropriate for the intended risk characterization;
c. Provides an evaluation of the overall level of confidence in the
estimate of exposure and dose and the conclusions drawn; and
d. Communicates the results of the exposure assessment to the risk
assessor, who can then use the exposure characterization, along with
the characterization of the other risk assessment elements, to develop
a risk characterization.
A number of considerations are relevant to exposure assessment for
neurotoxicants. An appropriate evaluation of exposure should consider
the potential for exposure via ingestion, inhalation, and dermal
penetration from relevant sources of exposures, including multiple
avenues of intake from the same source. On-going Agency activities that
support neurotoxicity exposure

[[Page 52052]]

assessment include characterizing cumulative risk and revising the
Guidelines for the Health Risk Assessment of Chemical Mixtures.
In addition, neurotoxic effects may result from short-term (acute),
high-concentration exposures as well as from longer term (subchronic),
lower level exposures. Neurotoxic effects may occur after a period of
time following initial exposure or be obfuscated by repair mechanisms
or apparent tolerance. The type and severity of effect may depend
significantly on the pattern of exposure rather than on the average
dose over a long period of time. For this reason, exposure assessments
for neurotoxicants may be much more complicated than those for longlatency
effects such as carcinogenicity. It is rare for sufficient data
to be available to construct such patterns of exposure or dose, and
professional judgment may be necessary to evaluate exposure to
neurotoxic agents.

VI. Risk Characterization

A. Overview

Risk characterization, the culmination of the risk assessment
process, consists of an integrative analysis and a risk
characterization summary. The integrative analysis (a) involves
integration of the toxicity information from the hazard
characterization and dose-response analysis with the human exposure
estimates, (b) provides an evaluation of the overall quality of the
assessment and the degree of confidence in the estimates of risk and
conclusions drawn, and (c) describes risk in terms of the nature and
extent of harm. The risk characterization summary communicates the
results of the risk assessment to the risk manager.
This summary should include but is not limited to a discussion of
the following elements:
a. Quality of and confidence in the available data;
b. Uncertainty analysis;
c. Justification of defaults or assumptions;
d. Related research recommendations;
e. Contentious issues and extent of scientific consensus;
f. Effect of reasonable alternative assumptions on conclusions and
estimates;
g. Highlight reasonable plausible ranges;
h. Reasonable alternative models; and
i. Perspective through analogy.
The risk manager can then use the risk assessment, along with other
risk management elements, to make public health decisions.
An effective risk characterization must fully, openly, and clearly
characterize risks and disclose the scientific analyses, uncertainties,
assumptions, and science policies that underlie decisions throughout
the risk assessment and risk management processes. The risk
characterization must feature values such as transparency in the
decision-making process; clarity in communicating with each other and
the public regarding environmental risk and the uncertainties
associated with assessments of environmental risk; and consistency
across program offices in core assumptions and science policies, which
are well grounded in science and reasonable.
The following sections describe these four aspects of the risk
characterization in more detail.

B. Integration of Hazard Characterization, Dose-Response Analysis and
Exposure Assessment

In developing the hazard characterization, dose-response analysis
and exposure portions of the risk assessment, the assessor must take
into account many judgments concerning human relevance of the toxicity
data, including the appropriateness of the various animal models for
which data are available and the route, timing, and duration of
exposure relative to expected human exposure. These judgments should be
summarized at each stage of the risk assessment process (e.g., the
biological relevance of anatomical variations may be established in the
hazard characterization process, or the influence of species
differences in metabolic patterns in the dose-response analysis). In
integrating the information from the assessment, the risk assessor must
determine if some of these judgments have implications for other
portions of the assessment and whether the various components of the
assessment are compatible.
The risk characterization should not only examine the judgments but
also explain the constraints of available data and the state of
knowledge about the phenomena studied in making them, including (1) the
qualitative conclusions about the likelihood that the chemical may pose
a specific hazard to human health, the nature of the observed effects,
under what conditions (route, dose levels, time, and duration) of
exposure these effects occur, and whether the health-related data are
sufficient to use in a risk assessment; (2) a discussion of the doseresponse
characteristics of the critical effects(s), data such as the
shapes and slopes of the dose-response curves for the various end
points, the rationale behind the determination of the NOAEL and LOAEL
and calculation of the benchmark dose, and the assumptions underlying
the estimation of the RfD or RfC; and (3) the estimates of the
magnitude of human exposure; the route, duration, and pattern of the
exposure; relevant pharmacokinetics; and the number and characteristics
of the population(s) exposed.
If data to be used in a risk characterization are from a route of
exposure other than the expected human exposure, then pharmacokinetic
data should be used, if available, to make extrapolations across routes
of exposure. If such data are not available, the Agency makes certain
assumptions concerning the amount of absorption likely or the
applicability of the data from one route to another (U.S. EPA, 1992).
The level of confidence in the hazard characterization should be
stated to the extent possible, including the appropriate category
regarding sufficiency of the health-related data. A comprehensive risk
assessment ideally includes information on a variety of end points that
provide insight into the full spectrum of potential neurotoxicological
responses. A profile that integrates both human and test species data
and incorporates a broad range of potential adverse neurotoxic effects
provides more confidence in a risk assessment for a given agent.
The ability to describe the nature of the potential human exposure
is important to predict when certain outcomes can be anticipated and
the likelihood of permanence or reversibility of the effect. An
important part of this effort is a description of the nature of the
exposed population and the potential for sensitive, highly susceptible,
or highly exposed populations. For example, the consequences of
exposure to the developing individual versus the adult can differ
markedly and can influence whether the effects are transient or
permanent. Other considerations relative to human exposures might
include the likelihood of exposures to other agents, concurrent
disease, and nutritional status.
The presentation of the integrated results of the assessment should
draw from and highlight key points of the individual characterizations
of component analyses performed under these Guidelines. The overall
risk characterization represents the integration of these component
characterizations. If relevant risk assessments on the agent or an
analogous agent have been done by EPA or other Federal agencies, these
should

[[Page 52053]]

be described and the similarities and differences discussed.

C. Quality of the Data Base and Degree of Confidence in the Assessment

The risk characterization should summarize the kinds of data
brought together in the analysis and the reasoning on which the
assessment is based. The description should convey the major strengths
and weaknesses of the assessment that arise from availability of data
and the current limits of our understanding of the mechanisms of
toxicity.
Health risk is a function of the hazard characterization, doseresponse
analysis, and exposure assessment. Confidence in the results
of a risk assessment is, thus, a function of confidence in the results
of the analysis of these elements. Each of these elements should have
its own characterization as a part of the assessment. Within each
characterization, the important uncertainties of the analysis and
interpretation of data should be explained, and the risk manager should
be given a clear picture of consensus or lack of consensus that exists
about significant aspects of the assessment. Whenever more than one
view is supported by the data and choosing between them is difficult,
all views should be presented. If one has been selected over the
others, the rationale should be given; if not, then all should be
presented as plausible alternative results.

D. Descriptors of Neurotoxicity Risk

There are a number of ways to describe risks. Several ways that are
relevant to describing risks for neurotoxicity are as follows:

Estimation of the Number of Individuals
The RfD or RfC is taken to be a chronic exposure level at or below
which no significant risk occurs. Therefore, presentation of the
population in terms of those at or below the RfD or RfC (``not at
risk'') and above the RfD or RfC (``may be at risk'') may be useful
information for risk managers. This method is particularly useful to a
risk manager considering possible actions to ameliorate risk for a
population. If the number of persons in the at-risk category can be
estimated, then the number of persons removed from the at-risk category
after a contemplated action is taken can be used as an indication of
the efficacy of the action.
Presentation of Specific Scenarios
Presenting specific scenarios in the form of ``what if?'' questions
is particularly useful to give perspective to the risk manager,
especially where criteria, tolerance limits, or media quality limits
are being set. The question being asked in these cases is, at this
proposed limit, what would be the resulting risk for neurotoxicity
above the RfD or RfC?
Risk Characterization for Highly Exposed Individuals
This measure is one example of the just-discussed descriptor. This
measure describes the magnitude of concern at the upper end of the
exposure distribution. This allows risk managers to evaluate whether
certain individuals are at disproportionately high or unacceptably high
risk.
The objective of looking at the upper end of the exposure
distribution is to derive a realistic estimate of a relatively highly
exposed individual or individuals. This measure could be addressed by
identifying a specified upper percentile of exposure in the population
and/or by estimating the exposure of the highest exposed individual(s).
Whenever possible, it is important to express the number of individuals
who comprise the selected highly exposed group and discuss the
potential for exposure at still higher levels.
If population data are absent, it will often be possible to
describe a scenario representing high-end exposures using upper
percentile or judgment-based values for exposure variables. In these
instances caution should be used not to compound a substantial number
of high-end values for variables if a ``reasonable'' exposure estimate
is to be achieved.
Risk Characterization for Highly Sensitive or Susceptible
Individuals
This measure identifies populations sensitive or susceptible to the
effect of concern. Sensitive or susceptible individuals are those
within the exposed population at increased risk of expressing the toxic
effect. All stages of nervous system maturation might be considered
highly sensitive or susceptible, but certain subpopulations can
sometimes be identified because of critical periods for exposure, for
example, pregnant or lactating women, infants, children.
In general, not enough is understood about the mechanisms of
toxicity to identify sensitive subgroups for all agents, although
factors such as nutrition, personal habits (e.g., smoking, alcohol
consumption, illicit drug abuse), or preexisting disease (e.g.,
diabetes, sexually transmitted diseases) may predispose some
individuals to be more sensitive to the neurotoxic effects of various
agents.
Other Risk Descriptors
In risk characterization, dose-response information and the human
exposure estimates may be combined either by comparing the RfD or RfC
and the human exposure estimate or by calculating the margin of
exposure (MOE). The MOE is the ratio of the NOAEL from the most
appropriate or sensitive species to the estimated human exposure level.
If a NOAEL is not available, a LOAEL may be used in calculating the
MOE. Alternatively, a benchmark dose may be compared with the estimated
human exposure level to obtain the MOE. Considerations for the
evaluation of the MOE are similar to those for the uncertainty factor
applied to the LOAEL/NOAEL or the benchmark dose. The MOE is presented
along with a discussion of the adequacy of the data base, including the
nature and quality of the hazard and exposure data, the number of
species affected, and the dose-response information.
The RfD or RfC comparison with the human exposure estimate and the
calculation of the MOE are conceptually similar but are used in
different regulatory situations. The choice of approach depends on
several factors, including the statute involved, the situation being
addressed, the data base used, and the needs of the decision maker. The
RfD or RfC and the MOE are considered along with other risk assessment
and risk management issues in making risk management decisions, but the
scientific issues that must be taken into account in establishing them
have been addressed here.
If the MOE is equal to or more than the uncertainty factor x any
modifying factor used as a basis for an RfD or RfC, then the need for
regulatory concern is likely to be small. Although these methods of
describing risk do not actually estimate risks per se, they give the
risk manager some sense of how close the exposures are to levels of
concern.

E. Communicating Results

Once the risk characterization is completed, the focus turns to
communicating results to the risk manager. The risk manager uses the
results of the risk characterization along with other technological,
social, and economic considerations in reaching a regulatory decision.
Because of the way in which these risk management factors

[[Page 52054]]

may affect different cases, consistent but not necessarily identical
risk management decisions must be made on a case-by-case basis. These
Guidelines are not intended to give guidance on the nonscientific
aspects of risk management decisions.

F. Summary and Research Needs

These Guidelines summarize the procedures that the U.S.
Environmental Protection Agency would use in evaluating the potential
for agents to cause neurotoxicity. These Guidelines discuss the general
default assumptions that should be made in risk assessment for
neurotoxicity because of gaps in our knowledge about underlying
biological processes and how these compare across species. Research to
improve the risk assessment process is needed in a number of areas. For
example, research is needed to delineate the mechanisms of
neurotoxicity and pathogenesis, provide comparative pharmacokinetic
data, examine the validity of short-term in vivo and in vitro tests,
elucidate the functional modalities that may be altered, develop
improved animal models to examine the neurotoxic effects of exposure
during the premating and early postmating periods and in neonates,
further evaluate the relationship between maternal and developmental
toxicity, provide insight into the concept of threshold, develop
approaches for improved mathematical modeling of neurotoxic effects,
improve animal models for examining the effects of agents given by
various routes of exposure, and address the synergistic or antagonistic
effects of mixtures of chemicals and neurotoxic response. Such research
will aid in the evaluation and interpretation of data on neurotoxicity
and should provide methods to assess risk more precisely.

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Winneki, G. (1995) Endpoints of developmental neurotoxicity in
environmentally exposed children. Toxicol. Lett. 77:127-136.
World Health Organization. (1986) Principles and methods for the
assessment of neurotoxicity associated with exposure to chemicals.
In: Environmental Health Criteria Document 60. Geneva: World Health
Organization.

[FR Doc. 95-24652 Filed 10-3-95; 8:45 am]
BILLING CODE 6560-50-P

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Proposed Guidelines for Neurotoxicity Risk Assessment

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