Thursday
May 14, 1998
Part HI
Environmental
Protection Agency
Guidelines For Neurotoxicity Risk
Assessment; Notice
2692
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Federal Register/Vol. 63, No. 93/Thursday, May 14, 1998/Notices
ENVIRONMENTAL PROTECTION
AGENCY
[FRL-6011-3]
RIN 2080-AA08
Guidelines for Neurotoxicity Risk
Assessment
AGENCY: Environmental Protection
Agency.
ACTION: Notice of availability of final
Guidelines for Neurotoxicity Risk
Assessment.
SUMMARY: The U.S. Environmental
Protection Agency (EPA) is today
publishing in final form a document
entitled Guidelines for Neurotoxicity
Risk Assessment (hereafter
"Guidelines"). These Guidelines were
developed as part of an interoffice
guidelines development program by a
Technical Panel of the Risk Assessment
Forum. The Panel was composed of
scientists from throughout die Agency,
and selected drafts were peer-reviewed
internally and by experts from
universities, environmental groups,
industry, and other governmental
agencies. The Guidelines are based, in
part, on recommendations derived from
various scientific meetings and
workshops on neurotoxicology, from
public comments, and from
recommendations of the Science
Advisory Board. An earlier draft
underwent external peer review in a
workshop held on June 2-3, 1992, and
received internal review by the Risk
Assessment Forum. The Risk
Assessment Subcommittee of the
Committee on the Environment and
Natural Resources of Office of Science
and Technology Policy reviewed the
proposed Guidelines during a meeting
held on August 15, 1995. The
Guidelines were revised and proposed
for public comment on October 4, 1995
(60 FR 52032-52056). The proposed
Guidelines were reviewed by the
Science Advisory Board on July 18,
1996. EPA appreciates the efforts of all
participants in the process, and has
tried to address their recommendations
in these Guidelines.
This notice describes the scientific
basis for concern about exposure to
agents that cause neurotoxicity, outlines
the general process for assessing
potential risk to humans because of
environmental contaminants, and
addresses Science Advisory Board and
public comments on the 1995 Proposed
Guidelines for Neurotoxicity Risk
Assessment (60 FR-.52032-52056). These
Guidelines are intended to guide
Agency evaluation of agents that are
suspected to cause neurotoxicity, in line
with the policies and procedures
established in the statutes administered
by the Agency.
DATES: The Guidelines will be effective
on April 30,1998.
ADDRESSES: The Guidelines will be
made available in several ways:
(1) The electronic version will be
accessible from EPA's National Center
for Environmental Assessment home
page on the Internet at http://
www.epa.gov/ncea.
(2) 3 Va" high-density computer
diskettes in WordPerfect format will be
available from ORD Publications,
Technology Transfer and Support
Division, National Risk Management
Research Laboratory, Cincinnati, OH;
Tel: 513-569-7562; Fax: 513-569-7566.
Please provide the EPA No.: EPA/630/
R-95/001Fa when ordering.
(3) This notice contains the full
document. Copies of the Guidelines will
be available for inspection at EPA
headquarters and regional libraries,
through the U.S. Government
Depository Library program, and for
purchase from the National Technical
Information Service (NITS), Springfield,
VA; telephone: 703-487-4650, fax: 703-
321-8547. Please provide the NTIS PB
No. (PB98-117831) when ordering.
FOR FURTHER INFORMATION CONTACT: Dr.
Hugh A. Tilson, Neurotoxicology
Division, National Health and
Environmental Effects Research
Laboratory, U.S. Environmental
Protection Agency, Research Triangle
Park, NC 27711, Tel: 919-541-2671;
Fax: 919-541-4849; E-mail:
tilson.hugh@epamail.epa.gov.
SUPPLEMENTARY INFORMATION : In its 1983
book Risk Assessment in the Federal
Government: Managing the Process, the
National Academy of Sciences
recommended that Federal regulatory
agencies establish "inference
guidelines" to promote consistency and
technical quality in risk assessment, and
to ensure that the risk assessment
process is maintained as a scientific
effort separate from risk management. A
task force within EPA accepted that
recommendation and requested that
Agency scientists begin to develop such
guidelines. In 1984, EPA scientists
began work on risk assessment
guidelines for carcinogenicity,
mutagenicity, suspect developmental
toxicants, chemical mixtures, and
exposure assessment. Following
extensive scientific and public review,
these first five guidelines were issued
on September 24, 1986 (51 FR 33992-
34054). Since 1986, additional risk
assessment guidelines have been
proposed, revised, reproposed, and
finalized. These guidelines continue the
process initiated in 1984. As with other
EPA guidelines (e.g., developmental
toxicity, 56 FR 63798-63826; exposure
assessment, 57 FR 22888-22938; and
carcinogenicity, 61 FR 17960-18011),
EPA will revisit these guidelines as
experience and scientific consensus
evolve.
These Guidelines set forth principles
and procedures to guide EPA scientists
in the conduct of Agency risk
assessments and to inform Agency
decision makers and the public about
these procedures. Policies in this
document are intended as internal
guidance for EPA. Risk assessors and
risk managers at EPA are the primary
audience, although these Guidelines
may be useful to others outside the
Agency. In particular, the Guidelines
emphasize that risk assessments will be
conducted on a case-by-case basis,
giving full consideration to all relevant
scientific information. This approach
means that Agency experts study
scientific information on each chemical
under review and use the most
scientifically appropriate interpretation
to assess risk. The Guidelines also stress
that this information will be fully
presented in Agency risk assessment
documents, and that Agency scientists
will identify the strengths and
weaknesses of each assessment by
describing uncertainties, assumptions,
and limitations, as well as the scientific
basis and rationale for each assessment.
The Guidelines are formulated in part to
bridge gaps in risk assessment
methodology and data. By identifying
these* gaps and the importance of the
missing information to the risk
assessment process, EPA wishes to
encourage research and analysis that
will lead to new risk assessment
methods and data.
Dated: April 30, 1998.
Carol M. Browner,
Administrator.
Contents
Part A: Guidelines for Neurotoxicity Risk
Assessment
List of Tables
1. Introduction
1.1. Organization of These Guidelines
1.2. The Role of Environmental Agents in
Neurotoxicity
1.3. Neurotoxicity Risk Assessment
1.4. Assumptions
2. Definitions and Critical Concepts
3. Hazard Characterization
3.1. Neurotoxicological Studies: Endpoints
and Their Interpretation
3.1.1. Human Studies
3.1.1.1. Clinical Evaluations
3.1.1.2. Case Reports
3.1.1.3. Epidemiologic Studies
3.1.1.4. Human Laboratory Exposure
Studies
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3,1.2. Animal Studies*"
3.1.2.1. Structural Endpoints of
Neurotoxiciry
3,1.2.2. Neurophysiological Endpoints of
Neurotoxicity
3.1.2.3. Neurochemical Endpoints of
Neurotoxicity
3.1.2.4. Behavioral Endpoints of
Neurotoxicity
3,1.3. Other Considerations
3.1.3.1. Pharmacokinetics
3.1.3.2. Comparisons of Molecular
Structure
3.1.3.3. Statistical Considerations
3.1.3.4. In Vitro Data in Neurotoxicology
3.1.3.5. Neuroendocrine Effects
3.2. Dose-Response Evaluation
3.3. Characterization of the Health-Related
Database
4, Quantitative Dose-Response Analysis
4.1. LOAEL/NOAEL and BMD
Determination
4.2. Determination of the Reference Dose or
Reference Concentration
5. Exposure Assessment
6. Risk Characterization
6.1. Overview
6.2. Integration of Hazard Characterization,
Dose-Response Analysis, and Exposure
Assessment
6.3.Qualityofthe Database and Degree of
Confidence in the Assessment
6.4. Descriptors of Neurotoxicity Risk
6.4.1. Estimation of the Number of
Individuals
6.4.2. Presentation of Specific Scenarios
6.4.3. Risk Characterization for Highly
Exposed Individuals
6.4.4. Risk Characterization for Highly
Sensitive or Susceptible Individuals
6.5.5. Other Risk Descriptors
6.5. Communicating Results
6.6. Summary and Research Needs
References
Part B: Response to Science Advisory Board
and Public Comments^-
I, Introduction
2. Response to Science Advisory Board
Comments
3. Response to Public Comments
List of Tables
Table 1. Examples of possible indicators of a
ncurotoxic effect
Table 2. Neurotoxicants and disorders with
specific neurological targets
TableS, Examples of neurophysiological
measures of neurotoxicity
Table 4. Examples of neurotoxicants with
known neurochemical mechanisms
Table 5. Examples of measures in a
representative functional observational
battery
Table 6. Examples of specialized behavioral
tests to measure neurotoxicity
Table 7. Examples of compounds or
treatments producing developmental
neurotoxicity
Table 8. Characterization of the health-
related database
Part A: Guidelines for Neurotoxicity
Risk Assessment
1. Introduction
These Guidelines describe the
principles, concepts, and procedures
that the U.S. Environmental Protection
Agency (EPA) will follow in evaluating
data on potential neurotoxicity
associated with exposure to
environmental toxicants. The Agency's
authority to regulate substances that
have the potential to interfere with
human health is derived from a number
of statutes that are implemented through
multiple offices within EPA. The
procedures outlined here are intended
to help develop a sound scientific basis
for neurotoxicity risk assessment,
promote consistency in the Agency's
assessment of toxic effects on the
nervous system, and inform others of
the approaches used by the Agency in
those assessments. This document is not
a regulation and is not intended for EPA
regulations. The Guidelines set forth
current scientific thinking and
approaches for conducting and
evaluating neurotoxic risk assessments.
They are not intended, nor can they be
relied upon, to create any rights
enforceable by any party in litigation
with the United States.
1.1. Organization of These Guidelines
This introduction (section 1)
summarizes the purpose of these
Guidelines within the overall
framework of risk assessment at EPA. It
also outlines the organization of the
guidance and describes several default
assumptions to be used in the risk
assessment process, as discussed in the
recent National Research Council report
"Science and Judgment in Risk
Assessment" (NRC, 1994).
Section 2 sets forth definitions of
particular terms widely used in the field
of neurotoxicology. These include
"neurotoxicity" and "behavioral
alterations." Also included in this
section are discussions concerning
reversible and irreversible effects and
direct versus indirect effects.
Risk assessment is the process by
which scientific judgments are made
concerning the potential for toxicity in
humans. The National Research Council
(NRC, 1983) has defined risk assessment
as including some or all of the following
components (paradigm): hazard
identification, dose-response
assessment, exposure assessment, and
risk characterization. In its 1994 report
"Science and Judgment in Risk
Assessment" the NRC extended its view
of the paradigm to include
characterization of each component
(NRC, 1994). In addition, it noted the
importance of an approach that is less
fragmented and more holistic, less
linear and more interactive, and that
deals with recurring conceptual issues
that cut across all stages of risk
assessment. These Guidelines describe a
more interactive approach by organizing
the process around the qualitative
evaluation of the toxicity data (hazard
characterization), the quantitative dose-
response analysis, the exposure
assessment, and the risk
characterization. In these Guidelines,
hazard characterization includes
deciding whether a chemical has an
effect by means of qualitative
consideration of dose-response
relationships, route, and duration of
exposure. Determining a hazard often
depends on whether a dose-response
relationship is present (Kimmel et al.,
1990). This approach combines the
information important in comparing the
toxicity of a chemical with potential
human exposure scenarios (section 3).
In addition, it avoids the potential for
labeling chemicals as "neurotoxicants"
on a purely qualitative basis. This
organization of the risk assessment
process is similar to that discussed in
the Guidelines for Developmental
Toxicity Risk Assessment (56 FR
63798), the main difference being that
the quantitative dose-response analysis
is discussed under a separate section in
these Guidelines.
Hazard characterization involves
examining all available experimental
animal and human data and the
associated doses, routes, timing, and
durations of exposure to determine
qualitatively if an agent causes
neurotoxicity in that species and under
what conditions. From the hazard
characterization and criteria provided in
these Guidelines, the health-related
database can be characterized as
sufficient or insufficient for use in risk
assessment (section 3.3). Combining
hazard identification and some aspects
of dose-response evaluation into hazard
characterization does not preclude the
evaluation and use of data for other
purposes when quantitative information
for setting reference doses (RfDs) and
reference concentrations (RfCs) is not
available.
The next step in the dose-response
analysis (section 4) is the quantitative
analysis, which includes determining
the no-observed-adverse-effect-level
(NOAEL) and/or the lowest-observed-
adverse-effect-level (LOAEL) for each
study and type of effect. Because of the
limitations associated with the use of
the NOAEL, the Agency is beginning to
use an additional approach, the
benchmark dose approach (BMD)
(Crump, 1984; U.S. EPA, 1995a),for
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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), ari oral or dermal RfD or an
inhalation RfC, based on neurotoxic
effects, is then derived. This RfD or RfC
is derived using the NOAEL or
benchmark dose divided by uncertainty
factors to account for interspecies
differences in response, intraspecies
variability, and other factors of study
design or the database. A statement of
the potential for human risk and the
consequences of exposure can come
only from integrating the hazard
characterization and dose-response
analysis with the human exposure
estimates in the final risk
characterization.
The section on exposure assessment
(section 5) identifies human populations
exposed or potentially exposed to an
agent, describes their composition and
size, and presents the types,
magnitudes, frequencies, and durations
of exposure to the agent. The exposure
assessment provides an estimate of
human exposure levels for particular
populations from all potential sources.
In risk characterization (section 6), the
hazard characterization, dose-response
analysis, and exposure assessment for
given populations are combined to
estimate some measure of the risk for
neurotoxicity. As part of risk
characterization, a summary of the
strengths and weaknesses of each
component of the risk assessment is
given, along with major assumptions,
scientific judgments and, to the extent
possible, qualitative and quantitative
estimates of the uncertainties. This
characterization of the health-related
database is always presented in
conjunction with information on the
dose, route, duration, and timing of
exposure as well as the dose-response
analysis including the RfD or RfC. If
human exposure estimates are available,
the exposure basis used for the risk
assessment is clearly described, e.g.,
highly exposed individuals or highly
sensitive or susceptible individuals. The
NOAEL may be compared to the various
estimates of human exposure to
calculate the margin(s) of exposure
(MOE). The considerations for judging
the acceptability of the MOE are similar
to those for determining the appropriate
size of the uncertainty factor for
calculating the RfD or RfC.
The Agency recently issued a policy
statement and associated guidance for
risk characterization (U.S. EPA, 1995b,
1995c), which is currently being
implemented throughout EPA. This
statement is designed to ensure that
critical information from each stage of a
risk assessment is used in forming
conclusions about risk and that this
information is communicated from risk
assessors to risk managers (policy
makers), from middle to upper
management, and from the Agency to
the public. Additionally, the policy
provides a basis for greater clarity,
transparency, reasonableness, and
consistency in risk assessments across
Agency programs.
Final neurotoxicity risk assessment
guidelines may reflect additional
changes in risk characterization
practices resulting from implementation
activities. Risk assessment is just one
component of the regulatory process
and defines the potential adverse health
consequences of exposure to a toxic
agent. The other component, risk
management, combines risk assessment
with statutory directives regarding
socioeconomic, technical, political, and
other considerations in order to decide
whether to control future exposure to
the suspected toxic agent and, if so, the
nature and level of control. One major
objective of these Guidelines is to help
the risk assessor determine whether the
experimental animal or human data
indicate the potential for a neurotoxic
effect. Such information can then be
used to categorize evidence that will
identify and characterize neurotoxic
hazards, as described in section 3.3,
Characterization of the Health-Related
Database, and Table 8 of these
Guidelines. Risk management is not
dealt with directly in these Guidelines
because the basis for decision making
goes beyond scientific considerations
alone, but the use of scientific
information in this process is discussed.
For example, the acceptability of the
MOE is a risk management decision, but
the scientific bases for establishing this
value are discussed here.
1.2. The Role of Environmental Agents
in Neurotoxicity
Chemicals are an integral part of life,
with the capacity to improve as well as
endanger health. The general population
is exposed to chemicals in air, water,
foods, cosmetics, household products,
and drugs used therapeutically or
illicitly. During daily life, a person
experiences a multitude of exposures to
potentially neuroactive substances,
singly and in combination, both
synthetic and natural. Levels of
exposure vary and may or may not pose
a hazard, depending on dose, route, and
duration of exposure.
A link between human exposure to
some chemical substances and
neurotoxicity has been firmly
established (Anger, 1986; OTA, 1990).
Because many natural and synthetic
chemicals are present in today's
environment, there is growing scientific
and regulatory interest in the potential
for risks to humans from exposure to
neurotoxic agents. If sufficient exposure
occurs, the effects resulting from such
exposures can have a significant adverse
impact on human health. It is not
known how many chemicals may be
neurotoxic in humans (Reiter, 1987).
EPA's TSCA inventory of chemical
substances manufactured, imported, or
processed in the United States includes
more than 65,000 substances and is
increasing yearly. An overwhelming
majority of the materials in commercial
use have not been tested for neurotoxic
potential (NRC, 1984).
Estimates of the number of chemicals
with neurotoxic properties have been
made for subsets of substances. For
instance, a large percentage of the more
than 500 registered active pesticide
ingredients affect the nervous system of
the target species to varying degrees. Of
588 chemicals listed by the American
Conference of Governmental Industrial
Hygienists, 167 affected the nervous
system or behavior at some exposure
level (Anger, 1984). Anger (1990)
estimated that of the approximately 200
chemicals to which 1 million or more
American workers are exposed, more
than one-third may have adverse effects
on the nervous system if sufficient
exposure occurs. Anger (1984) also'
recognized neurotoxic effects as one of
the 10 leading workplace disorders. A
number of therapeutic substances,
including some anticancer and antiviral
agents and abused drugs, can cause
adverse or neurotoxicological side
effects at therapeutic levels (OTA,
1990). The number of chemicals with
neurotoxic potential has been estimated
to range from 3% to 28% of all
chemicals (OTA, 1990). Thus,
estimating the risks of exposure to
chemicals with neurotoxic potential is
of concern with regard to their overall
impact on human health.
1.3. Neurotoxicity Risk Assessment
In addition to its primary role in
psychological functions, the nervous
system controls most, if not all, other
bodily processes. It is sensitive to
perturbation from various sources and
has limited ability to regenerate.There
is evidence that even small anatomical,
biochemical, or physiological insults to
the nervous system may result in
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adverse effects on human health.
Therefore, there is a need for consistent
guidance on how to evaluate data on
neurotoxic substances and assess their
potential to cause transient or persistent
and director indirect effects on human
health.
These Guidelines develop principles
and concepts in several areas. They
outline the scientific basis for evaluating
effects due to exposure to
neurotoxicants and discuss principles
and methods for evaluating data from
human and animal studies on behavior,
neurochemistry, neurophysiology, and
neuropathology. They also discuss
adverse effects on neurological
development and function in infants
and children following prenatal and
perinatal exposure to chemical agents.
They outline the methods for
calculating reference doses or reference
concentrations when neurotoxicity is
the critical effect, discuss the
availability of alternative mathematical
approaches to dose-response analyses,
characterize the health-related database
for neurotoxicity risk assessment, and
discuss the integration of exposure
information with results of the dose-
response assessment to characterize
risks. These Guidelines do not advocate
developing reference doses specific for
neurotoxicity, but rather support the use
of neurotoxicity as one possible
endpoint to develop reference doses.
EPA offices have published guidelines
for neurotoxicity testing in animals
(U.S. EPA, 1986.1987,1988a, 1991a).
The testing guidelines address the
development of new data for use in risk
assessment.
These neurotoxicity risk assessment
guidelines provide the Agency's first
comprehensive guidance on the use and
interpretation of neurotoxicity data, and
are part of the Agency's risk assessment
guidelines development process, which
was initiated in 1984. As part of its
neurotoxicity guidelines development
program, EPA has sponsored or
participated in several conferences on
relevant issues CTilson, 1990); these and
other sources (see references) provide
the scientific basis for these Guidelines.
This guidance is intended for use by
Agency risk assessors and is separate
and distinct from the recently published
document on principles of neurotoxicity
risk assessment (U.S. EPA, 1994). The
document on principles was prepared
under the auspices of the Subcommittee
on Risk Assessment of the Federal
Coordinating Council for Science,
Engineering, and Technology and was
not intended to provide specific
directives for how neurotoxicity risk
assessment should be performed. It is
expected that, like other EPA risk
assessment guidelines for noncancer
endpoints (U.S. EPA, 1991b), this
document will encourage research and
analysis leading to new risk assessment
methods and data, which in turn would
be used to revise and improve the
Guidelines and better guide Agency risk
assessors.
1.4. Assumptions
There are a number of unknowns in
the extrapolation of data from animal
studies to humans. Therefore, a number
of default assumptions are made that are
generally applied in the absence of data
on the relevance of effects to potential
human risk. Default assumptions should
not be applied indiscriminately. First,
all available mechanistic and
pharmacokinetic data should be
considered. If these data indicate that an
alternative assumption is appropriate or
if they obviate the need for applying an
assumption, such information should be
used in risk assessment. For example,
research in rats may determine that the
neurotoxicity of a chemical is caused by
a metabolite. If subsequent research
finds that the chemical is metabolized to
a lesser degree or not at all in humans,
then this information should be used in
formulating the default assumptions.
The following default assumptions form
the basis of the approaches taken in
these Guidelines:
(1) It is assumed that an agent that
produces detectable adverse neurotoxic
effects in experimental animal studies
will pose a potential hazard tdftumans.
This assumption is based on the
comparisons of data for known-human
neurotoxicants (Anger, 1990'; Kimmel et
al., 1990; Spencer and Schaumburg,
1980), which indicate that experimental
animal data are frequently predictive of
a neurotoxic effect in humans.
(2) It is assumed that behavioral,
neurophysiological, neurochemical, and
neuroanatomical manifestations are of
concern. In the past, the tendency has
been to consider only neuropathological
changes as endpoints of concern. Based
on data on agents that are known human
neurotoxicants (Anger, 1990; Kimmel et
al., 1990; Spencer and Schaumberg,
1980), there is usually at least one
experimental species that mimics the
types of effects seen in humans, but in
other species tested, the neurotoxic
effect may be different or absent. For
example, certain organophosphate
compounds produce a delayed-onset
neuropathy in hens similar to that seen
in humans, whereas rodents are
characteristically insensitive to these
compounds. A biologically significant
increase in any of the manifestations is
considered indicative of an agent's
potential for disrupting the structure or
function of the human nervous system.
(3) It is assumed that the neurotoxic
effects seen in animal studies may not
always be the same as those produced
in humans. Therefore, it may be difficult
to determine the most appropriate
species in terms of predicting specific
effects in humans. The fact that every
species may not react in the same way
is probably due to species-specific
differences in maturation of the nervous
system, differences in timing of
exposure, metabolism, or mechanisms
of action.
(4) It is also assumed that, in the
absence of data to the contrary, the most
sensitive species is used to estimate
human risk. This is based on the
assumption that humans are as sensitive
as the most sensitive animal species
tested. This provides a conservative
estimate of sensitivity for added
protection to the public. As with other
noncancer endpoints, it is assumed that
there is a nonlinear dose-response
relationship for neurotoxicants.
Although there may be a threshold for
neurotoxic effects, these are often
difficult to determine empirically.
Therefore, a nonlinear relationship is
assumed to exist for neurotoxicants.
These assumptions are "plausibly
conservative" (NRC, 1994) in that they
are protective of public health and are
also well founded in scientific
knowledge about the effects of concern.
2. Definitions and Critical Concepts
This section defines the key terms and
concepts that EPA will use in the
identification and evaluation of
neurotoxicity. The various health effects
that fall within the broad classification
of neurotoxicity are described and
examples are provided. Adverse effects
include alterations from baseline or
normal conditions that diminish an
organism's ability to survive, reproduce,
or adapt to the environment.
Neurotoxicity is an adverse change in
the structure or function of the central
and/or peripheral nervous system
following exposure to a chemical,
physical, or biological agent (Tilson,
1990). Functional neurotoxic effects
include adverse changes in somatic/
autonomic, sensory, motor, and/or
cognitive function. Structural
neurotoxic effects are defined as
neuroanatomical changes occurring at
any level of nervous system
organization; functional changes are
defined as neurochemical,
neurophysiological, or behavioral
effects. Chemicals can also be
categorized into four classes: Those that
act on the central nervous system, the
peripheral nerve fibers, the peripheral
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nerve endings, or muscles or other
tissues (Albert, 1973). Changes in
function can result from toxicity to
other specific organ systems, and these
indirect changes may be considered
adverse. For example, exposure to a
high dose of a chemical may cause
damage to the liver, resulting in general
sickness and a decrease in a functional
endpoint such as motor activity. In this
case, the change in motor activity could
be considered as adverse, but not
necessarily neurotoxic. A discussion
concerning problems associated with
risk assessment of high doses of
chemicals in the context of drinking
water and health was published by the
National Research Council (1986).
The risk assessor should also know
that there are different levels of concern
based on the magnitude of effect,
duration of exposure, and reversibility
of some neurotoxic effects. Neurotoxic
effects may be irreversible (the organism
cannot return to the state prior to
exposure, resulting in a permanent
change) or reversible (the organism can
return to the pre-exposure condition).
Clear or demonstrable irreversible
change in either the structure or
function of the nervous system causes
greater concern than do reversible
changes. If neurotoxic effects are
observed at some time during the
lifespan of the organism but are slowly
reversible, the concern is also high.
There is lesser concern for effects that
are rapidly reversible or "transient," i.e.,
measured in minutes, hours, or days,
and that appear to be associated with
the pharmacokinetics of the causal agent
and its presence in the body. Reversible
changes that occur in the occupational
setting or environment, however, may
be of high concern if, for example,
exposure to a short-acting solvent
interferes with operation of heavy
equipment in an industrial plant. The
context of the exposure should be
considered in evaluating reversible
effects. Setting of exposure limits is not
always associated with the
determination of a reference dose,
which is based on chronic dosing. Data
from acute or subacute dosing can be
used for health advisories or in studies
involving developmental exposures.
It should also be noted that the
nervous system is known for its reserve
capacity (Tilson and Mitchell, 1983).
That is, repeated insult to the nervous
system could lead to an adaptation.
There are, however, limits to this
capacity, and when these limits are
exceeded, further exposure could lead
to frank manifestations of neurotoxicity
at the structural or functional level. The
risk assessor should be aware that once
damaged, neurons, particularly in the
central nervous system, have a limited
capacity for regeneration. Reversibility
of effects resulting from cell death or
from the destruction of cell processes
may represent an activation of repair
capacity, decreasing future potential
adaptability. Therefore, even reversible
neurotoxic changes should be of
concern. Evidence of progressive effects
(those that continue to worsen even
after the causal agent has been
removed), delayed-onset effects (those
that occur at a time distant from the last
contact with the causal agent), residual
effects (those that persist beyond a
recovery period), or latent effects (those
that become evident only after an
environmental challenge or aging) have
a high level of concern.
Environmental challenges can include
stress, increased physical or cognitive
workload, pharmacological
manipulations, and nutritional
deficiency or excess. Evidence for
reversibility may depend on the region
of the nervous system affected, the
chemical involved, and organismic
factors such as the age of the exposed
population. Some regions of the nervous
system, such as peripheral nerves, have
a high capacity for regeneration, while
regions in the brain such as the
hippocampus are known for their ability
to compensate or adapt to neurotoxic
insult. For example, compensation is
likely to be seen with solvents (e.g., n-
hexane) that produce peripheral
neuropathy because of the repair
capacity of the peripheral nerve. In
addition, tolerance to some cholinergic
effects of cholinesterase-inhibiting
compounds may be due to
compensatory down-regulation of
muscarinic receptors. Younger
individuals may have more capacity to
adapt than older individuals, suggesting
that the aged may be at greater risk to
neurotoxic exposure.
Neurotoxic effects can be observed at
various levels of organization of the
nervous system, including
neurochemical, anatomical,
physiological, or behavioral. At the
neurochemical level, for example, an
agent that causes neurotoxicity might
inhibit macromolecule or transmitter
synthesis, alter the flow of ions across
cellular membranes, or prevent release
of neurotransmitter from the nerve
terminals. Anatomical changes may
include alterations of the cell body, the
axon, or the myelin sheath. At the
physiological level, a chemical might
change the thresholds for neural
activation or reduce the speed of
neurotransmission. Behavioral
alterations can include significant
changes in sensations of sight, hearing,
or touch; alterations in simple or
complex reflexes and motor functions;
alterations in cognitive functions such
as learning, memory, or attention; and
changes in mood, such as fear or rage,
disorientation as to person, time, or
place, or distortions of thinking and
feeling, such as delusions and
hallucinations. At present, relatively
few neurotoxic syndromes have been
thoroughly characterized in terms of the
initial neurochemical change, structural
alterations, physiological consequence,
and behavioral effects. Knowledge of
exact mechanisms of action is not,
however, necessary to conclude that a
chemically induced change is a
neurotoxic effect.
Neurotoxic effects can be produced by
chemicals that do not require
metabolism prior to interacting with
their sites in the nervous system
(primary neurotoxic agents) or those
that require metabolism prior to
interacting with their sites (secondary
neurotoxic agents). Chemically induced
neurotoxic effects can be direct (due to
an agent or its metabolites acting
directly on sites in the nervous system)
or indirect (due to agents or metabolites
that produce their effects primarily by
interacting with sites outside the
nervous system). For example,
excitatory amino acids such as domoic
acid damage specific neurons directly
by activating excitatory amino acid
receptors in the nervous system,
whereas carbon monoxide decreases
oxygen availability, which can
indirectly kill neurons. Other examples
of indirect effects include cadmium-
induced spasms in blood vessels
supplying the nervous system,
dichloroacetate-induced perturbation of
metabolic pathways, and chemically
induced alterations in skeletomuscular
function or structure and effects on the
endocrine system. Professional
judgment may be required in making
determinations about direct versus
indirect effects.
The interpretation of data as
indicative of a potential neurotoxic
effect involves the evaluation of the
validity of the database. This approach
and these terms have been adapted from
the literature on human psychological
testing (Sette, 1987; Sette and MacPhail,
1992), where they have long been used
to evaluate the level of confidence in
different measures of intelligence or
other abilities, aptitudes, or feelings.
There are four principal questions that
should be addressed: whether the effects
result from exposure (content validity);
whether the effects are adverse or
lexicologically significant (construct
validity); whether there are correlative
measures among behavioral,
physiological, neurochemical, and
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26931
morphological endpoints (concurrent
validity); and whether the effects are
predictive of what will happen under
various conditions (predictive validity).
Addressing these issues can provide a
useful framework for evaluating either
human or animal studies or the weight
of evidence for a chemical (Sette, 1987;
Sette and MacPhail, 1992). The next
sections indicate the extent to which
chemically induced changes can be
Interpreted as providing evidence of
neurotoxicity.
3. Hazard Characterization
3.1. Neurotoxicological Studies:
Endpoints and Their Interpretation
The qualitative characterization of
neurotoxic hazard can be based on
either human or animal data (Anger,
1984; Reiter. 1987; U.S. EPA, 1994).
Such data can result from accidental,
inappropriate, or controlled
experimental exposures. This section
describes many of the general and some
of the specific characteristics of human
studies and reports of neurotoxicity. It
then describes some features of animal
studies of neuroanatomical,
neurochemical, neurophysiological, and
behavioral effects relevant to risk
assessment. The process of
characterizing the sufficiency or
insufficiency of neurotoxic effects for
risk assessment is described in section
3.3. Additional sources of information
relevant to hazard characterization, such
as comparisons of molecular structure
among compounds and in vitro
screening methods, are also discussed.
The hazard characterization should:
a. Identify strengths and limitations of
the database:
Epidemiological studies (case
reports, cross-sectional, case-control,
cohort, or human laboratory exposure
studies);
Animal studies (including
structural or neuropathological,
neurochemical. neurophysiological,
behavioral or neurological, or
developmental endpoints).
b. Evaluate the validity of the
database:
Content validity (effects result from
exposure);
Construct validity (effects are
adverse or lexicologically significant);
Concurrent validity (correlative
measures among behavioral,
physiological, neurochemical, or
morphological endpoints);
Predictive validity (effects are
predictive of what will happen under
various conditions).
c. Identify and describe key
lexicological studies.
d. Describe the type of effects:
Structural (neuroanatomical
alternations);
Functional (neurochemical,
neurophysiological, behavioral
alterations).
e. Describe the nature of the effects
(irreversible, reversible, transient,
progressive, delayed, residual, or latent).
f. Describe how much is known about
how (through what biological
mechanism) the chemical produces
adverse effects.
g. Discuss other health endpoints of
concern.
h. Comment on any nonpositive data
in humans or animals.
I. Discuss the dose-response data
(epidemiological or animal) available for
further dose-response analysis.
j. Discuss the route, level, timing, and
duration of exposure in studies
demonstrating neurotoxicity as
compared to expected human
exposures.
k. Summarize the hazard
characterization:
Confidence in conclusions;
Alternative conclusions also
supported by the data;
Significant data gaps; and
Highlights of major assumptions.
3.1.1. Human Studies
It is well established that information
from the evaluation of human exposure
can identify neurotoxic hazards (Anger
and Johnson, 1985; Anger, 1990).
Prominent among historical episodes of
neurotoxicity in human populations are
the outbreaks of methylmercury
poisoning in Japan and Iraq and the
neurotoxicity seen in miners of metals,
including mercury, manganese, and lead
(Carson et al., 1987; Silbergeld and
Percival, 1987; OTA, 1990). In the past
decade, lead poisoning in children has
been a prominent issue of concern
(Silbergeld and Percival, 1987).
Neurotoxicity in humans has been
studied and reviewed for many
pesticides (Hayes, 1982; NRDC, 1989;
Ecobichon and Joy, 1982; Ecobichon et
al., 1990). Organochlorines,
organophosphates, carbamates,
pyrethroids, certain fungicides, and
some fumigants are all known
neurotoxicants. They may pose
occupational risks to manufacturing and
formulation workers, pesticide
applicators and farm workers, and
consumers through home application or
consumption of residues in foods.
Families of workers may also be
exposed by transport into the home
from workers' clothing. Data on humans
can come from a number of sources,
including clinical evaluations, case
reports, epidemiologic studies, and
human laboratory exposure studies. A
more extensive description of issues
concerning human neurotoxicology and
risk assessment has been published
elsewhere (U.S. EPA, 1993). A review of
the types of tests used to assess
cognitive and neurological function in
children, in addition to a discussion of
methodological issues in the design of
prospective, longitudinal studies of
developmental neurotoxicity in
humans, has recently been published
(Jacobsonandjacobson, 1996). Stanton
and Spear (1990) reviewed assessment
measures used in developmental
neurotoxicology for their comparability
in humans and laboratory animals and
their ability to detect comparable
adverse effects across species. At the
level of the various functional
assessments for sensory, motivational,
cognitive and motor function, and social
behavior, there was good agreement
across species among the neurotoxic
agents reviewed.
3.1.1.1. Clinical Evaluations
Clinical methods are used extensively
in neurology and neuropsychology to
evaluate patients suspected of having
neurotoxicity. An array of examiner-
administered and paper-and-pencil
tasks are used to assess sensory, motor,
cognitive, and affective functions and
personality states/traits.
Neurobehavioral data are synthesized
with information from
neurophysiological studies and medical
history to derive a working diagnosis.
Brain functional imaging techniques
based on magnetic resonance imaging or
emission tomography may also be useful
in helping diagnose neurodegenerative
disorders following chemical exposures
in humans (Omerand et al., 1994;
Callender et al., 1994). Clinical
diagnostic approaches have provided a
rich conceptual framework for
understanding the functions (and
malfunctions) of the central and
peripheral nervous systems and have
formed the basis for the development of
methods for measuring the behavioral
expression of nervous system disorders.
Human neurobehavioral toxicology has
borrowed heavily from neurology and
neuropsychology for concepts of
nervous system impairment and
functional assessment methods.
Neurobehavioral toxicology has adopted
the neurologic/neuropsychologic model,
using adverse changes in behavioral
function to assist in identifying
chemical-or drug-induced changes in
nervous system processes.
Neurological and neuropsychological
methods have long been employed to
identify the adverse health effects of
environmental workplace exposures
(Sterman and Schaumburg, 1980).
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Peripheral neuropathies (with sensory
and motor disturbances),
encephalopathies, organic brain
syndromes, extrapyramidal syndromes,
demyelination, autonomic changes, and
dementia are well-characterized
consequences of acute and chronic
exposure to chemical agents. The range
of exposure conditions that produce
clinical signs of neurotoxicity also has
been defined by these clinical methods.
It is very important to make external/
internal dose measurements in humans
to determine the actual dose(s) that can
cause unwanted effects.
Aspects of the neurological
examination approach limit its
usefulness for neurotoxicological risk
assessment. Information obtained from
the neurological exam is mostly
qualitative and descriptive rather than
quantitative. Estimates of the severity of
functional impairment can be reliably
placed into only three or four categories
(for example, mild, moderate, severe).
Much of the assessment depends on the
subjective judgment of the examiner.
For example, the magnitude and
symmetry of muscle strength are often
judged by having the patient push
against the resistance of the examiner's
hands. The endpoints are therefore the
absolute and relative amount of muscle
load sensed by the examiner in his or
her arms.
Compared with other methods, the
neurological exam may be less sensitive
in detecting early neurotoxicity in
peripheral sensory and motor nerves.
While clinicians'judgments are equal in
sensitivity to quantitative methods in
assessing the amplitude of tremor,
tremor frequency is poorly quantified by
clinicians. Thus, important aspects of
the clinical neurologic exam may be
insufficiently quantified and lack
sufficient sensitivity for detecting early
neurobehavioral toxicity produced by
environmental or workplace exposure
conditions. However, a neurological
evaluation of persons with documented
neurobehavioral impairment would be
helpful for identifying nonchemical
causes of neurotoxicity, such as diabetes
and cardiovascular insufficiency.
Administration of a
neuropsychological battery also requires
a trained technician, and interpretation
requires a trained and experienced
neuropsychologist. Depending on the
capabilities of the patient, 2 to 4 hours
may be needed to administer a full
battery; 1 hour may be needed for the
shorter screening versions. These
practical considerations may limit the
usefulness of neuropsychological
assessment in large field studies of
suspected neurotoxicity.
In addition to logistical problems in
administration and interpretation,
neuropsychological batteries and
neurological exams share two
disadvantages with respect to
neurotoxicity risk assessment. First,
neurological exams and
neuropsychological test batteries are
designed to confirm and classify
functional problems in individuals
selected on the basis of signs and
symptoms identified by the patient,
family, or other health professionals.
Their usefulness in detecting low base-
rate impairment in workers or the
general population is generally thought
to be limited, decreasing the usefulness
of clinical assessment approaches for
epidemiologic risk assessment.
Second, neurological exams and
neuropsychological test batteries were
developed to assess the functional
correlates of the most common forms of
nervous system dysfunction: brain
trauma, focal lesions, and degenerative
conditions. The clinical tests were
validated against these neurological
disease states. With a few notable
exceptions, chemicals are not believed
to produce impairment similar to that
from trauma or lesions; neurotoxic
effects are more similar to the effects of
degenerative disease. There has been
insufficient research to demonstrate
which tests designed to assess
functional expression of neurologic
disease are useful in characterizing the
modes of central nervous system
impairment produced by chemical
agents and drugs.
It should be noted that alternative
approaches are available that avoid
many of the limitations of clinical and
neurological and tradition^t-
neuropsychological method's.
Computerized behavioral assessment
systems designed for field testing of
populations exposed to chemicals in the
community or workplace have been
developed during the past decade. The
most widely used system is the
Neurobehavioral Evaluation System
(NES) developed by Baker et al. (1985).
Advantages of computerized tests
include (1) standardized administration
to eliminate intertester variability and
minimize subject-experimenter
interaction; (2) automated data
collection and scoring, which is faster,
easier, and less error-prone than
traditional methods; and (3) test
administration requires minimal
training and experience. NES tests have
proven sensitive to a variety of solvents,
metals, and pesticides (Otto, 1992).
Computerized systems available for
human neurotoxicity testing are
critically reviewed in Anger et al.
(1996).
3.1.1.2. Case Reports
The first type of human data available
is often the case report or case series,
which can identify cases of a disease
and are reported by clinicians or
discerned through active or passive
surveillance, usually in the workplace.
However, case reports involving a single
neurotoxic agent, although informative,
are rare in the literature; for example,
farmers are likely to be exposed to a
wide variety of potentially neurotoxic
pesticides. Careful case histories assist
in identifying common risk factors,
especially when the association between
the exposure and disease is strong, the
mode of action of the agent is
biologically plausible, and clusters
occur in a limited period of time.
Case reports can be obtained more
quickly than more complex studies.
Case reports of acute high-level
exposure to a toxicant can be useful for
identifying signs and symptoms that
may also apply to lower exposure. Case
reports can also be useful when
corroborating epidemiological data are
available.
3.1.1.3. Epidemiologic Studies
Epidemiology has been defined as
"the study of the distributions and
determinants of disease and injuries in
human populations" (Mausner and
Kramer, 1985). Knowing the frequency
of illness in groups and the factors that
influence the distribution is the tool of
epidemiology that allows the evaluation
of causal inference with the goal of
prevention and cure of disease
(Friedlander and Hearn, 1980).
Epidemiologic studies are a useful
means of evaluating the effects of
neurotoxic substances on human
populations, particularly if effects of
exposure are cumulative or exposures
are repeated. Such studies are less
useful in cases of acute exposure, where
the effects are short-term. Frequently,
determining the precise dose or
exposure concentration in
epidemiological studies can be difficult.
3.1.1.3.1. Cross-Sectional Studies.
In cross-sectional studies or surveys,
both the disease and suspected risk
factors are ascertained at the same time,
and the findings are useful in generating
hypotheses. A group of people are
interviewed, examined, and tested at a
single point in time to ascertain a
relationship between a disease and a
neurotoxic exposure. This study design
does not allow the investigator to
determine whether the disease or the
exposure came first, rendering it less
useful in estimating risk. These studies
are intermediate in cost and time
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26933
required to complete compared with
case reports and more complex
analytical studies, but should be
augmented with additional data.
3.1.1.3.2. Case-Control (Retrospective)
Studies.
Last (1986) defines a case-control
study as one that "starts with the
identification of persons with the
disease (or other outcome variable) of
interest, and a suitable control
population (comparison, reference
group) of persons without the disease."
He states that the relationship of an
"attribute" to the disease is measured by
comparing the diseased with the
nondiseased with regard to how
frequently the attribute is present in
each of the groups. The cases are
assembled from a population of persons
with and without exposure, and the
comparison group is selected from the
same population; the relative
distribution of the potential risk factor
(exposure) in both groups is evaluated
by computing an odds ratio that serves
as an estimate of the strength of the
association between the disease and the
potential risk factor. The statistical
significance of the ratio is determined
by calculating a p-value and is used to
approximate relative risk.
The case-control approach to the
study of potential neurotoxicants in the
environment provides a great deal of
useful information for the risk assessor.
In his textbook, Valciukas (1991) notes
that the case-control approach is the
strategy of choice when no other
environmental or biological indicator of
neurotoxic exposure is available. He
further states: "Considering the fact that
for the vast majority of neurotoxic
chemical compounds, no objective
biological indicators of exposure are
available (or if they are, their half-life is
too short to be of any practical value),
the case-control paradigm is a widely
accepted strategy for the assessment of
toxic causation." The case-control study
design, however, can be very
susceptible to bias. The potential
sources of bias are numerous and can be
specific to a particular study. Many of
these biases also can be present in cross-
sectional studies. For example, recall
bias or faulty recall of information by
study subjects in a questionnaire-based
study can distort the results. Analysis of
the case-comparison study design
assumes that the selected cases are
representative persons with the
diseaseeither all cases with the
disease or a representative sample of
them have been ascertained. It further
assumes that the control or comparison
group is representative of the
nonexposed population (or that the
prevalence of the characteristic under
study is the same in the control group
as in the general population). Failure to
satisfy these assumptions may result in
selection bias that may invalidate study
results.
An additional source of bias in case-
control studies is the presence of
confounding variables, i.e., factors
known to be associated with the
exposure and causally related to the
disease under study. These should be
controlled, either in the design of the
study by matching cases to controls on
the basis of the confounding factor, or
in the analysis of the data by using
statistical techniques such as
stratification or regression. Matching
requires time to identify an adequate
number of potential controls to
distinguish those with the proper
characteristics, while statistical control
of confounding factors requires a larger
study.
The definition of exposure is critical
in epidemiologic studies. In
occupational settings, exposure
assessment often is based on the job
assignment of the study subjects, but
can be more precise if detailed company
records allow the development of
exposure profiles. Positive results from
a properly controlled retrospective
study should weigh heavily in the risk
assessment process.
3.1.1.3.3. Cohort (Prospective, Follow-
Up) Studies.
In a prospective study design, a
healthy group of people is assembled
and followed forward in time and
observed for the development^
dysfunction. Such studies are''
invaluable for determining the time
course for development of dysfunction
(e.g., follow-up studies performed in
various cities on the effects of lead on
child development). This approach
allows the direct estimate of risks
attributed to a particular exposure, since
toxic incidence rates in the cohort can
be determined. Prospective study
designs also allow the study of chronic
effects of exposure. One major strength
of the cohort design is that it allows the
calculation of rates to determine the
excess risk associated with an exposure.
Also, biases are reduced by obtaining
information before the disease develops.
This approach, however, can be very
time-consuming and costly.
In cohort studies information bias can
be introduced when individuals provide
distorted information about their health
because they know their exposure status
and may have been told of the expected
health effects of the exposure under
study. More credence should be given to
those studies in which both observer
and subject bias are carefully controlled
(e.g., double-blind studies).
A special type of cohort study is the
retrospective cohort study, in which the
investigator goes back in time to select
the study groups and traces them over
time, often to the present. The studies
usually involve specially exposed.
groups and have provided much
assistance in estimating risks due to
occupational exposures. Occupational
retrospective cohort studies rely on
company records of past and current
employees that include information on
the dates of employment, age at
employment, date of departure, and
whether diseased (or dead in the case of
mortality studies). Workers can then be
classified by duration and degree of
exposure. Positive or negative results
from a properly controlled prospective
study should weigh heavily in the risk
assessment process.
3.1.1.4. Human Laboratory Exposure
Studies
Neurotoxiciry assessment has an
advantage not afforded to the evaluation
of other toxic endpoints, such as cancer
or reproductive toxicity, in that the
effects of some chemicals are short in
duration and reversible. This makes it
ethically possible to perform human
laboratory exposure studies and obtain
data relevant to the risk assessment
process. Information from experimental
human exposure studies has been used
to set occupational exposure limits,
mostly for organic solvents that can be
inhaled. Laboratory exposure studies
have contributed to risk assessment and
the setting of exposure limits for several
solvents and other chemicals with acute
reversible effects.
Human exposure studies sometimes
offer advantages over epidemiologic
field studies. Combined with
appropriate sampling of biological
fluids (urine or blood), it is possible to
calculate body concentrations, examine
toxicokinetics, and identify metabolites.
Bioavailability, elimination, dose-
related changes in metabolic pathways,
individual variability, time course of
effects, interactions between chemicals,
and interactions between chemical and
environmental/biobehavioral processes
(stressors, workload/respiratory rate) are
factors that are generally easier to
collect under controlled conditions.
Other goals of laboratory studies
include the in-depth characterization of
effects, the development of new
assessment methods, and the
examination of the sensitivity,
specificity, and reliability of
neurobehavioral assessment methods
across chemical classes. The laboratory
is the most appropriate setting for the
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study of environmental and
biobehavioral variables that affect the
action of chemical agents. The effects of
ambient temperature, task difficulty,
rate of ongoing behavior, conditioning
variables, tolerance/sensitization, sleep
deprivation, motivation, and so forth are
sometimes studied.
From a methodological standpoint,
human laboratory studies can be
divided into two categories: between-
subjects and within-subjects designs. In
the former, the neurobehavioral
performance of exposed volunteers is
compared with that of nonexposed
participants. In the latter, preexposure
performance is compared with
neurobehavioral function under the
influence of the chemical or drug.
Within-subjects designs have the
advantage of requiring fewer
participants, eliminating individual
differences as a source of variability,
and controlling for chronic mediating
variables, such as caffeine use and
educational achievement. A
disadvantage of the within-subjects
design is that neurobehavioral tests
must be administered more than once.
Practice on many neurobehavioral tests
often leads to improved performance
that may confound the effect of the
chemical/drug. There should be a
sufficient number of test sessions in the
pre-exposure phase to allow
performance on all tests to achieve a
relatively stable baseline level.
Participants in laboratory exposure
studies may have been recruited from
populations of persons already exposed
to the chemical/drug or from chemical-
naive populations. Although the use of
exposed volunteers has ethical
advantages, can mitigate against novelty
effects, and allows evaluation of
tolerance/sensitization, finding an
accessible exposed population in
reasonable proximity to the laboratory
can be difficult. Chemical-naive
participants are more easily recruited
but may differ significantly in important
characteristics from a representative
sample of exposed persons. Chemical-
naive volunteers are often younger,
healthier, and better educated than the
populations exposed environmentally,
in the workplace, or
pharmacotherapeutically.
Compared with workplace and
environmental exposures, laboratory
exposure conditions can be controlled
more precisely, but exposure periods are
much shorter. Generally only one or two
relatively pure chemicals are studied for
several hours, whereas the population of
interest may be exposed to multiple
chemicals containing impurities for
months or years. Laboratory studies are
therefore better at identifying and
characterizing effects with acute onset
and the selective effects of pure agents.
In all cases, the potential for participant
bias should be as carefully controlled
for as possible. Even the consent form
can lead to participant bias, as toxic
effects have been reported in some
individuals who were warned of such
effects in an informed consent form. In
addition, double-blind studies have
been shown to provide some control for
observer bias that may occur in single-
blind studies. More credence should be
given to those studies in which both
observer and subject bias are carefully
controlled (Benignus, 1993).
A test battery that examines multiple
neurobehavioral functions may be more
useful for screening and the initial
characterization of acute effects.
Selected neurobehavioral tests that
measure a limited number of functions
in multiple ways may be more useful for
elucidating mechanisms or validating
specific effects.
Both chemical and behavioral control
procedures are valuable for examining
the specificity of the effects. A
concordant effect among different
measures of the same neurobehavioral
function (e.g., reaction time) and a lack
of effect on some other measures of
psychomotor function (e.g., untimed
manual dexterity) would increase the
confidence in a selective effect on motor
speed and not on attention or another
nonspecific motor function. Likewise,
finding concordant effects among
similar chemical or drug classes along
with different effects from dissimilar
classes would support the specificity of
chemical effect. For example, finding
that the effects of a solvent were similar
to those of ethanol but not caffeine
would support the specificity of solvent
effects on a given measure of
neurotoxicity.
3.1.2. Animal Studies
This section provides an overview of
the major types of endpoints that may
be evaluated in animal neurotoxicity
studies, describes the kinds of effects
that may be observed and some of the
tests used to detect and quantify these
effects, and provides guidance for
interpreting data. Compared with
human studies, animal studies are more
often available for specific chemicals,
provide more precise exposure
information, and control environmental
factors better (Anger, 1984). For these
reasons, risk assessments tend to rely
heavily on animal studies.
Many tests that can measure some
aspect of neurotoxicity have been used
in the field of neurobiology in the past
50 years. The Office of Prevention,
Pesticides and Toxic Substances
(OPPTS) has published animal testing
guidelines that were developed in
cooperation with the Office of Research
and Development (U.S. EPA, 1991a).
While the test endpoints included in the
1991 document serve as a convenient
focus for this section, there are many
other endpoints for which there are no
current EPA guidelines. The goal of the
current document is to provide a
framework for interpreting data
collected in tests frequently used by
neurotoxicologists.
Five categories of endpoints will be
described: structural or
neuropathological, neurophysiological,
neurochemical, behavioral, and
developmental. Table 1 lists a number
of endpoints in each of these categories.
It is imperative for the risk assessor to
understand that the interpretation of the
indicators listed in Table 1 as
neurotoxic effects is dependent on the
dose at which such changes occur and
the possibility that damage to other
organ systems may contribute to or
cause such changes indirectly.
TABLE 1.EXAMPLES OF POSSIBLE INDICATORS OF A NEUROTOXIC EFFECT
Structural or neuropathological endpoints:
Gross changes in morphology, including brain weight.
Histologic changes in neurons or glia (neuronopathy, axonopathy, myelinopathy).
Neurochemical endpoints:
Alterations in synthesis, release, uptake, degradation of neurotransmitters.
Alterations in second-messenger-associated signal transduction.
Alterations in membrane-bound enzymes regulating neuronal activity.
Inhibition and aging of neuropathy enzyme.
Increases in glial fibrillary acidic protein in adults.
Neurophysiological endpoints:
Change in velocity, amplitude, or refractory period of nerve conduction.
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TABLE 1.EXAMPLES OF POSSIBLE INDICATORS OF A NEUROTOXIC EFFECTContinued
Change In latency or amplitude of sensory-evoked potential.
Change in electroencephalographic pattern.
Behavioral and neurological endpoints:
Increases or decreases in motor activity.
Changes In touch, sight, sound, taste, or smell sensations.
Changes In motor coordination, weakness, paralysis, abnormal movement or posture, tremor, ongoing performance
Absence or decreased occurrence, magnitude, or latency of sensorimotor reflex.
Altered magnitude of neurological measurement, including grip strength, hindlimb splay.
Seizures.
Changes In rate or temporal patterning of schedule-controlled behavior.
Changes in learning, memory, and attention.
Developmental endpoints:
Chemically induced changes in the time of appearance of behaviors during development.
Chemically induced changes in the growth or organization of structural or neurochemical elements.
3.1,2.1. Structural Endpoints of '
Neurotoxicity
Structural endpoints are typically
defined as neuropathological changes
evident by gross observation or light
microscopy, although most neurotoxic
changes will be detectable only at the
light microscopic level. Gross changes
in morphology can include discrete or
widespread lesions in nerve tissue. A
change in brain weight is considered to
be a biologically significant effect. This
is true regardless of changes in body
weight, because brain weight is
generally protected during
undernutrition or weight loss, unlike
many other organs or tissues. It is
inappropriate to express brain weight
changes as a ratio of body weight and
thereby dismiss changes in absolute
brain weight. Changes in brain weight
are a more reliable indicator of
alteration in brain structure than are
measurements of length or width in
fresh brain, because there is litde
historical data in the toxicology
literature.
Neurons are composed of a neuronal
body, axon, and dendritic processes.
Various types of neuropathological
lesions may be classified according to
the site where they occur (Spencer and
Schaumburg, 1980; WHO, 1986; Krinke,
1989; Griffin, 1990). Neurotoxicant-
induced lesions in the central or
peripheral nervous system may be
classified as a neuronopathy (changes in
the neuronal cell body), axonopathy
(changes in the axons), myelinopathy
(changes in the myelin sheaths), or
nerve terminal degeneration. Nerve
terminal degeneration represents a very
subtle change that may not be detected
by routine histopathology, but requires
detection by special procedures such as
silver staining or neurotransmitter-
specific immunohistochemistry. For
axonopathies, a more precise location of
the changes may also be described (i.e.,
proximal, central, or distal axonopathy).
In the case of some developmental
exposures, a neurotoxic chemical might
delay or accelerate the differentiation or
proliferation of cells or cell types.
Alteration in the axonal termination site
might also occur with exposure. In an
aged population, exposure to some
neurotoxicants might accelerate the
normal loss of neurons associated with
aging (Reuhl, 1991). In rare cases,
neurotoxic agents have been reported to
produce neuropathic conditions
resembling neurodegenerative disorders,
such as Parkinson's disease, in humans
(WHO, 1986). Table 2 lists examples of
such neurotoxic chemicals, their
putative site of action, the type of
neuropathology produced, and the
disorder or condition that each typifies.
Inclusion of any chemical in any of the
following tables is for illustrative
purposes, i.e., it has been reported that
the chemical will produce a neurotoxic
effect at some dose; any individual
chemical listed may also adversely
affect other organs at lower doses. It is
important that the severity of each
structural union be graded objectively
and the grading criteria reported.
TABLE 2.NEUROTOXICANTS AND DISORDERS WITH SPECIFIC NEUROLOGICAL TARGETS
Site of action
Neuron ceil body
Nerve terminal
Schwann cell myelin
Centra-peripheral distal axon
Central axons
Proximal axon
Neurotoxic change
Neuronopathy
Terminal destruction . ..
Myelinopathy
Distal axonopathy
Central axonopathy
Proximal axonopathy
Neurotoxic chemical
Methylmercury
Quinolinic acid
3-Acetylpyridine
1-Methyl-4-phenyl 1,2,
3 6-tetrahydro-
pyridine (MPTP) (dopaminergic)
Hexachlorophene
Acrylamide, carbon disulfide, n-
hexane.
Clioquinol
B,B'-lminodipropionitriIe
Corresponding neurodegenerative
disorder
Minamata disease.
Huntington's disease.
Cerebellar ataxia.
Parkinson's disease.
Congenital hypomyelinogenesis.
Peripheral neuropathy.
Subacute myeloopticoneuro-pathy.
Motor neuron disease.
Alterations in the structure of the
nervous system (i.e., neuronopathy,
axonopathy. myelinopathy, terminal
degeneration) are regarded as evidence
of a neurotoxic effect. The risk assessor
should note that pathological changes in
many cases require time for the
perturbation to become observable,
especially with evaluation at the light
microscopic level. Neuropathological
studies should control for potential
differences in the area(s) and section(s)
of the nervous system sampled; in the
age, sex, and body weight of the subject;
and in fixation artifacts (WHO, 1986).
Concern for the structural integrity of
nervous system tissues derives from
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their functional specialization and lack
of regenerative capacity.
Within general class of nervous
system structural alteration, there are
various histological changes that can
result after exposure to neurotoxicants.
For example, specific changes in nerve
cell bodies include chromatolysis,
vacuolization, and cell death. Axons can
undergo swelling, degeneration, and
atrophy, while myelin sheath changes
include folding, edematous splitting,
and demyelination. Although terminal
degeneration does occur, it is not
readily detectable by light microscopy.
Many of these changes are a result of
complex effects at specific subcellular
organelles, such as the axonal swelling
that occurs as a result of neurofilament
accumulation in acrylamide toxicity.
Other changes may be associated with
regenerative or adaptive processes that
occur after neurotoxicant exposure.
3.1.2.2. Neurophysiological Endpoints
of Neurotoxicity
Neurophysiological studies measure
the electrical activity of the nervous
system. The term "neurophysiology" is
often used synonymously with
"electrophysiology" (Dyer, 1987).
Neurophysiological techniques provide
information on the integrity of defined
portions of the nervous system. Several
neurophysiological procedures are
available for application to
neurotoxicological studies. Examples
are listed in Table 3. They range in scale
from procedures that employ
microelectrodes to study the function of
single nerve cells or restricted portions
of them, to procedures that employ
macroelectrodes to perform
simultaneous recordings of the summed
activity of many cells. Microelectrode
procedures typically are used to study
mechanisms of action and are frequently
performed in vitro. Macroelectrode
procedures are generally used in studies
to detect or characterize the potential
neurotoxic effects of agents of interest
because of potential environmental
exposure. The present discussion
concentrates on macroelectrode
neurophysiological procedures because
it is more likely that they will be the
focus of decisions regarding critical
effects in risk assessment. All of the
procedures described below for use in
animals also have been used in humans
to determine chemically induced
alterations in neurophysiological
function.
TABLE 3.EXAMPLES OF NEUROPHYSIOLOGICAL MEASURES OF NEUROTOXICITY
System/function
Procedure
Representative agents
Retina
Visual pathway
Visual function.
Auditory pathway
Auditory function
Somatosensory pathway.
Somatosensory function ,
Spinocerebellar pathway .
Mixed nerve
Motor axons
Sensory axons
Neuromuscular
General central nervous system/level of arous-
al.
Electroretinography (ERG)
Flash-evoked potential (FEP)
Pattern-evoked potential (PEP) (pattern size
and contrast).
Brain stem auditory evoked potential (BAER)
(clicks).
BAER (tones) ,
Somatosensory provoked .-.
Sensory-evoked potential (SEP) (tactile)
SEP recorded from cerebellum
Peripheral nerve compound action potential
(PNAP).
PNAP isolate motor components
PNAP isolate sensory components
Electromyography (EMG)
Electroencephalography (EEG)
Developmental lead.
Carbon disulfide.
Carbon disulfide.
Aminoglycoside, antibiotics, toluene, styrene.
Aminoglycoside, antibiotics, toluene, styrene.
Acrylamide, n-hexane.
Acrylamide, n-hexane.
Acrylamide, n-hexane.
Triethyltin.
Triethyltin.
Triethyltin.
Dithiobiuret.
Toluene.
3.1.2.2.1. Nerve Conduction Studies.
Nerve conduction studies, generally
performed on peripheral nerves, can be
useful in investigations of possible
peripheral neuropathy. Most peripheral
nerves contain mixtures,of individual
sensory and motor nerve-fibers, which
may or may not be differentially
sensitive to neurotoxicants. It is possible
to distinguish sensory from motor
effects in peripheral nerve studies by
measuring activity in sensory nerves or
by measuring the muscle response
evoked by nerve stimulation to measure
motor effects. While a number of
endpoints can be recorded, the most
critical variables are nerve conduction
velocity, response amplitude, and
refractory period. It is important to
recognize that damage to nerve fibers
may not be reflected in changes in these
endpoints if the damage is not
sufficiently extensive. Thus, the
interpretation of data from such studies
may be enhanced if evaluations such as
nerve pathology and/or other structural
measures are also included.
Nerve conduction measurements are
influenced by a number of factors, the
most important of which is temperature.
An adequate nerve conduction study
will either measure the temperature of
the limb under study and
mathematically adjust the results
according to well-established
temperature factors or will control limb
temperature within narrow limits.
Studies that measure peripheral nerve
function without regard for temperature
are not adequate for risk assessment.
In well-controlled studies, statistically
significant decreases in nerve
conduction velocity are indicative of a
neurotoxic effect. While a decrease in
nerve conduction velocity is indicative
of demyelination, it frequently occurs
later in the course of axonal degradation
because normal conduction velocity
may be maintained for some time in the
face of axonal degeneration. For this
reason, a measurement of normal nerve
conduction velocity does not rule out
peripheral axonal degeneration if other
signs of peripheral nerve dysfunction
are present.
Decreases in response amplitude
reflect a loss of active nerve fibers and
may occur prior to decreases in
conduction velocity in the course of
peripheral neuropathy. Hence, changes
in response amplitude may be more
sensitive measurements of axonal
degeneration than is conduction
velocity. Measurements of response
amplitude, however, can be more
variable and require careful application
of experimental techniques, a larger
sample size, and greater statistical
power than measurements of velocity to
detect changes. The refractory period
refers to the time required after
stimulation before a nerve can fire again
and reflects the functional status of
nerve membrane ion channels.
Chemically induced changes in
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26937
refractory periods in_a well-controlled
study indicate a neurotoxic effect.
In summary, alterations in peripheral
nerve response amplitude and refractory
period in studies that are well
controlled for temperature are indicative
of a neurotoxic effect. Alterations in
peripheral nerve function are frequently
associated with clinical signs such as
numbness, tingling, or burning
sensations or with motor impairments
such as weakness. Examples of
compounds that alter peripheral nerve
function in humans or experimental
animals include acrylamide, carbon
disulfide. n-hexane, lead, and some
organophosphates.
3.1.2.2.2. Sensory, Motor, and Other
Evoked Potentials. Evoked potential
studies are electrophysiological
procedures that measure the response
elicited from a defined stimulus such as
a tone, a light, or a brief electrical pulse.
Evoked potentials reflect the function of
the system under study, including
visual, auditory, or somatosensory;
motor, involving motor nerves and
innervated muscles; or other neural
pathways in the central or peripheral
nervous system (Rebert, 1983; Dyer,
1985; Mattsson and Albee, 1988;
Mattsson etal., 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,
%vhen such information is available.
Such correlative information
strengthens the confidence in
electrophysiological outcomes. In the
absence of such supportive information,
the extent to which evoked potential
studies provide convincing evidence of
neurotoxicity is a matter of professional
judgment on a case-by-case basis.
Judgments should consider the nature,
magnitude, and duration of such effects,
along with other factors discussed
elsewhere in this document.
Data are in the form of a voltage
record collected over time and can be
quantified in several ways. Commonly,
the latency (time from stimulus onset)
and amplitude (voltage) of the positive
and negative voltage peaks are
identified and measured. Alternative
measurement schemes may involve
substitution of spectral phase or
template shifts for peak latency and
spectral power, spectral amplitude, root-
mean-square, or integrated area under
the curve for peak amplitude. Latency
measurements are dependent on both
the velocity of nerve conduction and the
time of synaptic transmission. Both of
these factors depend on temperature, as
discussed in regard to nerve conduction,
and similar caveats apply for sensory
evoked potential studies. In studies that
are well controlled for temperature,
increases in latencies or related
measures can reflect deficits in nerve
conduction, including demyelination or
delayed synaptic transmission, and are
indicators of a neurotoxic effect.
Decreases in peak latencies, like
increases in nerve conduction velocity,
are unusual, but the neural systems
under study in sensory evoked
potentials are complex, and situations
that might cause a peak measurement to
occur earlier are conceivable. Two such
situations are a reduced threshold for
spatial or temporal summation of
afferent neural transmission and a
selective loss of cells responding late in
the peak, thus making the measured
peak occur earlier. Decreases in peak
latency should not be dismissed
outright as experimental or statistical
error, but should be examined carefully
and perhaps replicated to assess
possible neurotoxicity. A decrease in
latency is not conclusive evidence of a
neurotoxic effect.
Changes in peak amplitudes or
equivalent measures reflect changes in
the magnitude of the neural population
responsive to stimulation. Both
increases and decreases in amplitude
are possible following exposure to
chemicals. Whether excitatory or
inhibitory neural activity is translated
into a positive or negative deflection in
the sensory evoked potential is
dependent on the physical orientation
of the electrode with respect to the
tissue generating the response, which is
frequently unknown. Comparisons
should be based on the absolute change
in amplitude. Therefore, either increases
or decreases in amplitude may be
indicative of a neurotoxic effect.
Within any given sensory system, the
neural circuits that generate various
evoked potential peaks differ as a
function of peak latency. In general,
early latency peaks reflect the
transmission of afferent sensory
information. Changes in either the
latency or amplitude of these peaks are
considered convincing evidence of a
neurotoxic effect that is likely to be
reflected in deficits in sensory
perception. The later-latency peaks, in
general, reflect not only the sensory
input but also the more nonspecific
factors such as the behavioral state of
the subject, including such factors as
arousal level, habituation, or
sensitization (Dyer, 1987). Thus,
changes in later-latency evoked
potential peaks should be interpreted in
light of the behavioral status of the
subject and would generally be
considered evidence of a neurotoxic
effect.
3.1.2.2.3. Seizures/Convulsions. Some
neurotoxicants (e.g., lindane,
pyrethroids, trimethyltin,
dichlorodiphenyltrichloroethane [DDT])
produce observable convulsions. When
convulsionlike behaviors are observed,
as described in the behavioral section
on convulsions, neurophysiological
recordings can provide additional
information to help interpret the results.
Recordings of brain electrical activity
that demonstrate seizurelike activity are
indicative of a neurotoxic effect.
In addition to producing seizures
directly, chemicals may also alter the
frequency, severity, duration, or
threshold for eliciting seizures through
other means by a phenomenon known
as "kindling." Such alterations can
occur after acute exposure or after
repeated exposure to dose levels below
the acute threshold. In experiments
demonstrating changes in sensitivity
following repeated exposures to the test
compound, information regarding
possible changes in the pharmacokinetic
distribution of the compound is
required before the seizure
susceptibility changes can be
interpreted as evidence of neurotoxicity.
Increases in susceptibility to seizures
are considered adverse.
3.1.2.2.4. Electroencephalography
(EEC). EEC analysis is used widely in
clinical settings for the diagnosis of
neurological disorders, and less often
for the detection of subtle toxicant-
induced dysfunction (WHO, 1986;
Eccles, 1988). The basis for using EEC
in either setting is the relationship
between specific patterns of EEC
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 EEC. Dissociation of EEC
activity and behavior can, however,
occur after exposure to certain
chemicals. Normal patterns of transition
between sleep stages or between
sleeping and waking states are known to
remain disturbed for prolonged periods
of time after exposure to some
chemicals. Changes in the pattern of the
EEC can be elicited by anesthetic drugs
and stimuli producing arousal (e.g.,
lights, sounds). In studies with
toxicants, changes in EEC pattern can
sometimes precede alterations in other
objective signs of neurotoxicity (Dyer,
1987).
EEC studies should be done under
highly controlled conditions, and the
data should be considered on a case-by-
case basis. Chemically induced seizure
activity detected in the EEC pattern is
evidence of a neurotoxic effect.
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3.1.2.3. Neurochemical Endpoints of
Neurotoxicity "
Many different neurochemical
endpoints have been measured in
neurotoxicological studies, and some
have proven useful in advancing the
understanding of mechanisms of action
of neurotoxic chemicals (Bondy, 1986;
Mailman, 1987; Morell and Mailman,
1987; Costa, 1988; Silbergeld, 1993).
Normal functioning of the nervous
system depends on the synthesis and
release of specific neurotransmitters and
activation of their receptors at specific
presynaptic and postsynaptic sites.
Chemicals can interfere with the ionic
balance of a neuron, act as a
cytotoxicant after transport into a nerve
terminal, block reuptake of
neurotransmitters and their precursors,
act as a metabolic poison, overstimulate
receptors, block transmitter release, and
inhibit transmitter synthetic or catabolic
enzymes. Table 4 lists several chemicals
that produce neurotoxic effects at the
neurochemical level (Bondy, 1986;
Mailman, 1987; Morell and Mailman,
1987; Costa, 1988).
TABLE 4.EXAMPLES OF NEUROTOXICANTS WITH KNOWN NEUROCHEMICAL MECHANISMS
Site of action
Neurotoxicants acting on ionic balance:
Inhibit sodium entry
Block closing of sodium channel .
increase permeability to sodium
Increase intracellular calcium
Synaptic neurotoxicants
Uptake blockers
Metabolic poisons
Hyperactivation of receptors
Blocks transmitter release
Inhibition of transmitter degradation
Blocks axonal transport
Examples
Tetrodotoxin.
p,p'-DDT, pyrethroids.
Batrachotoxin.
Chlorodecone.
MPTP.
Hemicholinium.
Cyanide.
Domoic acid.
Botulinum toxin.
Pesticides of the organophosphate and carbamate classes.
Acrylamide.
As stated previously, any
neurochemical change is potentially
neurotoxic. Persistent or irreversible
chemically induced neurochemical
changes are indicative of neurotoxicity.
Because the ultimate functional
significance of some biochemical
changes is not known at this time,
neurochemical studies should be
interpreted with reference to the
presumed neurotoxic consequence(s) of
the neurochemical changes. For
example, many neuroactive agents can
increase or decrease neurotransmitter
levels, but such changes are not
indicative of a neurotoxic effect. If,
however, these neurochemical changes
may be expected to have
neurophysiological, neuropathological,
or neurobehavioral correlates, then the
neurochemical changes could be
classified as neurotoxic effects.
Some neurotoxicants, such as the
organophosphate and carbamate
pesticides, are known to inhibit the
activity of a specific enzyme,
acetylcholinesterase (for a review see
Costa, 1988), which hydrolyzes the
neurotransmitter acetylcholine.
Inhibition of the enzyme in either the
central or peripheral nervous system
prolongs the action of the acetylcholine
at the neuron's synaptic receptors and is
thought to be responsible for the range
of effects these chemicals produce,
although it is possible that these
compounds have other modes of action
(Eldefrawi et al., 1992; Greenfield et al.,
1984; Small, 1990).
There is agreement that objective
clinical measures of cholinergic
overstimulation (e.g., salivation,
sweating, muscle weakness, tremor,
blurred vision) can be used to evaluate
dose-response and dose-effect
relationships and define the presence
and absence of effects. A given
depression in peripheral and central
cholinesterase activity may or may not
be accompanied by clinical
manifestations. A depression in RBC
and/or plasma cholinesterase activity
may or may not be accompanied by
clinical manifestations. It should be
noted, however, that reduction in
cholinesterase activity, even if the
aiiticholinesterase exposure is not
severe enough to precipitate clinical
signs or symptoms, may imjaair the
organism's ability to adapt to additional
exposures to anticholinesterase
compounds. Inhibition of RBC and/or
plasma cholinesterase activity is a
biomarker of exposure, as well as a
reflection of cholinesterase inhibition in
other peripheral tissues (e.g.,
neuromuscular junction, peripheral
nerve, or ganglia) (Maxwell et al., 1987;
Nagymajtenyi et al., 1988; Padilla et al.,
1994), thereby contributing to the
overall hazard identification of
cholinesterase-inhibiting compounds.
The risk assessor should also be aware
that tolerance to the cholinergic
overstimulation may be observed
following repeated exposure to
cholinesterase-inhibiting chemicals. It
has been reported, however, that
although tolerance can develop to some
effects of cholinesterase inhibition, the
cellular mechanisms responsible for the
development of tolerance may also lead
to the development of other effects, i.e.,
cognitive dysfunction, not present at the
time of initial exposure (Bushnell et al.,
1991). These adaptive biochemical
changes in the tolerant animal may
render it supersensitive to subsequent
exposure to cholinergically active
compounds (Pope et al., 1992).
In general, the risk assessor should
understand that assessment of
cholinesterase-inhibiting chemicals
should be done on a case-by-case basis
using a weight-of evidence approach in
which all of the available data (e.g.,
brain, blood, and other tissue
cholinesterase activity, as well as the
presence or absence of clinical signs) is
considered in the evaluation. Generally,
the toxic effects of anticholinesterase
compounds are viewed as reversible,
but there is human and experimental
animal evidence indicating that there
may be residual, if not permanent,
effects of exposure to these compounds
(Steenland et al., 1994; Tandon et al.,
1994; Stephens et al., 1995).
A subset of organophosphate agents
also produces organophosphate-induced
delayed neuropathy (OPIDN) after acute
or repeated exposure. Inhibition and
aging of neurotoxic esterase (or
neuropathy enzymes) are associated
with agents that produce OPIDN
(Johnson, 1990; Richardson, 1995). The
conclusion that a chemical may produce
OPIDN should be based on at least two
of three factors: (1) Evidence of a
clinical syndrome, (2) pathological
lesions, and (3) neurotoxic esterase
(NTE) inhibition. NTE inhibition is
necessary, but not sufficient, evidence
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26939
of the potential to produce OPIDN when
there Is at least 55%-70% inhibition
after acute exposure (Ehrich et al., 1995)
and at least 45% inhibition following
repeated exposure.
Chemically induced injury to the
central nervous system may be
accompanied by hypertrophy of
astrocytes. In some cases, these
astrocytic changes can be seen light
microscopically with
immunohistochemical stains for glial
flbrillary acidic protein (GFAP), the
major intermediate filament protein in
astrocytes. In addition, GFAP can be
quantified by an immunoassay, which
has been proposed as a marker of
astrocyte reactivity (O'Callaghan, 1988).
Immunohistochemical stains have the
advantage of better localization of GFAP
increases, whereas immunoassay
evaluations are superior at detecting and
quantifying changes in GFAP levels and
establishing dose-response
relationships. The ability to detect and
quantify changes in GFAP by
immunoassay is improved by dissecting
and analyzing multiple brain regions.
The interpretation of a chemical-
induced change in GFAP is facilitated
by corroborative data from the
neuropathology or neuroanatomy
evaluation. A number of chemicals
known to injure the central nervous
system, including trimethyltin,
methylmercury, cadmium, 3-
acetylpyridine, and
methylphenyltetrahydropyridine
(MPTP), have been shown to, increase
levels of GFAP. Measures of GFAP are
now included as an optional test in the
Neurotoxicity Screening Battery (U.S.
EPA. 1991a).
Increases in GFAP above control
levels may be seen at dosages below
those necessary to produce damage seen
by standard microscopic or
histopathological techniques. Because
increases in GFAP reflect an astrocyte
response in adults, treatment-related
increases in GFAP are considered to be
evidence that a neurotoxic effect has
occurred. There is less agreement as to
how to interpret decreases in GFAP
relative to an appropriate control group.
The absence of a change in GFAP
following exposure does not mean that
the chemical is devoid of neurotoxic
potential. Known neurotoxicants such
as cholinesterase-inhibiting pesticides,
for example, would not be expected to
increase brain levels of GFAP.
Interpretation of GFAP changes prior to
weaning may be confounded by the
possibility that chemically induced
increases in GFAP could be masked by
changes in the concentration of this
protein associated with maturation of
the central nervous system, and these
data may be difficult to interpret.
3.1.2.4. Behavioral Endpoints of
Neurotoxicity
Behavior reflects the integration of the
various functional components of the
nervous system. Changes in behavior
can arise from a direct effect of a
toxicant on the nervous system, or
indirectly from its effects on other
physiological systems. Understanding
the interrelationship between systemic
toxicity and behavioral changes (e.g.,
the relationship between liver damage
and motor activity) is extremely
important. The presence of systemic
toxicity may complicate, but does not
preclude, interpretation of befegvioral
changes as evidence of neurotoxicity. In
addition, a number of behaviors (e.g.,
schedule-controlled behavior) may
require a motivational component for
successful completion of the task. In
such cases, experimental paradigms
designed to assess the motivation of an
animal during behavior might be
necessary to interpret the meaning of
some chemical-induced changes in
behavior.
EPA's testing guidelines developed
for the Toxic Substances Control Act
and the Federal Insecticide, Fungicide
and Rodenticide Act describe the use of
functional observational batteries (FOB),
motor activity, and schedule-controlled
behavior for assessing neurotoxic
potential (U.S. EPA, 1991a). Examples
of measures obtained in a typical FOB
are presented in Table 5. There are
many other measures of behavior,
including specialized tests of motor and
sensory function and of learning and
memory (Tilson, 1987; Anger, 1984).
TABLE 5.EXAMPLES OF MEASURES
IN A REPRESENTATIVE FUNCTIONAL
OBSERVATIONAL BATTERY
Home cage
and open field
Arousal
Autonomic
signs.
Convulsions,
tremors.
Gait
Mobility
Posture
Rearing.
Stereotypy.
Touch re-
sponse.
Manipulative
Approach re-
sponse.
Click re-
sponse.
Foot splay.
Grip strength
Righting re-
flex.
Tail pinch re-
sponse.
Physiological
Body tem-
perature.
Body weight.
TABLE 6.EXAMPLES OF SPECIALIZED BEHAVIORAL TESTS To MEASURE NEUROTOXICITY
Function
Procedure
Representative agents
Motor Function
Weakness .
Incoordina'ion
Tren>or
Myodonic spasms ...
Grip strength, swimming endurance, suspen-
sion rod, discriminative motor function.
Rotorod, gait assessments, righting reflex
Rating scale spectral analysis
Rating scale
n-Hexane, methyl.
n-Butylketone, carbaryl.
3-Acetylpyridine, ethanol.
Chlordecone, Type 1.
pyrethroids, DDT.
DDT, Type II pyrethroids.
Sensory Function
Auditory . ...
Visual . . . ...
Sofnatosensory
Pain sensitivity .
Olfactory . ซ
Discrimination conditioning
Reflex modification.
Discrimination conditioning
Discrimination conditioning
Discrimination conditioning
Discrimination conditioning
Toluene, trimethyltin.
Methylmercury.
Acrylamide.
Parathion.
3-Methylindole, methylbromide.
Cognitive Function
Habituation
Startle reflex
Diisopropylfluorophosphate.
Pre/neonatal methylmercury.
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TABLE 6..EXAMPLES OF SPECIALIZED BEHAVIORAL TESTS To MEASURE NEUROTOXICITYContinued
Function
Classical conditioning
Instrumental conditioning
Procedure
Nictitating membrane
Conditioned flavor
aversion
Passive avoidance
Olfactory conditioning.
One-way avoidance
Two-way avoidance
Y-maze avoidance
Biel water maze
Morris water maze
Radial arm ma?A
Delayed matching to sample
Repeated acquisition
Representative agents
Aluminum
Carbaryl
Trimethyltin IDPN
Neonatal trimethyltin
Chlordecone
Pre/neonatal lead
Hypervitaminosis A.
Styrene
DFP
Trimethyltin
DFP.
Carbaryl
At the present time, there is no^ clear .
consensus concerning the use of specific
behavioral tests to assess chemical-
induced sensory, motor, or cognitive
dysfunction in animal models. The risk
assessor should also know that the
literature is clear that a number of other
behaviors besides those listed in Tables
1,5, and 6 could be affected by
chemical exposure. For example,
alterations in food and water intake,
reproduction, sleep, temperature
regulation, and circadian rhythmicity
are controlled by specific regions of the
brain, and chemical-induced alterations
in these behaviors could be indicative of
neurotoxicity. It is reasonable to assume
that an NOAEL or LOAEL could be
based on one or more of these
endpoints.
The following sections describe, in
general, behavioral tests and their uses
and offer guidance on interpreting data.
3.1.2.4.1. Functional Observational
Battery (FOB). An FOB is designed to
detect and quantify major overt
behavioral, physiological, and
neurological signs (Gad, 1982;
O'Donoghue, 1989; Moser, 1989). A
number of batteries have been
developed, each consisting of tests
generally intended to evaluate various
aspects of sensorimotor function (Tilson
and Moser, 1992). Many FOB tests are
essentially clinical neurological
examinations that rate the presence or
absence, and in many cases the severity,
of specific neurological signs. Some
FOBs in animals are similar to clinical
neurological examinations used with
human patients. Most FOBs have
several components or tests. A typical
FOB is summarized in Table 5 and
evaluates several functional domains,
including neuromuscular (i.e.,
weakness, incoordination, gait, and
tremor), sensory (i.e., audition, vision,
and somatosensory), and autonomic
(i.e., pupil response and salivation)
function.
The relevance of statistically
significant test results from an FOB is
judged according to the number of signs
affected, the dose(s) at which effects are
observed, and the nature, severity, and
persistence of the effects and their
incidence in relation to control animals.
In general, if only a few unrelated
measures in the FOB are affected, or the
effects are unrelated to dose, the results
may not be considered evidence of a
neurotoxic effect. If several neurological
signs are affected, but only at the high
dose and in conjunction with other
overt signs of toxicity, including
systemic toxicity, large decreases in
body weight, decreases in body
temperature, or debilitation, there is less
persuasive evidence of a direct
neurotoxic effect. In cases where several
related measures in a battery of tests are
affected and the effects appear to be
dose dependent, the data are considered
to be evidence of a neurotoxic effect,
especially in the absence of systemic
toxicity. The risk assessor should be
aware of the potential for a number of
false positive statistical findings in these
studies because of the large number of
endpoints customarily included in the
FOB.
FOB data can be grouped into one or
more of several neurobiological
domains, including neuromuscular (i.e.,
weakness, incoordination, abnormal
movements, gait), sensory (i.e., auditory,
visual, somatosensory), and autonomic
functions CTilson and Moser, 1992).
This statistical technique may be useful
when separating changes that occur on
the basis of chance or in conjunction
with systemic toxicity from those
treatment-related changes indicative of
neurotoxic effects. In the case of the
developing organism, chemicals may
alter the maturation or appearance of
sensorimotor reflexes. Significant
alterations in or delay of such reflexes
is evidence of a neurotoxic effect.
Examples of chemicals that affect
neuromuscular function are
3-acetylpyridine, acrylamide, and
triethyltin. Organophosphate and
carbamate insecticides produce
autonomic dysfunction, while
organochlorine and pyrethroid
insecticides increase sensorimotor
sensitivity, produce tremors and, in
some cases, cause seizures and
convulsions (Spencer and Schaumburg,
1980).
3.1.2.4.2. Motor Activity. Motor
activity represents a broad class of
behaviors involving coordinated
participation of sensory, motor, and
integrative processes. Assessment of
motor activity is noninvasive and has
been used to evaluate the effects of
acute and repeated exposure to
neurotoxicants (MacPhail et al., 1989).
An organism's level of activity can,
however, be affected by many different
types of environmental agents,
including non-neurotoxic agents. Motor
activity measurements also have been
used in humans to evaluate disease
states, including disorders of the
nervous system (Goldstein and Stein,
1985).
Motor activity is usually quantified as
the frequency of movements over a
period of time. The total counts
generated during a test period will
depend on the recording mechanism
and the size and configuration of the
testing apparatus. Effects of agents on
motor activity can be expressed as
absolute activity counts or as a
percentage of control values. In some
cases, a transformation (e.g., square root)
may be used to achieve a normal
distribution of the data. In these cases,
the transformed data and not raw data
should be used for risk assessment
purposes. The frequency of motor
activity within a session usually
decreases and is reported as the average
number of counts occurring in each
successive block of time. The EPA's
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26941
Office of Prevention pesticides and
Toxic Substances guidelines (U.S. EPA,
199la), for example, call for test
sessions of sufficient duration to allow
motor activity to approach steady-state
levels during the last 20 percent of the
session for control animals. A sum of
the counts in each epoch will add up to
the total number of counts per session.
Motor activity can be altered by a
number of experimental factors,
including neurotoxic chemicals.
Decreases in activity could occur
following high doses of non-neurotoxic
agents (Kotsonis and Klaassen, 1977;
Landauer et al., 1984). Examples of
neurotoxic agents that decrease motor
activity include many pesticides (e.g.,
carbamates. chlorinated hydrocarbons,
organophosphates, and pyrethroids),
heavy metals (lead, tin, and mercury),
and other agents (3-acetylpyridine,
acrylamide, and 2,4-dithiobiuret). Some
neurotoxicants (e.g., toluene, xylene,
triadimefon) produce transient increases
in activity by presumably stimulating
neurotransmitter release, while others
(e.g., trimethyltin) produce persistent
increases in motor activity by destroying
specific regions of the brain (e.g.,
hippocampus).
Following developmental exposures,
neurotoxic effects are often observed as
a change in the ontogenetic profile or
maturation of motor activity patterns.
Frequently, developmental exposure to
neurotoxic agents will produce an
increase in motor activity that persists
into adulthood or that results in changes
in other behaviors. This is evidence of
a neurotoxic effect. Like other organ
systems, the nervous system may be
differentially sensitive to toxicants in
groups such as the young. For example,
toxicants introduced to the developing
nervous system may kill stem cells and
thus cause profound-effects on adult
structure and function. Moreover,
toxicants may have greater access to the
developing nervous system before the
blood-brain barrier is completely formed
or before metabolic detoxifying systems
are functional.
Motor activity measurements are
typically used with other tests (e.g.,
FOB) to help detect neurotoxic effects.
Agent-induced changes in motor
activity associated with other overt
signs of toxicity (e.g., loss of body
weight, systemic toxicity) or occurring
in non-dose-related fashion are of less
concern than changes that are dose
dependent, are related to structural or
other functional changes in the nervous
system, or occur in the absence of life-
threatening toxicity.
13.1.2.4.3. Schedule-Controlled
Operant Behavior. Schedule-controlled
operant behavior (SCOB) involves the
maintenance of behavior (e.g.,
performance of a lever-press or key-peck
response) by reinforcement. Different
rates and patterns of responding are
controlled by the relationship between
response and subsequent reinforcement.
SCOB provides a measure of
performance of a learned behavior (e.g.,
lever press or key peck) and involves
training and motivational variables that
should be considered in evaluating the
data. Agents may interact with sensory
processing, motor output, motivational
variables (i.e., related to reinforcement),
training history, and baseline
characteristics (Rice, 1988; Cory-
Slechta, 1989). Qualitatively, rates and
patterns of SCOB display cross-species
generality, but the quantitative measures
of rate and pattern of performance can
vary within and between species.
In laboratory animals, SCOB has been
used to study a wide range of
neurotoxicants, including
methylmercury, many pesticides,
organic and inorganic lead, triethyltin,
and trimethyltin (MacPhail, 1985;
Tilson, 1987; Rice, 1988). The primary
SCOB endpoints for evaluation are
response rate and the temporal pattern
of responding. These endpoints may
vary as a function of the contingency
between responding and reinforcement
presentation (i.e., schedule of
reinforcement). Schedules of
reinforcement that have been used in
toxicology studies include fixed ratio
and fixed interval schedules. Fixed ratio
schedules engender high rates of
responding and a characteristic pause
after delivery of each reinforcement.
Fixed interval schedules engender a
relatively low rate of responding during
the initial portion of the interval and
progressively higher rates near the end
of the interval. For some schedules of
reinforcement, the temporal pattern of
responding may play a more important
role in defining the performance
characteristics than the rate of
responding. For other schedules, the
reverse may be true. For example, the
temporal pattern of responding may be
more important than rate of responding
for defining performance on a fixed
interval schedule. For a fixed ratio
schedule, more importance might be
placed on the rate of responding than on
the post-reinforcement pause.
The overall qualitative patterns are
important properties of the behavior.
Substantial qualitative changes in
operant performance, such as
elimination of characteristic response
patterns, can be evidence of an adverse
effect. Most chemicals, however, can
disrupt operant behavior at some dose,
and such adverse effects may be due
either to neurotoxic or non-neurotoxic
mechanisms. Unlike large qualitative
changes in operant performance, small
quantitative changes are not adverse.
Some changes may actually represent an
improvement, e.g., an increase in the
index of curvature with a decrease in
fixed interval rate of responding.
Assessing the toxicological importance
of these effects requires considerable
professional judgment and evaluation of
converging evidence from other types of
toxicological endpoints. While most
chemicals decrease the efficiency of
responding at some dose, some agents
may increase response efficiency on
schedules requiring high response rates
because of a stimulant effect or an
increase in central nervous system
excitability. Agent-induced changes in
responding between reinforcements
(i.e., the temporal pattern of responding)
may occur independently of changes in
the overall rate of responding.
Chemicals may also affect the reaction
time to respond following presentation
of a stimulus. Agent-induced changes in
response rate or temporal patterning
associated with other overt signs of
toxicity (e.g., body weight loss, systemic
toxicity, or occurring in a non-dose-
related fashion) are of less concern than
changes that are dose dependent, related
to structural or other functional changes
in the nervous system, or occur in the
absence of life-threatening toxicity.
3.1.2.4.4. Convulsions. Observable
convulsions in animals are indicative of an
adverse effect. These events can reflect
central nervous system activity comparable
to that of epilepsy in humans and could be
defined as neurotoxicity. Occasionally, other
toxic actions of compounds, such as direct
effects on muscle, might mimic some
convulsionlike behaviors. In some cases,
convulsions or convulsionlike behaviors may
be observed in animals that are otherwise
severely compromised, moribund, or near
death. In such cases, convulsions might
reflect an indirect effect of systemic toxicity
and are less clearly indicative of
neurotoxicity. As discussed in the section on
neurophysiological measures, electrical
recordings of brain activity could be used to
determine specificity of effects on the
nervous system.
3.1.2.4.5. Specialized Tests for
Neurotoxicity. Several procedures have
been developed to measure agent-
induced changes in specific
neurobehavioral functions such as
motor, sensory, or cognitive function
(Tilson, 1987; Cory-Slechta, 1989).
Table 6 lists several behavioral tests, the
neurobehavioral functions they were
designed to assess, and agents known to
affect the response. Many of these tests
in animals have been designed to assess
neural functions in humans using
similar testing procedures.
A statistically or biologically
significant chemically induced change
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In any measure in Table 6 may be
evidence of an adverse effect. However,
judgments of neurotoxicity may involve
not only the analysis of changes seen
but the structure and class of the
chemical and other available
neurochemical, neurophysiological, and
neuropathological evidence. In general,
behavioral changes seen across broader
dose ranges indicate more specific
actions on the systems underlying those
changes, i.e., the nervous system.
Changes that are not dose dependent or
that are confounded with body weight
changes and/or other systemic toxicity
may be more difficult to interpret as
neurotoxic effects.
3.1.2.4.5.1. Motor Function.
Neurotoxicants commonly affect motor
function. These effects can be
categorized generally into (1) weakness
or decreased strength, (2) tremor, (3)
incoordination, and (4) spasms,
myoclonia, or abnormal motor
movements (Tilson, 1987; Cory-Slechta,
1989). Specialized tests used to assess
strength include measures of grip
strength, swimming endurance,
suspension from a hanging rod, and
discriminative motor function. Rotorod
and gait assessments are used to
measure coordination, while rating
scales and spectral analysis techniques
can be used to quantify tremor and other
abnormal movements.
3.1.2.4.5.2. Sensory Function. Gross
perturbations of sensory function can be
observed in simple neurological
assessments such as the hot plate or tail
flick test. However, these tests may not
be sufficiently sensitive to detect subtle
sensory changes. Psychophysical
procedures that study the relationship
between a physical dimension (e.g.,
intensity, frequency) of a stimulus and
behavior may be necessary to quantify
agent-induced alterations in sensory
function. Examples of psychophysical
procedures include discriminated
conditioning and startle reflex
modification.
3.1.2.4.5.3. Cognitive Function.
Alterations in learning and memory in
experimental animals should be inferred
from changes in behavior following
exposure when compared with that seen
prior to exposure or with a nonexposed
control group. Learning is defined as a
relatively lasting change in behavior due
to experience, and memory is defined as
the persistence of a learned'behavipr
over time. Table 6 lists several examples
of learning and memory tests and
representative neurotoxicants known to
affect these tests. Measurement of
changes in learning and memory should
be separated from other changes in
behavior that do not involve cognitive
or associative processes (i.e., motor
function, sensory capabilities,
motivational factors). In addition, any
apparent toxicant-induced change in
learning,or memory should ideally be
demonstrated over a range of stimulus
and response conditions and testing
conditions. In developmental exposures,
it should be shown that the animals
have matured enough to perform the
specified task. Developmental
neurotoxicants can accelerate or delay
the ability to learn a response or may
interfere with cognitive function at the
time of testing. Older animals frequently
perform poorly on some types of tests,
and it should be demonstrated that
control animals in this population are
capable of performing the procedure.
Neurotoxicants might accelerate age-
related dysfunction or alter motivational
variables that are important for learning
to occur. Further, it is not the case that
a decrease in responding on a learning
task is adverse while an increase in
performance on a learning task is not. It
is well known that lesions in certain
regions of the brain can facilitate the
acquisition of certain types of behaviors
by removing preexisting response
tendencies (e.g., inhibitory responses
due to stress) that moderate the rate of
learning under normal circumstances.
Apparent improvement in
performance is not either adverse or
beneficial until demonstrated to be so
by converging evidence with a variety of
experimental methods. Examples of
procedures to assess cognitive function
include simple habituation, classical
conditioning, and operant (or
instrumental) conditioning, including
tests for spatial learning and memory.
3.1.2.4.5.4. Developmental
Neurotoxicity. Although the previous
discussion of various neurotoxicity
endpoints and tests applie^S) studies in
which developmental exposures are
used, there are particular issues of
importance in the evaluation of
developmental neurotoxicity studies.
This section underscores the importance
of detecting neurotoxic effects following
developmental exposure because an
NRC (1993) report has indicated that
infants and children may be
differentially sensitive to environmental
chemicals such as pesticides. Exposure
to chemicals during development can
result in a spectrum of effects, including
death, structural abnormalities, altered
growth, and functional deficits (U.S.
EPA, 1991b). A number of agents have
been shown to cause developmental
neurotoxicity when exposure occurred
during the period between conception
and sexual maturity (e.g., Riley and
Vorhees, 1986; Vorhees, 1987).
Table 7 lists several examples of
agents known to produce developmental
neurotoxicity in experimental animals.
Animal models of developmental
neurotoxicity have been shown to be
sensitive to several environmental
agents known to produce developmental
neurotoxicity in humans, including
lead, ethanol, x-irradiation,
methylmercury, and polychlorinated
biphenyls (PCBs) (Kimmel et al., 1990;
Needleman, 1990; Jacobsonet al., 1985;
Needleman, 1986). In many of these
cases, functional deficits are observed at
dose levels below those at which other
indicators of developmental toxicity are
evident or at minimally toxic doses in
adults. Such effects may be transient,
but generally are considered adverse.
Developmental exposure to a chemical
could result in transient or reversible
effects observed during early
development that could reemerge as the
individual ages (Barqne et al., 1995).
TABLE 7.EXAMPLES OF COMPOUNDS
OR TREATMENTS PRODUCING DE-
VELOPMENTAL NEUROTOXICITY
Alcohols
Antimitotics .
Insecticides.
Metals
Polyhalogenated hy-
drocarbons.
Methanol, ethanol.
X-radiation,
azacytidine.
DDT, chlordecone.
Lead, methylmercury,
cadmium.
PCBs, PBBs.
Testing for developmental
neurotoxiciry has not been required
routinely by regulatory agencies in the
United States, but is required by EPA
when other information indicates the
potential for developmental
neurotoxicity (U.S. EPA, 1986, 1988a,
1988b, 1989, 1991a, 1991b). Useful data
for decision making may be derived
from well-conducted adult
neurotoxicity studies, standard
developmental toxicity studies, and
multigeneration studies, although the
dose levels used in the latter may be
lower than those in studies with shorter
term exposure.
Important design issues to be
evaluated for developmental
neurotoxiciry studies are similar to
those for standard developmental
toxicity studies (e.g., a dose-response
approach with the highest dose
producing minimal overt maternal or
perinatal toxicity, with number of litters
large enough for adequate statistical
power, with randomization of animals
to dose groups and test groups, with
litter generally considered as the
statistical unit). In addition, the use of
a replicate study design provides added
confidence in the interpretation of data.
A pharmacological/physiological
challenge may also be valuable in
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26943
evaluating neurological function and
"unmasking" effects not otherwise
detectable. For example, a challenge
with a psychomotor stimulant such as
d-amphetamine may unmask latent
developmental neurotoxicity (Hughes
and Sparber. 1978; Adams and Buelke-
Sam, 1981;Buelke-Sametal., 1985).
Direct extrapolation of developmental
neurotoxicity to humans is limited in
the same way as for other endpoints of
toxicity, i.e., by the lack of knowledge
about underlying toxicological
mechanisms and their significance (U.S.
EPA. 1991b). However, comparisons of
human and animal data for several
agents known to cause developmental
neurotoxicity in humans showed many
similarities in effects (Kimmel et al.,
1990). As evidenced primarily by
observations in laboratory animals,
comparisons at the level of functional
category (sensory, motivational,
cognitive, motor function, and social
behavior) showed close agreement
across species for the agents evaluated,
even though the specific endpoints used
to assess these functions varied
considerably across species (Stanton
and Spear. 1990). Thus, it can be
assumed that developmental
neurotoxicity effects in animal studies
indicate the potential for altered
neurobehavioral development in
humans, although the specific types of
developmental effects seen in
experimental animal studies will not be
the same as those that may be produced
in humans. Therefore, when data
suggesting adverse effects in
developmental 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% or less)
may be more sensitive to the effects of
an agent on behavioral endpoints 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, or 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 mild
maternal toxicity (e.g., 10%-20%
reduction in weight gain during
gestation and lactation). At doses
causing moderate maternal toxicity (i.e.,
20% or more 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
(e.g., alcohol) are known to produce
adverse developmental effects at
minimally toxic doses in adult humans
(Coles etal., 1991).
Although interpretation of
developmental neurotoxicity data may
be limited, it is clear that functional
effects should 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 have been reviewed
byWinneke(1995).
3.1.3. Other Considerations
3.1.3.1. 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. The vast
majority of the central nervous system is
served by blood vessels with blood-
brain barrier properties, which exclude
most ionic and nonlipid-soluble
chemicals from the brain and spinal
cord. The brain contains several
structures called circumventricular
organs (CVOs) that are served by blood
vessels lacking blood-brain barrier
properties. Brain regions adjacent to
these CVOs are thus exposed to
relatively high levels of many
neurotoxicants. 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 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.
3.1.3.2. 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 (S AR) of some chemical
classes have been studied, including
hexacarbons, organophosphates,
carbamates, and pyrethroids. Therefore,
class relationships or SAR may help
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predict neurotoxicityjDr 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 (1995), for
example, reported that the behavioral
effects of protorypic cholinesterase-
inhibiting pesticides differed
qualitatively in a battery of behavioral
tests.
3.1.3.3. 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. The risk
assessor should be aware that some
neurotoxicants may induce a greater
variability in biologic response, rather
than a clear shift in mean or other
parameters (Laties and Evans, 1980;
Glowa and MacPhail, 1995). 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 should 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
endpoints. 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.
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, can be 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 endpoints within a series of
evaluations, some type of correction for
multiple observations is warranted
(Winer, 1971).
3.1.3.4. 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, including primary cell
cultures, cell lines, and cloned cells,
produce data for evaluating potential
and known neurotoxic substances.
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
endpoints, 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 endpoints 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.
3.1.3.5. Neuroendocrine Effects
Neuroendocrine dysfunction may
occur because of a disturbance in the
regulation and modulation of
neuroendocrine feedback systems. One
major indicator of neuroendocrine
function is secretion of hormones from
the pituitary. Hypothalamic control of
anterior pituitary secretions is also
involved in a number of important
bodily functions. Many types of
behaviors (e.g., reproductive behaviors,
sexually dimorphic behaviors in
animals) are dependent on the integrity
of the hypothalamic-pituitary system,
which could represent a potential site of
neurotoxicity. Pituitary secretions arise
from a number of different cell types in
this gland, and neurotoxicants could
affect these cells directly or indirectly.
Morphological changes in cells
mediating neuroendocrine secretions
could be associated with adverse effects
on the pituitary or hypothalamus and
could ultimately affect behavior and the
functioning of the nervous system.
Biochemical changes in the
hypothalamus may also be used as
indicators of potential adverse effects on
neuroendocrine function. Finally, the
development of the nervous system is
intimately associated with the presence
of circulating hormones such as thyroid
hormone (Porterfield, 1994). The nature
of the nervous system deficit, which
could include cognitive dysfunction,
altered neurological development, or
visual deficits, depends on the severity
of the thyroid disturbance and the
specific developmental period when
exposure to the chemical occurred.
3.2. Dose-Response Evaluation
Dose-response evaluation is a critical
part of the qualitative characterization
of a chemical's potential to produce
neurotoxicity and involves the
description of the dose-response
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26945
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 adverse
effects when compared with controls.
The lack of a dose-response relationship
in the data may suggest that the effect
is not related to the putative neurotoxic
effect or that the study was not
appropriately controlled. Much of the
focus is on identifying the critical
effect(s) observed at the LOAEL and the
NOAEL 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 database
characterized as having sufficient
evidence for use in a risk assessment
(see section 3.3). The risk assessor
should be aware of possible problems
associated with estimating a NOAEL in
studies involving a small number of test
subjects and that have a poor dose-
response relationship.
In addition to identifying the NOAEL/
LOAEL or BMD, the dose-response
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 endpoints showing
responses may be at levels of
organization below the whole organism
(e.g., neurochemical or
electrophysiological endpoints). The
adversity of such effects can be disputed
(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 dose-effect continuum,
and the likelihood for progression of the
effect with continued exposure.
3.3. Characterization of the Health-
Related Database
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 dose-
response 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 in order to
estimate the risks of those hazards from
anticipated or estimated exposures.
Several examples using a weight-of-
evidence approach similar to that
described in these Guidelines have been
described elsewhere (Tilson et al., 1995;
Tilsonetal., 1996).
TABLE 8.CHARACTERIZATION OF THE HESBTH-RELATED DATABASE
Sufficient evidence
Sufficient human evidence
Sufficient experimental ani-
mal evidence/limited
human data.
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 evi-
dence.
This category includes agents for which there is sufficient evidence from epidemiologic studies, e.g., case control
and cohort studies, to judge that some neurotoxic effect is associated with exposure. A case series in conjunc-
tion 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, often much 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.)
This category includes agents for which there is sufficient evidence from experimental animal studies and/or lim-
ited 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 adverse neurotoxic effect in a single ap-
propriate, well-executed study in a single experimental animal species. The minimum evidence needed to judge
that a potential hazard does not exist would include data from an appropriate number of endpoints from more
than one study and two species showing no adverse neurotoxic effects at doses that were minimally toxic in
terms of producing an adverse effect. Information on pharmacokinetics, mechanisms, or known properties of the
chemical class may also strengthen the evidence.
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JTABLE 8.CHARACTERIZATION OF THE HEALTH-RELATED DATABASEContinued
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
databases from studies in animals or humans that are limited by study design or conduct (e.g., inadequate con-
duct or report of clinical signs). Many genera! toxicity studies, for example, are considered insufficient in terms o
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 pro-
viding 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 informa-
tion on SAR or data from in vitro tests. Although such information would be insufficient by itself to proceed fur-
ther 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 SAR 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 database 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 endpoints 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 electrophysiblbgical
changes may be regarded as adverse
because of their known or presumed
relation to neuropathological and/or
neurobehavioral consequences. In the
absence of supportive information, a
professional judgment should 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 tri-ortho-cresyl phosphate, are the
most common and striking examples 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
neurological 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
.only a single endpoint needs to be
found to demonstrate a hazard, but
many endpoints 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 endpoint 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 endpoints 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
structure-activity considerations, data
from other toxicity studies, or 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
database is sufficient to indicate a
potential neurotoxic hazard is not the
end of analysis. The circumstances of
expression of the 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."
4. Quantitative Dose-Response Analysis
This section describes several
approaches (including the LOAEL/
NOAEL and BMD) for determining the
reference dose (RfD) or reference
concentration (RfC). The NOAEL or
BMD/uncertainty factor approach
results in an 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:
Describe how the RfD/RfC was
calculated;
Discuss the confidence in the
estimates;
Describe the assumptions or
uncertainty factors used; and
Discuss the route and level of
exposure observed, as compared to
expected human exposures.
4.1. LOAEL/NOAEL and 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 endpoint from a
single study (the critical study) and
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26947
Ignores both the slope.of the dose-
response function and baseline
variability in the endpoint 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, it
has been proposed that mathematical
curve-fitting techniques (Crump, 1984;
Gaylor and Slikker, 1990;Glowa, 1991;
Glowa and MacPhail. 1995; U.S. EPA.
1995a) be compared with the NOAEL
procedure in calculating the RfD or RfC.
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% or 10% increase in
response. These guidelines suggest that
the use of the BMD should be explored
in specific situations. The Agency is
currently developing guidelines for the
use of the BMD in risk assessment.
Many neurotoxic endpoints 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. The risk assessor
should be aware of the importance of
trying to reconcile findings from several
studies that seem to report widely
divergent results. 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; Glowa and MacPhail,
1995). Categorical regression analysis
has been proposed because 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 3.2). Dose-
response analyses should consider the
uncertainty that U-shaped 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.
4.2. 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 an 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) database
for several agents are based on
neurotoxicity endpoints 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 endpoints observed.
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
database, a modifying factor of up to 10
may be applied, depending on the
confidence one has in the database.
Uncertainty factors of less than 10 can
be used, depending upon the
availability of relevant information.
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.
5. 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:
Provides a statement of the purpose,
scope, level of detail, and approach
used in the exposure assessment;
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;
Provides an evaluation of the
overall level of confidence in the
estimate of exposure and dose and the
conclusions drawn; and
Communicates the results of the
exposure assessment to the risk
assessor, who can then use the exposure
characterization, along with the hazard
and dose/response characterizations, 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 exposure, including
multiple avenues of intake from the
same source.
In addition, neurotoxic effects may
result from short-term (acute), high-
concentration exposures as well as from
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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 long-latency 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.
6. Risk Characterization
6.1. Overview
Risk characterization is the
summarization step of the risk
assessment process and consists of an
integrative analysis and a 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 ฉ 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 in a
complete, informative, and useful
format.
This summary should include, but is
not limited to, a discussion of the
following elements:
Quality of and confidence in the
available data;
Uncertainty analysis;
Justification of defaults or
assumptions;
Related research recommendations;
Contentious issues and extent of
scientific consensus;
Effect of reasonable alternative
assumptions on conclusions and
estimates;
Highlights of reasonable plausible
ranges;
Reasonable alternative models; and
Perspectives through analogy.
The risk manager can then use the
derived risk to make public health
decisions.
An effective risk characterization
should 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
should feature values such as
transparency in the decision-making
process; clarity in communicating with
the scientific community 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.
6.2. 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 risk assessor should
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
should 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 dose-response
characteristics of the critical effects,
data such as the shapes and slopes of
the dose-response curves for the various
endpoints, 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 populationฎ 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
endpoints 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 in order 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
be described and the similarities and
differences discussed.
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26949
6.3. Quality of the Database 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.
A health risk assessment is only as
good as its component parts, i.e., hazard
characterization, dose-response
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.
6.4. Descriptors of Neurotoxicity Risk
There are a number of ways to
describe risks. Several relevant ways for
neurotoxicity are as follows:
6.4.1. Estimation of the Number of
Individuals
The RfD orRfC 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.
6.4.2. 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 exposure limit, what
would be the resulting risk for
neurotoxicity above the RfD or RfC?
6.4.3. 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 in
order not to compound a substantial
number of high-end values for variables
if a "reasonable" exposure estimate is to
be achieved.
6.4.4. Risk Characterization fcirHighly
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, or children. The aged
population is considered to be at
particular risk because of the limited
ability of the nervous system to
regenerate or compensate to neurotoxic
insult.
In general, not enough is understood
about the mechanisms of toxiciry to
identify sensitive subgroups for all
agents, although factors such as
nutrition (e.g., vitamin B), personal
habits (e.g., smoking, alcohol
consumption, illicit drug abuse), or
preexisting disease (e.g., diabetes,
neurological diseases, sexually
transmitted diseases, polymorphisms for
certain metabolic enzymes) may
predispose some individuals to be more
sensitive to the neurotoxic effects of
specific agents. Gender-related
differences in response to
neurotoxicants have been noted, but
these appear to be related to gender-
dependent toxicodynamic or
toxicokinetic factors.
In general, it is assumed that an
uncertainty factor of 10 for
intrapopulation variability will be able
to accommodate differences in
sensitivity among various
subpopulations, including children and
the elderly. However, in cases where it
can be demonstrated that a factor of 10
does not afford adequate protection,
another uncertainty factor may be
considered in conducting the risk
assessment.
6.4.5. 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
database, 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 database
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 should be
taken into account in establishing them
have been addressed here.
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If the MOE is equalib or more than
the uncertainty factor multiplied by any
modifying factor used as a basis for an
RiD 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.
6.5. 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
may affect different cases, consistent but
not necessarily identical risk
management decisions should be made
on a case-by-case basis. These
Guidelines are not intended to give
guidance on the nonscientific aspects of
risk management decisions.
6.6. 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, determine the effects of
recurrent exposures over prolonged
periods of time, and address the
synergistic or antagonistic effects of
mixed exposures 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.
Additional research is needed to
determine the most appropriate dose-
response approach to be: used in
neurotoxicity risk assessments.
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Part B: Response to Science Advisory
Board and Public Comments
1. Introduction
A notice of availability for public
comments of these Guidelines was
published in the Federal Register in
October 1995. Twenty-five responses
were received. These Guidelines were
presented to the Environmental Health
Committee of the Science Advisory
Board (SAB) on July 18, 1996. The
report of the SAB was provided to the
Agency in April" 1997. The SAB and
public comments were diverse and
represented varying perspectives. Many
of the comments were favorable and
expressed agreement with positions
taken in the proposed Guidelines. Some
comments addressed items that were
more pertinent to testing guidance than
risk assessment guidance or were
otherwise beyond the scope of these
Guidelines. Some of the comments
concerned generic points that were not
specific to neurotoxicity issues. Others
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addressed topics thatJiave not been
developed sufficientiy and should be
viewed as research issues. There were
conflicting views about the need to
provide additional detailed guidance
about decision making in the evaluation
process as opposed to promoting
extensive use of scientific judgment.
Many public comments provided
specific suggestions for clarification of
details and corrections of factual
material in the Guidelines.
2. Response to Science Advisory Board
Comments
The SAB found the Guidelines
"* * * to be quite successful, and, all
things considered, well suited to its
intended task." However,
recommendations were made to
improve specific areas.
The SAB recommended that EPA
keep hazard identification as an
identifiable qualitative step in the risk
assessment process and that steps
should be taken to decouple the
qualitative step of hazard identification
from the more quantitatively rigorous
steps of exposure evaluation and dose-
response assessment. These Guidelines
now Include a hazard characterization
Step that clearly describes a qualitative
evaluation of hazard within the context
of the dose, route, timing and duration
of exposure. This step is clearly
differentiated from the quantitative
dose-response analysis, which describes
approaches for determining an RfD or
RfC.
The SAB supported the presumption
that what appears to be reversible
neurotoxicity. especially when arising
from gestational or neonatal exposure
and observed before adulthood, should
not be dismissed as of little practical
consequence. They may be indices of
silent toxicity that emerge later in life or
may suggest more robust and enduring
responses in aged individuals. These
Guidelines explain the concept of
functional reserve and advise caution in
instances where reversibility is seen and
in cases where exposure to a chemical
may result in delayed-onset
neurotoxiciry. These Guidelines also
indicate that reversibility may vary with
the region of the nervous system
damaged, the neurotoxic agent involved,
and organismic factors such as age.
The SAB restated previous positions
concerning cholinesterase-inhibiting
chemicals. Agent-induced clinical signs
of cholinergic dysfunction could be
used to evaluate dose-response and
dose-effect relationships and define the
presence and absence of given effects in
risk assessment. The SAB also indicated
that inhibition of RBC and plasma
cholinesterase activity could serve as a
biomarker of exposure to cholinesterase-
inhibiting agents and thereby
corroborate observations concerning the
presence of clinical effects associated
with cholinesterase inhibition. The SAB
also indicated that reduced brain
cholinesterase activity should be
assessed in the context of the biological
consequences of the reduction. These
Guidelines indicate that inhibition of
cholinesterase in the nervous system
reduces the organism's level of
"reserve" cholinesterase and, therefore,
limits the subsequent ability to respond
successfully to additional exposures and
that prolonged inhibition could lead to
adverse functional changes associated
with compensatory neurochemical
mechanisms. In general, an attempt was
made to coordinate these Guidelines
with the views of a recently convened
Scientific Advisory Panel regarding the
risk assessment of cholinesterase-
inhibiting pesticides (Office of Pesticide
Programs, Science Policy on the Use of
Cholinesterase Inhibition for Risk
Assessments of Organophosphate and
Carbamate Pesticides, 1997).
The SAB indicated that the
Guidelines were inclusive of the major
neurotoxicity endpoints of concern. No
additional neurochemical,
neurophysiological, or structural
endpoints were suggested. Comments
indicated that there was no need to
consider endocrine disrupters
differently from other potential
neurotoxic agents.
The SAB found that the descriptions
of the endpoints used in human and
animal neurotoxicological assessments
were thorough and well documented.
Several sections, particularly
concerning some of the neurochemical
and neurobehavioral measures, were
corrected for factual errors or supported
with more detailed descriptions.
The SAB recommended that the use
of the threshold assumption should
occur after an evaluation of likely
biological mechanisms and available
data to provide evidence that linear
responses would be expected. A strict
threshold is not always clear in the
human population because of the wide
variation in background levels for some
functions. Cumulative
neurotoxicological effects might also
alter the response of some individuals
within a special population, which
might allow the Agency to characterize
the risk to the sensitive population.
Although the SAB did not disagree with
the Guidelines' assumption of a
threshold as a default for neurotoxic
effects, it was suggested that the term
"nonlinear dose-response curve for most
neurotoxicants" be substituted for the
term "threshold." The Neurotoxicity
Risk Assessment Guidelines have been
amended to harmonize their treatment
of the issue of threshold with the
presentation and position taken with
other guidelines.
The SAB also recommended that the
topic of susceptible populations be
expanded to include the elderly and
other groups. The elderly could be at
increased risk of toxic effects for a
number of reasons, including a decline
in the reserve capacity with aging,
changes in the ability to detoxify or
excrete xenobiotics with age, and the
potential to interact with medicines or
other compounds that could synergize
interactions with toxic chemicals. The
SAB also indicated diat other
populations should be considered,
including those with chronic and
debilitating conditions, groups of
workers with potential exposure to
chemicals that may be neurotoxic,
individuals with genetic
polymorphisms that could affect
responsiveness to certain
neurotoxicants, and individuals that
may experience differential exposure
because of their proximity to chemicals
in the environment or diet. The
Guidelines have been modified to
emphasize the possible presence of all
of these susceptible populations. When
specific information on differential risk
is not available, the Agency will
continue to apply a default uncertainty
factor to account for potential
differences in susceptibility.
The SAB recommended that the ;
benchmark dose (BMD) was not ready
for immediate incorporation into
adjustment-factor-based safety
assessment or to serve as a substitute or
replacement for the more familiar
NOAEL or LOAEL. The SAB also
recommended that research and
development on the BMD should be
aggressively encouraged and actively
supported. The BMD could be a
replacement for the NOAEL or LOAEL
after the appropriate research has been
conducted.
3. Response to Public Comments
In addition to numerous supportive
statements, several issues were
indicated, although each issue was
raised by only a few commentators. The
public comment supported the SAB
recommendation that there was no clear
consensus concerning replacing the
NOAEL approach with the BMD to
calculate RfDs and RfCs for
neurotoxicity endpoints. There was also
support for ensuring that dose-response
and other experimental design
information be considered in
interpreting the results of hazard
identification studies before proceeding
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to quantitative dose^esponse analysis.
Public comment also supported the
position that .reversibility cannot be
ignored in neurotoxicity risk assessment
and that the risk assessor should exert
caution in interpreting reversible effects,
especially where an apparent transient
effect is cited to support evidence for
relatively benign effects. The public
comment also supported the use of
clinical signs in the risk assessment of
cholinesterase-inhifoiting compounds
and the finding that inhibition of brain
cholinesterase was an adverse effect.
The Guidelines emphasize the
importance of brain cholinesterase
inhibition, particularly in cases of
repeated exposure. The public comment
agreed with the SAB that RBC and
plasma cholinesterase activity are
biomarkers of exposure. It was
recommended that the Guidelines
incorporate additional information
addressing the neuroendocrine system
as a potential target site, and a section
has been added that defines the
vulnerable components of the
neuroendocrine system and the
behavioral, hormonal, and physiological
endpoints that may be indicative of a
direct or indirect effect on the
neuroendocrine system.
Public comment strongly endorsed
the default assumption that there is a
threshold for neurotoxic effects. The
Guidelines, however, reflect the
argument of the SAB that the term
"nonlinear dose-response curve for most
neurotoxicants" be substituted for
"threshold" in order to be consistent
with the presentation and positions
taken by other risk assessment
guidelines.
The public comments made a number
of recommendations to improve the
Guidelines with regard to consistency of
language between text and tables,
improve the clarity of some of the
tables, and improve the description of
some of the endpoints used in animal
studies. A number of factual errors were
corrected, including the description of
the blood-brain barrier and the degree of
inhibition of neurotoxic esterase
associated with organophosphate-
induced delayed-onset neuropathy.
Therefore, a number of changes have
been made in the Guidelines to clarify
and correct specific passages, but every
effort was made to maintain the original
intent concerning the use and
interpretation of results from various
neurotoxicological endpoints. Finally,
the public comment agreed with the
SAB that factors such as nutrition,
personal habits, age, or preexisting
disease may predispose some
individuals to be differentially sensitive
to neurotoxic chemicals. The risk
characterization section has been
expanded to reflect these potentially
sensitive subpopulations.
[FR Doc. 98-12303 Filed 5-13-98; 8:45 am]
BILLING CODE 6560-50-P
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