EPA/630/Z-96/001
Wednesday
October 4, 1995
Part II
Environmental
Protection Agency
Proposed Guidelines for Neurotoxteity
Risk Assessment; Notice
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52032
Federal Register
October 4. 1995 -' Notices
ENVIRONMENTAL PROTECTION
AGENCY
[FRL-&306-2]
Proposed Guidelines for Neurotoxicity
Risk Assessment
AGENCY: Environmental Protection
Agency (EPA).
ACTION: Proposed guidelines for
Neurotoxicity Risk Assessment and
request for comments.
SUMMARY: The U.S. Environmental
Protection Agency (EPA; Agency) is
today issuing proposed guidelines for
assessing the risks for neurotoxicity
from exposure to environmental agents.
As background information for this
guidance, this notice describes the
scientific basis for concern about
exposure to agents that causa
nourotoxicity and outlines the general
process for assessing potential risk to
humans because of environmental
contaminants.
These proposed Guidelines for
Neurotoxicity Risk Assessment
(hereafter "Guidelines") are intended to
guide Agency evaluation of agents that
are suspected to causa neurotoxicity in
line with the policies and procedures
established in the statutes administered
by the EPA. The Guidelines were
developed as part of an interoffice
guidelines development program under
the auspices of the Risk Assessment
Forum, within EPA's Office of Research
and Development Draft Guidelines
wore developed by an Agency work
group composed of scientists from
throughout the Agency, and selected
drafts were peer reviewed internally and
by experts from universities,
environmental groups, industry, and
other governmental agencies. A
subsequent draft has undergone peer
review in a workshop held on June 2-
3,1992, and has received internal
review by the Concordance and
Oversight Subcommittees of the Risk
Assessment Ebrum. Most recently, the
Committee od the Environment and
Natural Resources of,the Office of
Science and Technology Policy
reviewed the guidelines at a meeting
held on August IS, 1995. The proposed
Guidelines are based, in part, on
recommendations derived from these
reviews and on those made at various
scientific meetings and workshops on
naurotoxicology.
The public is invited to comment, and
public comments will be considered in
EPA decisions in formulating the final
Guidelines. Commenters are asked to
focus on several special issues,
particularly, (1) the issue of
compensation and recovery of function
in neurotoxico logical studies and how
to account for compensation in
neurotoxicology risk assessment; (2) the
use of blood and/or brain
acetylcholinesterase activity as an
indication of neurotoxicity for risk •
assessment; (3) endpoints indicative of
neurotoxicity that may not be covered
by these guidelines, i.e., endocrine
disruption or neuroendocrine*mediated
neurotoxicity; and (4) the possibility of
no threshold for some neurotoxic
agents.
The EPA Science Advisory Board
(SAB) also will review these proposed
Guidelines at a meeting to be
announced in a future Federal Register.
Agency staff will prepare summaries of
the public and SAB comments, analyses
of major issues presented by
commenters, and Agency responses to
those comments. Appropriate comments
will be incorporated, and the revised
Guidelines will be submitted to the Risk
Assessment Forum for review. The
Agency will consider comments from
the public, the SAB. and the Risk
Assessment Forum in its
recommendations to the EPA
Administrator.
DATES: The Proposed Guidelines are
being made available for a 120-day
public review and comment period.
Comments must be in writing and must
be postmarked by February 1.1996.
Please submit one unbound original
with pages consecutively numbered.
and three copies. If there are
attachments, include an index
numbered coasecutively with
comments, and three copies.
FOR FURTHER INFORMATION CONTACT: Dr.
Hugh A. Tilson. Tel: 919-541-2671;
Fax: 919-541-4849.
ADDRESSES: Comments on the proposed
Guidelines may be mailed or delivered
to: Dr. Hugh A. Tilson. Neurotoxicology
Division (MD-74B), National Health and
Environmental Effects Research
Laboratory, U.S. Environmental
Protection Agency, Research Triangle
Park. NC 27711. Please note that all
comments received in response to this
notice will be placed in a public record.
Commenters should not send any item
of personal information, such as
medical information or home address, if
they do not wish it to be part of the
public record.
SUPPLEMENTARY INFORMATION: In its 1983
book. Risk Assessment in the Federal
Government: Managing the Process, the
National Academy of Sciences
recommended that Federal regulatory
agencies establish "inference
guidelines" (1) to promote consistency
and technical quality in risk assessment,
and' (2) to ensure that the risk
assessment process is maintained as a
scientific effort separate from risk
management. A task force within EPA
accepted that recommendation and
requested that Agency scientists begin
to develop such guidelines.
In 1984, EPA scientists began work on
risk assessment guidelines for
carcinogenicity,.mutagenicity, suspect
developmental toxicants, chemical
mixtures, and exposure assessment.
Following extensive scientific and
public review, these first five guidelines
were issued on September 24,1986 (51
FR 33992-34054). Since 1986,
additional risk assessment guidelines
have been proposed for mole and female
reproductive risk (53 FR 24834-847; 53
FR 24850-869), and two of the 1986
guidelines, suspect developmental
toxicants (56 FR 63798-826) and
exposure assessment (57 FR 22888-
938}, have been revised, reproposed,
and finalized.
The Guidelines proposed today
continue the guidelines development
process initiated in 1984. These
Guidelines set forth principles and
procedures to guide EPA scientists in
the conduct of Agency risk assessments
and to inform Agency decision makers
and the public about these procedures.
In particular, the Guidelines emphasize
that risk assessments will be conducted
on a case-by-case basis, giving full
consideration to all relevant scientific
information. This case-by-case approach
means that Agency experts study
scientific information on each chemical
under review and use the most
scientifically appropriate interpretation
t« 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 pan
to bridge gaps in risk assessment
methodology and data. By identifying
these gaps and the importance of the
missing information to the risk
assessment process. EPA wishes to
encourage research and analysis that
will lead to-new risk assessment
methods and data.
Dated: September 25,199S.
Cirol M. Browner.
Administrator.
Propped Guideline* far Neorutoxicity Risk
I. Introduction
A. Organization of These Guidelines
B. The Role of Environmental Agents in
Neurotoxicity
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Federal Register / Vol. 60. No. 192 / Wednesday. October 4. 1995
C. N'eurotoxicsty Riss Assessment
D. Assumptions
II. Definitions and Critical Concent?
in. Hazard Characterization P
A. Neurotoxicological Studies: End Points
and Their Interpretation
1. Human Studies
a. Clinical Evaluations
b.- Case Reports
c. Epidemiologic Studies
(1) Cross-sectional studies
(24 Case-control (retrospective) studies
(3) Cohort (prospective, follow-up) studies
d. Human Laboratory Exposure Studies
2. Animal Studies
a. Structural End Points of Neurotoxicity
b. Neurophysiological End Point* of
Neurotoxicity
(1 j Nerve conduction studies
(2) Sensory, motor, and other evoked
potentials
(3) Seizures/convulsions
(4) Electroencephalography (EEC)
c. Neurochemical End Points of
Neurotoxicity
d. Behavioral End Points of Neurotoxicity
(1) Functional observational battery
(2) Motor activity
, (3) Schedule-controlled operant behavior
(4) Convulsions
(5) Specialized tests for neurotoxicity
(a) Motor function
(b) S«nsory function
(c) Cognitive function .
e. Developmental Neurotoxicity
3. Other Considerations
a. Pharmacokinetics
b. Comparisons of Molecular Structure
c. Statistical Considerations
d. In Vitro Data in Neurotoxicology
. B. Dose-Response Evaluation
C Characterization of the Health-Related
DataBase
IV. Dose-Response Analysis
A. LOABL/NOAEL and Benchmark DOM
(BMD) Determination
B. Determination of the Reference Dose or
Reference Concentration
V. Exposure Assessment
VI Risk Characterization
A. Overview
B. Integration of Hazard Characterization.
Dose-Response Analysis, and Exposure
Assessment
C Quality of the Data Base and Degree of
Confidence in the Assessment
D. Descriptors of Neurotoxicity Risk
1. Estimation of the Number of Individuals
2. Presentation of Specific Scenarios
3. Risk Characterization for Highly
Exposed Individual*
4. Risk Characterization for Highly
Sensitive or Susceptible Individuals •
5. Other Risk Descriptor*
E. Communicating Results
F. Summary and Research Needs
vn. References
List of Table*
Table 1. Examples of possible indicators of a
neurotoxic effect
Table 2. Neurotoxicant* and diseases with
specific neuronal targets
Table 3. Examples of neurophysiological
measures of neurotoxicity
Table 4. Examples of neurotoxicants with
known neurochemical mechanisms
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Table 5. Summary of measures in a
representative functional observational
battery, and the type of data produced bv
each , - *
Table 6. Examples of specialized behavioral
tests to measure neurotoxicity
Table 7. Examples of developmental
neurotoxicants
Table 8. Characterization of the Health- •
Related Database
I. Introduction
These proposed Guidelines describe
the principles, concepts, and procedures
that the U.S. Environmental Protection
Agency (EPA; Agency) would follow in
evaluating data on potential
neurotoxicity associated with exposure
to environmental toxicants. The
Agency's authority to regulate
substances that have the potential to
interfere with human health is derived
from a number of statutes that are
implemented through multiple offices
within the EPA. The procedures
outlined here are intended to help
develop a sound scientific basis for
neurotoxicity risk assessment, promote
consistency in the Agency's assessment
of toxic effects on the nervous system.
and inform others of the approaches
used by the Agency in those
assessments.
A. Organization of These Guidelines
This Introduction (section I)
summarizes the purpose of these
proposed Guidelines within the overall
framework of risk assessment at the
EPA. It also outlines the organization of
the guidance and describes several
default assumptions to be used in the
risk assessment process as discussed in
the recent National Research Council
report "Science and Judgment in Risk
Assessment {NRC, 1994)."
Section H sets forth definitions of
particular terms widely used in the field
of neurotoxicology. These include
"neurotoxicity" and "behavioral
alterations." Also included in this
section are discussions concerning
reversible and irreversible effects and
direct versus indirect effects.
Risk assessment is the process by
which scientific judgments are made
concerning the potential for toxicity to
occur in humans. The National Research
Council (NRC. 1983) has defined risk
assessment as including some or all of
the following components (paradigm):
hazard identification, dose-response
assessment, exposure assessment, and
nsk characterization, to its 1994 report
"Science and Judgment in Risk
Assessment" the NRC extended its view
of the paradigm to include
characterization of each component
(NRC 1994). In addition, it noted the
importance of an approach that is less
fragmented and more holistic, less
linear and more interactive, and one
that deals with recurring conceptual
issues that cut across all stages of risk
assessment. These Guidelines prooose a
more interactive approach by organizing
the process around components that
focus on evaluation of the toxicity data
(hazard characterization), the
quantitative dose-response analysis the
exposure assessment, and the risk
characterization. This is done because,
in practice, hazard identification for
tteurotoxicity and other noncancer
health effects is usually done in
conjunction with an evaluation of dose-
response relationships in the studies
used to identify the hazard. Determining
a hazard often depends on whether a
dose-response relationship is present
(Kunmel et al.. 1990). Thus, the hazard
characterization provides an evaluation
of a hazard within the context of the
dose, route, duration, and timing of
exposure. This approach combines the
information important in comparing the
toxicity of a chemical to potential
human exposure scenarios (Section V)
Secondly, it avoids the potential for
labeling chemicals as "neurotoxicants"
on a purely qualitative basis. This
organization of the risk assessment
process is similar to that discussed in
the Guidelines for Developmental
Toxiriry 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 if an
agent causes neurotoxicity in that
species and under what conditions.
From the hazard characterization and
criteria provided in these Guidelines,
the health-related data base can be
characterized as sufficient or
insufficient for use in risk assessment
(section m.C). Combining hazard
identification and some aspects of dose-
response evaluation into hazard
characterization does not preclude the
evaluation and use of data when
quantitative information for setting
reference doses (RfDs) and reference
concentrations (RfCs) are not available.
The next step, the dose-response
analysis (section IV) is the quantitative
analysis, and includes determining the
no-observed-adverse-effect-level
(NQAEL) 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, i.e., the
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Benchmark dose approach (Crump,
1984: U.S. EPA. 1995a). for more
quantitative dose-response evaluation
when sufficient data are available. The
benchmark dose approach takes into-
account the variability in the data and
the slope of the dose-response curve,
and provides a more consistent basis for
calculation of the RfD or RfC. If data are
considered sufficient for risk
assessment, and if neurotoxicity is the
effect occurring at the lowest dose level
(i.e.. the critical effect), an oral or
dermal RfD or an inhalation RfC, based
on neurotoxic effects, is then derived.
This RfD or RfC is derived using the
NOAEL or benchmark dose divided by
uncertainty factors to account for
interspecies differences in response,
intraspecies variability and other factor >.
of study design or the data base. A.
statement of the potential for human
risk and the consequences of exposure
can come only from integrating the
hazard characterization and dose-
response analysis with the human
exposure estimates in the final risk
characterization. *
The section on exposure assessment
(section V) identifies human
populations exposed or potentially
exposed to an agent, describes their
composition and size, and presents the
types, magnitudes, frequencies, and
durations of exposure to the agent; The
exposure assessment provides an
estimate of human exposure levels for
particular populations from all potential
sources.
In risk characterization (section VI).
the hazard characterization, dose-
response analysis, and the exposure
assessment for given populations are
combined to estimate some measure of
the risk for neurotoxicity. As part of risk
characterization, a summary of the
strengths and weaknesses of each
component of the risk assessment is
given along with major assumptions,
scientific judgments, and. to the extent
possible, qualitative and quantitative
estimates of the uncertainties. This
characterization of the health-related
data bast, 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.
I995c), which is currently being
implemented throughout EPA. This
policy statement is designed to ensure
that critical information from each stage
of a risk assessment is used in forming
conclusions about risk and that this
information is communicated from risk
assessors- to risk managers (policy
makers), from middle to upper
management, and from the Agency to
the public. Additionally, the policy
provides a basis for greater clarity,
transparency, reasonableness, and
consistency in risk assessments across
Agency programs. Final neurotoxicity
risk assessment guidelines may reflect
additional changes in risk
characterization practices resulting from
implementation activities.
Risk assessment is just one
component of the regulatory process
and defines the potential adverse health
consequences of exposure to a toxic
agent. The other component risk
management, combines risk assessment
with statutory directives regarding
socioeconomic, technical, political, and
other considerations, to decide whether
to control future exposure to the
suspected toxic agent and, if so. the
nature and level of control. One major
objective of these risk assessment
Guidelines is to help the risk assessor
determine whether the experimental
animal or human data indicate the
potential for a neurotoxic effect Such.
information can then be used
subsequently to categorize evidence to
identify and characterize neurotoxic
hazards as described in section OL3.C.
Characterization of the Health-Related
Data Base, and Table 8 of these
Guidelines. Risk management is not ,
dealt with directly in these Guidelines
because the basis for decision making
goes beyond scientific considerations
alone, but the use of scientific
information in this process is discussed.
For example, the acceptability of the
MOE is a risk management decision, but
the scientific bases for establishing this
value are discussed here.
B. The Role of Environmental Agents in
Neurotoxicity .
Chemicals are an integral part of life.
with the capacity to improve as well as
endanger health. The general population
is exposed to chemicals with neurotoxic
properties in air. water, foods,
cosmetics, household products, and
drugs used therapeutically or illicitly.
Naturally occurring neurotoxins. such
as animal and plant toxins, present
additional hazards. During daily life, a
person experiences a multitude of
exposures, both voluntary and
unintentional, to neuroactive
substances, singly and in combination.
Levels of exposure vary and may or may
not pose a hazard depending on dose.
route, and duration of exposure.
A link between human exposure to
some chemical substances and'
neurotoxicity has been firmly
established (Anger, 1986; OTA, 1990).
Because many natural and synthetic
chemicals are present in today's
environment, there is growing scientific
and regulatory interest in the potential
for risks to humans from exposure to #
neurotoxic agents. If sufficient exposure
occurs, the effects resulting from such
exposures can have a significant adverse
impact on human health. It is not
known how many chemicals may be
neurotoxic in humans (Reiter, 1987).
The EPA's inventory of toxic chemicals
is greater than 63,000 and increasing
yearly. An overwhelming majority of the
materials in commercial use have not
been tested for their neurotoxic
potential (NRC, 1984). Estimates of the
number of chemicals with neurotoxic
properties have been made for subsets of
substances. For instance, a large
percentage of the more than 500
registered active pesticide ingredients
are neurotoxic to varying degrees. Of
588 chemicals listed by the American
Conference of Governmental Industrial
Hygienists. 167 affected the nervous
system or behavior at some exposure
level (Anger, 1984). Anger (1990)
estimated that of the approximately 200
chemicals to which one million or more
American workers are exposed, more
than one-third may have adverse effects
on the nervous system, if sufficient
exposure occurs. Anger (1984) also
recognized neurotoxic effects as one of
the 10 leading workplace disorders. A
number of therapeutic substances,
including some anticancer and antiviral
agents and abused drugs, can cause
adverse or neurotoxicological side
effects at therapeutic levels (OTA.
1990). Thus, estimating the risks of
exposure to chemicals with neurotoxic
pptential is of concern with regard to
the overall impact of these exposures on
human health.
C. Neurotoxicity Risk Assessment
In addition to its primary role in
cognitive functions, the nervous system
controls most, if not all. other bodily
processes. It is sensitive to perturbation
from various sources and has limited
ability to regenerate. There is evidence
that even small anatomical,
biochemical, or physiological insults to
the nervous system may result in
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52035
adverse effects on human health.
Therefore, there is a need for consistent
guidance on how to evaluate data on
neurotoxic substances and assess to
what degree, if any, they have the
potential to cause transient or
persistent, direct or indirect effects on
human health.
To help address these needs, these
Guidelines develop principles and
concepts in several areas. First, these
Guidelines outline the scientific basis
for evaluating effects due to exposure to
neurotoxicants and discuss principles
and methods for evaluating data from
human and animal studies on behavior,
neurochemistry. neurophysiology, and
neuropathology. This guidance
document also discusses adverse effects
on neurological development and
function in infants" and children
following prenatal and perinatal
exposure to chemical agents. Other
sections of these Guidelines outline the
method for calculating reference doses
or reference concentrations when
neurotoxicity is the critical effect.
discuss the availability of alternative
mathematical approaches to dose-
response analyses, characterize the
health-related data base for
neurotoxicity risk assessment, and
discuss integration of exposure
information with the results of the dose-
response assessment to characterize
risks of exposures of concern. These
Guidelines do not advocate developing
reference doses specific for
neurotoxicity. but rather the use of
neurotoxicity as one possible end point
to develop reference doses.
EPA offices have published guidelines
for neurotoxicity testing in animals
(U.S. EPA. 1986.1987,1988af 1991a).
The testing guidelines address the
development of new data for us* in risk
assessment. These proposed
neurotoxicity risk assessment
Guidelines provide the Agency's first
comprehensive guidance on the use and
interpretation of neurotoxicity data.
These proposed Guidelines are part of
the Agency's risk assessment guidelines
development process, which was
initiated in 1984. As part of its
neurotoxicity guidelines development
program, the EPA has sponsored or
participated in several conferences on
relevant issues (Tilson. 1990); these and
other sources (see references) provide
the scientific basis for these proposed
risk assessment Guidelines. This
guidance is intended for use by Agency
risk assessors and is separate and
distinct from the recently published
document on principles of neurotoxicity
risk assessment (U.S. EPA. 1993). The
document on principles was prepared
under the auspices of the Subcommittee
on Risk Assessment of the Federal
Coordinating CSonal for Science,
Engineering, and Technology and was
not intended to provide specific
directives for how neurotoxicity risk
assessment should be performed.
It is expected that, like other EPA risk
assessment guidelines (U.S. EPA,
199lb), this document will encourage
research and analysis leading to new
risk assessment methods and data,
which in turn would be used to revise
and improve the Guidelines and better
guide Agency risk assessors.
D. Assumptions
There are a number of unknowns in
the extrapolation of data from animal
studies to humans. Therefore, a number
of default assumptions are made that are
generally applied in the absence of data
on the relevance of effects to potential
human risk. Default assumptions should
not be applied indiscriminantly. First.
all available mechanistic and
pharmacokinetic data should be
considered. If these data indicate that an
alternative assumption is appropriate or
obviate the need for applying an
assumption, such information should be
used in the risk assessment of that
agent The following default
assumptions form the basis of the
approaches taken in these Guidelines.
It is assumed that an agent that
produces detectable adverse neurotoxic
effects in experimental animal studies
will pose a potential hazard to humans.
This assumption is based on the
comparisons of data for known human
neurotoxicants (Anger. 1990; Kimmel et
al., 1990: Spencer and Schaumburg,
1980). which indicate that experimental
animal data are frequently predictive of
a neurotoxic effect in humans.
It is assumed that behavioral,
neurophysiological, neurochemical. and
neuroanatomical manifestations are of
concern. In the past, the tendency has
been to consider only neuropathological
changes as end points of concern. Based
on the data on agents that are known .
human neurotoxicants (Anger, 1990;
Kimmel et aL, 1990; Spencer and
Schaumberg. 1980), there is usually at
least one experimental species that
mimics the types of effects seen in
humans, but in other species tested, the
type of neurotoxic effect may be
different or absent Thus, a biologically
significant increase in any of the • •
manifestations is considered indicative
of an agent's potential for disrupting the
structure or function of the human
nervous system.
It is assumed that the types of
neurotoxic effects seen in animal
studies may not always be the same as
those produced in humans. Therefore, it
may be difficult to determine which will
be the most appropriate species in terms
of predicting the specific types of effects
seen in humans. The fact that every
species may not react in the same way
is probably due to species-specific
differences in maturation of the nervous
system, differences in timing of
exposure, metabolism, or mechanisms
of action.
It is assumed that the most
appropriate species will be used when
data are available to estimate human
risk. In the absence of such data, the
most sensitive species is used, based on
the fact that for the majority of known
human neurotoxicants, humans are as
sensitive or more so than the most r
sensitive animal species tested. /
In general, a threshold is assumed for
the dose-response curva for most
neurotoxicants. This is based on the
known capacity of the. nervous-system
to compensate for or to repair a certain
amount of damage at the cellular, tissue.
or organ level. In addition, because of
the multiplicity of cells in the nervous
system, multiple insults at the
molecular or cellular level may be
required to produce an effect on the
whole organism.
These assumptions are "plausibly
conservative" (NRC, 1994) in that they
are protective of public health and are
also well-founded in scientific
knowledge about the effects of concern.
n. Definitions and Critical Concepts
This section defines the key terms aud
concepts that the EPA will use in the
identification and evaluation of
neurotoxicity. The various health effects
that fall within the broad classification
of neurotoxicity are described and
examples are provided.
Adverse effects include alterations
from baseline that diminish an
organism's ability to survive, reproduce.
or adapt to the environment.
Neurotoxicity is an adverse change in
the structure or function of the central
and/or peripheral nervous system
following exposure to a chemical.
physical, or biological agent (Tilson.
1990). Neurotoxic effects include
changes in somatic/autonomic. sensory,
motor, and/or cognitive function.
Structural effects are defined as
neuroanatomical changes occurring at
any level of nervous system
organization; functional changes are
defined as neurochemical.
neurophysiological. or behavioral
alterations. Changes in function can also
.result from toxicity tq other specific
organ systems, and these indirect
changes may be considered adverse but
not necessarily neurotoxic.
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The nsk assessor also should know
that there are different levels of concern
based on the magnitude of effect and
reversibility of some neurotoxic effects.
Neuiotoxic effects may be irreversible,
i.e., cannot return to the state prior to
exposure, resulting in a permanent
change in. the organism, or reversible,
i.e., can return to the pre-exposure
condition, allowing the organism to
return to its state prior to exposure.
Clear or demonstrable irreversible
change in either the structure or
function of the nervous system causes
greater concern than do reversible
changes. If neurotoxic effects are
observed at some time during the life
span of the organism but are slowly
reversible, the concern is also high.
There is lesser concern for effects that
are rapidly reversible or transient, i.e.,
measured in minutes, hours, or days,
and appear to be associated with the
pharmacokinetics of the causal agent
and its presence in the body. Reversible
changes that occur in the occupational
setting or environment, however, may
be of high concern if, for example,
exposure to a short-acting solvent
interferes with operation of heavy
equipment in an industrial plant The
context of the exposure should be
considered in evaluating reversible
effects. The risk assessor should note
that once damaged, neurons,
particularly in the central nervous
system, have a limited capacity for
regeneration. Reversibility of effects
resulting from cell death or from the
destruction of cell processes may
represent an activation of repair
capacity, decreasing future potential
adaptability. Therefore, even reversible
r.eurotoxic changes should be of
concern. Evidence of progressive effects,
i.e.. those that continue to worsen even
after the causal agent has been removed;
or delayed effects, i.e., those that occur
at a time distant from the last contact
with the causal agent; or residual
effects, i.e., those that persist beyond a
recovery period: or latent offsets, i.e.,
those that become evident only after an
environmental challenge or aging, have
a high level of concern. Environmental
challenges can include stress, increased
physical or cognitive workload.
pharmacological manipulations, and
nutritional deficiency or excess.
Neurotoxic effects can be observed at
various levels of organization of the
nervous system, including
neurochemicaJ, anatomical,
physiological, or behavioral. At the
neurochemical level, for example, an'
agent that causes neurotoxicity might
inhibit rnacromolecule or transmitter
synthesis, alter the flow of ions across
cellular membranes, or prevent release
of neurotransmitter from the nerve
terminals. Anatomical changes may
include alterations of the cell body, the
axon. or the myelin sheath. At the
physiological level, a chemical might
change the thresholds for neural
activation or reduce the speed of
neurotransmission. Behavioral
alterations can include significant
changes in sensations of sight, hearing,
or touch; alterations in simple or
complex reflexes and motor functions;
alterations in cognitive functions such
as learning, memory or attention; and
changes in mood, such as fear or rage,
disorientation as to person, time, or
place, or distortions of thinking and
feeling, such as delusions and
hallucinations. At present, relatively
few neurotoxic syndromes have been
thoroughly characterized in terms of the
initial neurochemical change, structural
alterations, physiological consequence,
and behavioral effects. Knowledge of
exact mechanisms of action is not.
however, necessary to conclude that a
chemically induced change is a
neurotoxic effect,
Neurotoxic effects can be produced by
chemicals that do not require
metabolism prior to interacting with
their target sites in the nervous system,
i.e.. primary neurotoxic agents, or those
that require metabolism prior to
interacting with their target sites in the
nervous system, i.e., secondary
neurotoxic agents. Chemically induced
neurotoxic effects can be direct, i.e., due
to an agent or its metabolites acting
directly on target sites in the nervous
system, or indirect, i.e., due to agents or
metabolites that produce their effects
primarily by interacting with target sites
outside the nervous system, which
subsequently affect target sites in the
nervous system. Excitatory amino acids
such as domoic add damage specific
neurons directly by activating excitatory
amino acid receptors in the nervous
system, while carbon monoxide
decreases oxygen availability, which
indirectly kills neurons. Other examples
of indirect effects of chemicals that
could lead to altered structure and/or
function of the nervous system include
cadmium-induced spasms in blood
vessels supplying the nervous system.
dichloroacetate-induced perturbation of
metabolic pathways, and chemically
induced alterations in skeletomuscular
function or structure and effects on the
endocrine system. Professional
judgment may be required in making
determinations about direct versus
indirect effects.
The interpretation of data as
indicative of a 'potential neurotoxic
effect involves the evaluation of the
validity of the data base. This approach
and these terms have been adapted from
the literature on human psychological
testing (Sette, 1987; Sette and MacPhail,
1992) where they have long been used
to evaluate the level of confidence in
different measures of intelligence or
other abilities, aptitudes, or feelings.
There are four principal questions that
should be addressed: whether the effects
result from exposure (content validity);
whether the effects are adverse or
toxicologicaily significant (construct
validity); whether there are correlative
measures among behavioral,
physiological, neurochemical, and
morphological end points (concurrent j
validity); and whether the effects are a
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.
m. Hazard Characterization
A. Neurotoxicological Studies: End
Points and Their Interpretation
Identification and characterization of
neurotoxic hazard can be based on
either human or animal data (Anger,
1984; Reiter, 1987; U.S. EPA. 1993).
Such data can result from accidental,
inappropriate, or controlled
experimental exposures. This section
describes many of the general and some
of the specific characteristics of human
studies and reports of neurotoxicity. It
then describes some features of animal
studies of neuroanatomical.
neurochemical, neurophysiological. and
behavioral effects relevant to risk
assessment The process of
characterizing the sufficiency or
insufficiency of neurotoxic effects for
risk assessment is described in section
m.C Additional sources of information
relevant to hazard characterization, such
as comparisons of molecular structure
among compounds and in vitro
screening methods, are also discussed.
The hazard characterization should:
a. Identify strengths and limitations of
the database:
•^-Epidemiological studies (case reports,
cross-sectional, case-control, cohort.
or human laboratory exposure
studies);
—Animal studies including (structural
or neuropathological, deurochemical,
neurophysiological, behavioral or
• neurological, or developmental end
points).
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32037
b. Evaluate die vilicity of ihe
database:
—Content validity (effects result from
exposure):
—Construct validity (effects are adverse
or toxico logically- significant);
—Concurrent validity (correlative
measures among behavioral,
physiological, neurochemical, or
morphological end points);
—Predictive validity (effects are-
predictive of what will happen under
various conditions).
c. Identify and describe key
lexicological studies.
d. Describe the type of effects:
—Structural fneuroanatomical
• alternations);
—Functional (neurochemicar,
neurophysiological. behavioral
alterations).
e. Describe the nature of the effects
(irreversible, reversible, transient,
progressive, delayed, residual, or latent
effects).
f. Describe how. much is known about
how (through what biological
mechanism) the chemical produces
adverse effects.
g. Discuss other health end points of
concern.
h. Comment on any non-positive data
in humans or animals.
i. Discuss the dose-response data
(epidemiological qr animal) available for
further dose-response analysis.
j. Discuss the route, level, timing, and.'
duration of exposure in studies '
demonstrating neurotoxicity as '
compared to expected human "
exposures.
k. Summarize the hazard
characterization:
—Confidence in conclusions;.
—Alternative conclusions also
supported by the data;
—Significant data gaps; and
—Highlight of major assumptions.
1. Human Studies
It is well established that information
from the evaluation of human exposure
can identify neurotoxic hazards (Anger
and Johnson, 1985; Anger. 199O).
Prominent among historical episodes of
neurotoxicity in human.populations are
the outbreaks of methylmercury
poisoning in Japan and Iraq and the
neurotoxicity seen in miners of metals,
including mercury, manganese, and lead
(Carson et al.. 1987; Silbergeld and
Percival. 1987; OTA. 1990). In the last
decade, lead poisoning in children has '
been a prominent issue of concern
(Silbergeld and Percival. 1987).
Neurotoxicity in humans has been
studied and reviewed for many
pesticides (Hayes, 1982; NRDC, 1989:
Ecobichon and Joy, 1982; Ecobichon et
al., 1990). Organochlorines,
organophosphates, 'carbamates,^
pyrethroids. ceriSba fungicidesV'and
some fumigants ;are all- known
neurof oxicants. 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 rUnirat evaluations, case
reports, and epidemiologic studies. A
more extensive description of issues
concerning human neurotoxicology and
risk assessment has been published
elsewhere (U.S. EPA. 1993).
a. Clinical Evaluations. Clinical
methods are used extensively in
neurology and neuropsychology to
evaluate patients suspected of having
neurotoxicity. An extensive array of
examiner-administered and paper-and-
pencil tasks are used to assess sensory,
motor,, cognitive, and affective functions
and personality states/traits.
Neurobehavioral data are synthesized
with information from neurophysiologic
studies and medical history to derive a
working diagnosis. Brain imaging''
techniques based on magnetic resonance
imaging or emission tomography may
also be useful in helping, diagnose
neurodegenerative disorders following
chemical exposures in humans (Omerod
et al., 1994; Callender et al., 1994).
Clinical diagnostic approaches have
provided a rich conceptual framework
for understanding the functions (and
malfunctions) of the central and
peripheral nervous systems and have
formed the basis for die development of
methods for measuring the behavioral
expression of nervous system disorders.
Human neurobehaviorai toxicology has
borrowed heavily from neurology and
neuropsychology for concepts of
nervous system impairment and
functional assessment methods.
Neurobehavioral toxicology has adopted
the neurologic/neuropsycnologic model,
using adverse changes in behavioral
function to assist in identifying
chemically or drug-induced changes in
nervous system processes.
Neurologic and neuropsychologic
methods have long been employed to
identify the adverse health effects of
environmental workplace exposures
(Sterman and Schaumburg. 1980).
Peripheral neuropathies-(with sensory
and motor disturbances),
encephalopathies, organic brain
syndromes, extrapyramidal syndromes.
demyelination, autonomic changes, and
dementia are well-characterized
consequences of acute and chronic
exposure to chemical agents. The range
of exposure conditions that produce
clinical signs of neurotoxicity also has
been defined by these clinical methods.
It is very important to make external/
internal dose measurements in humans
to determine the actual dose(s) that can
cause unwanted effects.
Aspects of the neurologic examination
approach limit its usefulness for
neurotoxicologicrisk assessment.
Information obtained from the*
neurologic exam is mostly qualitative
and descriptive rather than quantitative.
Estimates of the severity of functional
impairment can be reliably placed intrf
only three or four categories (for J
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 o ften
judged by having the patient push
against the resistance of the examiner's1
hands. The end points are therefore the
absolute and relative amount of muscle
load sensed by the examiner in his or
her arms.
Compared with other methods, the
neurologic exam may be less sensitive
in detecting early neurotoxicity in
peripheral sensory and motor nerves."'
While clinicians' judgments are equal in
sensitivity to quantitative methods in
assessing the amplitude of tremor,
tremor frequency is poorly quantified by
clinicians. Thus, important aspects of
the clinical neurologic exam may be
insufficiently quantified and lack
sufficient sensitivity for detecting early
neurobehaviorai toxicity produced by
environmental 'or workplace exposure
conditions. However, a neurologic
evaluation of persons with documented
neurobehaviorai impairment would be
helpful for identifying nonchemical
causes of neurotoxicity, such as diabetes
and cardiovascular insufficiency.
Administration of a
neuropsychological battery also requires
a trained technician, and interpretation
requires a trained and experienced
neuropsychologist Depending on the
capabilities of the patient, 2 to 4 hours
may be needed to administer a full
battery; 1 hour may be needed for the
shorter screening versions. These
practical considerations may limit the
usefulness of neuropsychological
assessment in large field studies of
suspected neurotoxicity.
In addition to logistical problems in
administration and interpretation.
neuropsychological batteries and
neurologic exams share'two
disadvantages with respect to
neurotoxicity risk assessment. First,
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52C38 Federal Register / Vol. 60. N'o. 192 / Wednesday. October 4. 1995 , Notices
neurologic exams and
neuropsychological test batteries are
designed to confirm and classify
functional problems in individuals
selected on the basis of signs and
symptoms identified by the patient.
family, or other health professionals.
Their usefulness in detecting low base-
rate impairment in workers or the
general population is generally thought
to be limited, decreasing the usefulness
of clinical assessment approaches for
epidemiologic risk assessment
Second, neurologic exams and
neuropsychologic test batteries were
developed to assess the functional
correlates of the most common forms of
nervous system dysfunction: brain
trauma, focal lesions, and degenerative
conditions. The rfiniral tests were
validated against these neurologic
disease state*. With a few notable
exceptions, rh«mir»l« are not believed
to produce impairment similar to that
from trauma or lesions; nsurotoxic
effects are more similar to the effects of
degenerative disease. There Has been.
insufficient research to demonstrate
which tests designed to assess
functional expression of neurologic
disease are useful in characterizing the
modes of central nervous system
impairment produced by chemical
agents and drugs.
b. Case reports. The first type of
human data available is often the case
report or case eeries, which can identify
cases of a disease and are reported by
clinicians or discerned through active or
passive surveillance, usually in the
workplace. However, case reports where
exposure involved, a single neurotoxic
agent, although informative, are rare in
the literature: for example, fanners an
likely to be-exnoeed to a wide variety of
potentially neurotoxic pesticide*. .
Careful case histories assist in
identifying common risk factors.
especially when the association between
tha exposure and disease is strong, the
mode of action of tha agent is
biologically plausible, and clusters.
occur in a limited period of time.
Case reports are inexpensive
compared with epidemiologic studies
and can be obtained more quickly than
more complex studies. However, they
provide little information about disease
frequency or population at risk, but
their importance has been dearly
demonstrated, particularly in accidental
poisoning or acute exposure to high
levels of toxicant They remain an
important source of index cases of new
diseases and for surveillance.
a Epidemiologic Studies.
Epidemiology has been defined as "the
study of the distributions and
determinants of disease and injuries in
human populations" [Mausner and
Kramer. 1985). Knowing the frequency
of illness in groups and the factors that
influence the distribution is the tool of
epidemiology that allows the evaluation
of causal inference with the goal of
prevention and cure of disease
(Friedlander and Hearn, 1980),
Epidemiologic studies are a means of
evaluating the effects of neurotoxic
substances on human populations, but
such studies are limited because they
must be performed shortly alter
exposure if the effect is acute. Most
often these effects an suspected to be a
result of occupational exposures due to
tha increased opportunity for exposure
to industrial and other chemicals.
Frequently, determining the precise
dose or exposure concentration can be
difficult in epidemiological studies.
(1) Cross-sectional studies. In cross-
sectional studies or surveys, both the
disease and suspected risk factors an
ascertained at the same time, and the
findings are useful in generating
hypotheses. A group of people an
interviewed, examined, and tested at a
single point in H<»K» to ascertain a
relationship between a Himae* and a
neurotoxic exposure. This study design
does not allow the investigator to
determine whether the disease or the
exposure came first, rendering it less
useful in estimating risk. These studies
are. intermediate in cost and time
required to complete compared with
case reports and man complex
analytical studies but should b*
augmented with additional data.
(2) Case-control (retrospective)
studies. Last (1986} defines a case-
control study as one that "starts with
the identification of persons with the
disease (or other outcome variable) of
interest, and a suitable control
population (comparison, reference
group) of persons without the disease."
He states that the relationship of an
"attribute!* to the disease is measured by
comparing the diseased with the
nondiseased with regard to how
frequently the attribute is present in
each of the groups. The cases an
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-Hfe is
too short to be of any practical value),
the case-control paradigm is a widely
accepted strategy for the assessment of
toxic causation." Tha case-control study
design, however, can be very t
susceptible to bias. The potential f
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 ia a questionnaire-based
study caa distort the results of the
study. Analysis of tha case-comparison
study design assumes that the selected
cases are representative persons with
the disease—either all cases with the
disease or a representative sample of
them have been ascertained. It further
assnme« that the control or comparison
group is representative of the
nondiseased population (or that the
prevalence of the characteristic under
study is the same in the control group
as ia the general population). Failure to
satisfy these assumptions may result in
selection bias, but violation of
assumptions does not necessarily
invalidate the study results.
An additional source of bias in case-
control studies is the presence of
confounding variables, i.e., factors
known to be associated with the
exposure and causally related to the
disease under study. These must be .
controlled either in the design of the
study by matching cases to controls on
the basis of the confounding factor or in
the analysis of the data by using
statistical techniques such as
stratification or regression. Matching
requires time to identify an adequate
number of potential controls to
distinguish those with the proper
characteristics, while statistical control
of confounding factors requires a larger
study.
The definition of exposure is critical
in epidemiologic studies. In
occupational settings, exposure
assessment often is based on th.e 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
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Federal Register-/ Vol. 60, N'o« 192.' Wednesday. Octoger
52t)39
study should-wei§h heavily in the risk
assessment process.
(3) Cohort (prospective, followup)
studies. In a prospective study design, a
healdiy group of people is assembled
and followed forward in time and
observed for the development of
disease. Such studies are invaluable for
determining the time course for
development of disease (e.g., followup
studies performed in. various cities on
the effects of lead on child
development). This approach allows the
direct estimate of risks attributed to a
particular exposure since disease
incidence rates in the cohort can be
determined. Prospective study designs
also allow die study of chronic effects
of exposure. One major strength of the
cohort design is that it allows the
calculation of rates to determine the
excess risk associated with an exposure.
Also, biases are reduced by obtaining
information before the disease develops.
This approach, however, can be very
time-consuming and costly.
In cohort studies information bias can
be introduced when individuals provide
distorted information about their health
because they know their exposure status
and may have been told of the expected
health effects of the exposure under
study.
A special type of cohort study is the
retrospective cohort study in which the
investigator goes back in time to select
the study groups and traces them over
time, often to the present. The studies
usually involve specially exposed
groups and have provided much
assistance in estimating risks due to
occupational exposures. Occupational
retrospective cohort studies rely on
company records of past and current
employees that include information on
the dates of employment, age at
employment, date of departure, and
whether diseased (or dead in the case of
mortality studies). Workers can then be
classified by duration and degree of
exposure. Positive results from a
properly controlled prospective study-
should weigh heavily in the risk
assessment process;
d. Human Laboratory Exposure
Studies. Neurotoxicity assessment has
an advantage not afforded the
evaluation of other toxic end points,
such as cancer or reproductive toxicity,
in that the effects of some chemicals are
short in duration and reversible. This
makes it ethically possible to perform
human laboratory exposure studies and
obtain data relevant to the risk
assessment process.. Information from
experimental human exposure studies
has been used to set occupational
exposure limits, mostly for organic
solvents that can be inhaled. Laboratory
exposure studies have contributed to
risk assessment,arid the setting of
exposure- liniffl for'several solvents and
other chemicals'with acute reversible
effects. -'''• - " ' .
Human exposure studies sometime
offer advantages over epidemiologic
field studies. Combined with "^
appropriate sampling-of biologic fhiids
(urine or blood), it is possible to ••'<•-• - •
calculate-body concentrations-, exantine
toxicokinetics, and identify metabolites.
Bioavailabillty, 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 Tate)'are
factors that are generally easier to ^' -••"•
collect under controlled conditions.••'• •
Other goals of laboratory studies
include the indepth characterization of
effects, the development of new
assessment methods, and tha
examination of the sensitivity,
specificity, and reliability of
neurobehavioral assessment methods
across chemical classes. The laboratory
is die most appropriate setting for the
study of environmental and
biobehavioral variables that affect the *
action of chemical agents. The effects of
ambient temperature, task difficulty.
rate of ongoing behavior, conditioning
variables, tolerancs/sensitiziition, sleep
deprivation, motivation, and so forth are
sometimes studied.
From a.methodologic standpoint, '
human laboratory studies can be
divided'into two categories—between-
subjects-and within-subjects designs. In
the former, the neurobehavioral
performance of exposed volunteers is
compared with that of nonexposed .
participants. In the latter, preexposure
performance is compared with -
neurobehavioral function under the
influence of the chemical or drug.
Within-subjects designs havo 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-subfects
design is that neurobehavioral tests
must be administered more than once.
Practice on many neurobehavioral tests
often leads to unproved performance
that may confound the effect of the
chemical/drug; There should be a
sufficient number of test sessions in the
pre-exposure phase of the study to allow
performance on all tests to achieve a
relatively stable baseline level.
'Participants in laboratory exposure
studies may have been recruited from
populations of persons^afready exposed
to the chemical)drug or from naive "
populations. Although the use of
exposed volunteers has ethical
advantages, can mitigate against novelty
effects", and allows evaluation of
tolerance/sensitization. finding an
accessible exposed population in-
reasonable proximity to the laboratory is
difficult. Naive participants are more
easily recruited but may differ
significantly in important characteristics
from a representative sample of exposed
persons. Naive volunteers are often
younger, healthier, and better educated
than the populations exposed
environmentally, in the workplace, or
pharmacotherapeuticaUy. "' s
' Compared with workplace arid//:
environmental exposures, laboratory
exposure conditions can be controlled
more precisely, but exposure periods are
much shorter. Generally only one or two
relatively pure chemicals'are studied for
several hours while the population of
interest may be exposed to multiple
chemicals containing impurities for
months or years. Laboratory studies are
therefore better at identifying and
characterizing effects with acute onset
and the selective effects of pure agents.
Neurobehavioral test methods may
have been selected according to several-
strategies. A test battery that examines
multiple neurobehavioral functions may
be more useful for screening and the
initial characterization of acute effects.
Selected neurobehavioral tests that •
measure a more limited number of
functions in multiple ways may be more
useful for elucidating mechanisms or
validating specific effects.
Both chemical and behavioral control
procedures are valuable for examining
the specificity of the effects. A
concordant effect among different
measures of the same neurobehavioral
function (e.g., reaction time) and a lack
of effect on some other measures of
psychomotor function (e.g., untuned
manual dexterity) would increase the
confidence in a selective effect on motor
speed and not on attention or
nonspecific motor function. Likewise.
finding concordant effects among
similar chemical or drug classes along
with different effects from dissimilar
classes would support the specificity of
chemical effect. For example, finding
that the effects of a solvent were similar
to those of ethanol but not caffeine
would support the specificity of solvent
effects on a given measure of
neurotoxicity.
2. Animal Studies
This section provides an overview of
the major types of end points that may
be evaluated in animal neurotoxicity
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Federal Register , Vol. so. No. 192 / Wednesdav. Oc:ober 4. 1995
studies, describes :r.e Kinds of effects
that may be ooserved and some of the
tests used to detect and quantify these
effects, and provides guidance for
interpreting data. Compared with
human studies, animal studies are more
often available for specific chemicals,
provide more precise exposure
information, and control environmental
factors better (Anger. 1984). For these
reasons, risk assessments tend to rely
heavily on animal studies.
Many tests that can measure some
aspect of neurotoxicity have been used
in the field of neurobiology in the last
50 years. The Office of Prevention.
Pesticides and Toxic Substances
(OPPTS) has published animal testing
guidelines that were developed in
cooperation with the Office of Research
and Development (U.S. EPA. 1991a).
While the test end points included serve
as a convenient focus for this section,
there are many other end points for
which there are no current EPA
guidelines. The goal of this document is
to provide a framework for interpreting
data collected with tests frequently used
by neurotoxicplogists.
Five categories of end points will be
described: Structural or
neuropathological, neurophysiological,
neurochemical. behavioral, and
developmental end points. Table 1 lists
a number of end points in each of these
categories.
Table 1.—Example* of Possible Indicators of
a Neurotoxic Effect.
I. Structural or Neuropathological End Points
1. Gross changes in morphology, including
brain'weight
2. Hemorrhage in nerve tissue
3. Breakdown of neurons, giial cells
4. Accumulation, proliferation, or
rearrangement of structural elements
5. dial fibriliary acidic protein increases
(in adults]
II. Neurochamieal End Points
1. Alterations in synthesis, release, uptake,
degradation of neurotransmitters
2. Alterations in second messenger
associated signal transduction
3.'Alterations in membrane-bound
enzymes regulating neuronal activity
4. Inhibition of neuropathy target enzyme
in. Neurophysiological End Points
1. Change in velocity, amplitude, or
refractory period of nerve conduction
2. Change in latency or amplitude of
sensory-evoked potential
3. Change in electroeucephalographic
pattern
IV. Behavioral and Neurological End Points
1. Increases or decreases in motor activity
2. Changes in touch, sight, sound, taste, or
smell sensations
3. Changes in motor coordination.
weakness, paralysis, abnormal
movement or posture, tremor, ongoing
performance
4. Absence or decreased occurrence.
magnitude, or latency of sensorimotor
reflex '
5. Altered magnitude of neurological
measurement, including grip strength,
hindlimb splay
6. Seizures
7. Changes in rate or temporal patterning
of schedule-controlled behavior
8. Changes in laming, memory,
intelligence, attention
V. Developmental End Points
1. Chemically induced changes in the time
of appearance of behaviors during
development
2. Chemically induced changes in the
growth or organization of structural or
neurochemical elements.
a. Structural End Points of
Neurotoxicity. Structural end points are
typically denned as neuropathological
changes measured through gross
observation or with the aid of a
microscope. Gross changes in
morphology can include discrete or
widespread lesions in nerve tissue.
Changes in brain size (weight, width, or
length) are considered to be indicative
of neurotoxic events. This is true
regardless of changes in body weight.
because brain size is generally protected
during undemutrition or .weight loss.
unlike many other organs or tissues. It
is inappropriate to express brain weight
changes as a ratio of body weight and
thereby dismiss changes in absolute
brain weight. The risk assessor should
be aware that a unit of measurement
that is biologically meaningful should
be used for analysis. Brain length
' measurements, for example, expressed
to 1 or 10 micron units is biologically
meaningless. The same is true for brain
width.
Neurons are composed of aneuronal
body, axon, and dendritic processes.
Various types of neuropathological
lesions may be classified according to,
the site where they occur (WHO, 198a;
Krinke, 1989: Griffin, 1990). H
-Neurodegenerative lesions in the central
or peripheral nervous system may be
classified as a neuronopathy (changes in
the neuronal cell body), axonopathy
(changes in the axons), myelinopathy
(changes in the myelin sheaths), or
terminal degeneration. For
axonopathies, a more precise location of
the changes may also be described (i.e.,
' proximal, central, or distal axonopathy).
In the case of some developmental
exposures, a neurotoxic chemical might
delay or accelerate the differentiation or
proliferation of cells or cell types.
Alteration in the axonal termination site
might also occur with exposure. In an
aged population, exposure to some
neurotoxicants might accelerate the
normal loss of neurons associated with
aging (Reuhl, 1991). In rare cases,
neurotoxic agents have been reported to
produce neuropathic conditions
resembling neurodegenerative disorders
in humans such as Parkinson's disease
(WHO, 1986). Table 2 lists examples of
such neurotoxic chemicals, their
putative site of action, the type of
neuropathology produced, and the
disease or condition that each typifies.
TABLE 2.—NEUROTOXICANTS AND DISEASES WITH SPECIFIC NEURONAL TARGETS
Site of action
Neuron cell body •• .
Nerve terminal
Schwann cell Myelin —
Centra>penpheral distal
axon.
Central axons
Proximal axon . .
Neuropathology
Neuronopathy
Terminal destruction .„.
MyeKnopathy .
Distal axonopathy
Central axonopattiy ..
Proximal axonopathy ...
Neurotoxicant
.Metfiyhnercury Qunofnic acid 3-
Acetylpyridine.
1-MethyM-pnenyi-1 ,2,3,6-
tetrahydropyriolne (oopamtoergic).
HexacNorophene
Acrylamide Carbon dsulfide n-Hexane .....
Clioqutnol
B.BMrninodiproptonitrihi .
Corresponding neurodegenerative disease or
condition
Minamata disease, Huntington's disease, Cere-
bellar ataxia.
Parkinson's Disease.
Congenital hypomyelinogenesis.
Peripheral neuropathy.
Suoacute rnyeJoopticoneuropathy
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
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microscopic level. N'europathological
studies should control for potential
differences in the area(s) and section(s)
of the nervous system sampled, in the
age, sex, and body weight of the subject,
and in fixation artifacts (WHO, 1986).
Concern for the structural integrity of
nervous system tissues derives from its
functional specialization and the lack of
regenerative capacity in the central
nervous system.
In general, chemical effects can lead
to two types of structural alteration at
the cellular level: the breakdown of
cells, in whole or in part, or the
accumulation, proliferation, or
rearrangement of structural elements •
(e.g., intermediate filaments,
microtubules) or organelles (e.g.,
mitochondria). Some changes may be
associated with regenerative processes
that reflect adaptive changes associated
with exposure to a toxicant.
Chemically induced injury to the
central nervous system may be
associated with astrocytic hypertrophy.
Such changes may be seen using
immunocytochemical techniques
visualized by light microscopy or
quantified more precisely by
radioimmunoassay (RIA) procedures.
Assays of glial fibrillary acidic protein
(GFAP), the major intermediate filament
protein of astrocytes, have been
proposed as a biomarker of this
response (O'Callaghan. 1988). The
interpretation of a-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
methyl pheny Itetrahy dro pyridine-
(MPTP), have been shown to increase
levels of GFAP. Measures of GFAP are
now included in the Neurotoxicity Test
Battery testing guidelines (U.S. EPA,
1991a).
Increases in GFAP above control
.levels may be seen at dosages below
those necessary to produce damage seen
by standard microscopic or
histopathological techniques. Because
increases in GFAP reflect an astrocyte
response in adults, treatment-related
increases in GFAP are considered to be
evidence that a neurotoxic effect has
occurred. Decreases in GFAP are not
clearly interpretable as indicative of
neurotoxicity. The absence of a change
in GFAP following exposure does not
necessarily mean that the chemical is
devoid of neurotoxic potential Known
neurotoxicants such as cholinesterase-
inhibiting pesticides, for example.
would not be expected to increase brain
levels of GFAP. Interpretation of GFAP
changes prior to weaning is confounded
by the possibility that chemically
induced increases in GFAP may be
masked by changes in the concentration
of this protein associated with
maturation of the central nervous
system, and these data may be difficult
to interpret
b. Neurophysiological End Points of
Neurotoxicity. Neurophysiological
studies are those that measure the
electrical activity of the nervous svstem.
The term "neurophysiology" is often
used synonymously with,
"electrophysiology" (Dyer, 1987).
Neurophysiological techniques provide
information on the integrity of defined
portions of the nervous system. Several
. neurophysiological procedures are
available for application to
neurotoxicological studies. Examples of
neurophysiological measures, of
neuro.toxicity 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 macroelectrbdes
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
macroeiectrode 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 NEUROTOXICTTY
Systenvfunction
Retina : _
Visual pathway
Visual function
Audtory pathway _
Audrtory function
Somatosensory pathway
Somatosensory function ...
Spinocerebellar pathway
Mixed nerve ..._.
Motor axons ; .„ „.'
Sensory axons
Neuromuscular „„_
General central nervous system/level of arous-
al. • .
Procedure
Etectroretinograpny (ERG) ._
Flash evoked potential (FEP)
and contrast).
Brain «tftnt aitfftrw ounfcA/4 rwt***i4iAi /QACO*
(Cfcta).
BAER (tones)
Somatosentory... evokacv potential (SEP)
(shocks).
SEP (tactile) „
SEP recorded from cerebellum
(PNAP).
PNAP isolate motor components
PNAP isolate sensory components
Etectromyograpriy (EMG). H-reftex, M-re-
spons*.
Electroenceprialography (EEG)
Representative agents
Oevetoprrwntal leadi.
Carbon dsurrkto. • • •
Carbon <£sur5de.
AnwogrycosuJe, Antibiotics, Toluene, styrene.
Ammogrycoside, Antibiotics, Toluene, styrene.
Aerytamide, rvHexane.
Afifvianwte rvHAvji/^
Acrytamide rhHexarw.
Trwtnyltin.
Triethyltin.
Otthiobiuret
Anesthetics.
(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 ihotor nerve fibers, which may or
may not be differentially sensitive to
neurotoxicants. It is possible to
distinguish sensory from, motor effects
in peripheral nerve studies by
measuring activity in purely sensory
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r.erves such as the su.-ai nerve or by
measuring the muscle response evoked
by nerve stimulation to measure motor
et'fects. While a number of end points
can be recorded, the most critical
variables are (1) nerve conduction
velocity. (2) response amplitude, and (3)
refractory period.
.Verve 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 control limb
temperature within narrow limits.
Studies that measure peripheral nerve
function without regard for temperature
are not adequate for risk assessment.
In well-controlled studies, statistically
significant decreases in nerve
conduction velocity are indicative of a
neurotoxic effect. While a decrease in
nerve conduction velocity is indicative
of demyelination. it frequently occurs
later in the course of axonal degradation
because normal conduction velocity
may be maintained for some time in the
face of axonal degeneration. For this
reason, a measurement of normal nerve
conduction velocity does not rule out
peripheral axonal degeneration if other
signs of peripheral nerve dysfunction
are present.
Decreases in response amplitude
reflect a loss of active nerve fibers and
may occur prior to decreases in
conduction velocity in the course of
peripheral neuropathy. Hence, changes
in response amplitude may be more
sensitive measurements of axonal
degeneration than conduction velocity.
Measurements of response amplitude,
however, can be more variable and
require careful application of
experimental techniques, a larger
sample size, and greater statistical
power than measurements of velocity to
detect changes. The refractory period
refers to the time required after
stimulation before a nerve can fire again
and provides a measure reflecting the
functional status of nerve membrane ion
channels. Chemically induced changes
in refractory periods in a well-
controlled study indicate a neurotoxic
effect.
In summary, alterations in peripheral
nerve response amplitude and refractory
period in studies that are well
controlled for temperature are indicative
of a neurotoxic effect. Alterations in
peripheral nerve function are frequently
associated with clinical signs such as
numbness, tingling, or burning
sensations or with motor impairments
such as weakness. Examples of
compounds that alter peripheral nerve
function Ln humans or experimental
animals include acrylamide, carbon
disulfide. n-hexane, lead, and some
organophosphates.
(2) Sensory, motor, and other evoked
potentials. Evoked potential studies are
electrophysiologicai 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, 1986: Mattsson et al., 1992;
Boyes, 1992,1993). Evoked potential
studies should be interpreted with
respect to the known or presumed
neural generators of the responses, and
their likely relationships with
behavioral outcomes, when such
information is available. Such
correlative information strengthens the
confidence in electrophysiologicai
outcomes. In the absenca 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 sueh
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 t
the magnitude of the neural population tj
responsive to stimulation. Both >]
increases and decreases in amplitude
are possible following exposure to.
chemicals. Whether excitatory or
inhibitory neural activity is translated
into a positive or negative deflection in
the sensory evoked potential is
dependent on the physical orientation
of the electrode with respect to the
tissue generating the response, which is
frequently unknown. Comparisons
should be based on the absolute change
in amplitude. Therefore, either increases .
or decreases in amplitude may be
indicative of a neurotoxic effect.
Within any given sensory system, the
neural circuits that generate various
evoked potential peaks differ as a
function of peak latency. In general,
early latency peaks reflect the
transmission-of afferent sensory
information. Changes in either the
latency or amplitude of these peaks are
considered convincing evidence of a
neurotoxic effect that is likely to be
reflected in deficits in sensory
perception. The later-latency peaks, in
general, reflect not only the sensory
input but also the more nonspecific
factors such as the behavioral state of
the subject, including such factors as
arousal level, habituation, or
sensitization (Dyer, 1987). Thus,
changes in later-latency evoked
potential peaks must be interpreted in
light of the behavioral status of the
subject and would generally be
considered evidence of a neurotoxic
effect.
(3) Seizures/convulsions.
Neurophy siological recordings of brain
electrical activity that demonstrate
seizure-like activity are indicative of a
neurotoxic effect Occasionally,
behaviors resembling convulsions might
follow actions outside the nervous
system, such as direct effects on muscle.
When convulsion-like behaviors are
observed, as described in the behavioral
section, neurophysiological recordings
can determine if these behaviors
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52043
originate from seizure activity in the
brain.
In addition to producing seizures
directly, neurotoxicants also may alter
the frequency, severity, duration, or
threshold for eliciting seizures produced
through other means. Such changes can
occur after acute exposure or after
repeated exposure to dose levels below
the acute threshold and are considered
to be neurotoxic effects. Examples of
agents that produce convulsions include
lindane, DDT (dichloro-diphenyl-
trichloroethane), pyrethroids, and
trimethyltin.
(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 EEG in either setting
is the relationship between specific
patterns of EEG waveforms and specific
behavioral states. Because states of
alertness and stages of sleep are
associated with distinct patterns of
electrical activity in the brain, jt.is.
generally thought" that arousal; level can
be evaluated by monitoring the EEG.
Dissociation of EEG activity and
behavior can. however, occur after
exposure to certain chemicals. Normal
patterns of transition between sleep
stages or between sleeping and waking _
states are known to remain disturbed for
prolonged periods of time after exposure-
to some chemicals. Changes in the
pattern of the EEG can be elicited by
stimuli producing arousal (e.g., lights,
sounds) and anesthetic drugs. In studies
with toxicants, changes in EEG pattern
can sometimes precede alterations in
other objective signs of neurotoxicity
(Dyer, 1987).
EEG studies must be done under
highly controlled conditions, and the
data must be considered on a case-by-
case basis. Chemically induced seizure
activity detected in the EEG pattern is
evidence of a neurotoxic effect,
c. Neurochemical End Points of
Neurotoxicity, Many different
aeurochemical end points have been
measured in neurotoxicological studies,
and some have proven useful in
advancing the understanding of
mechanisms of action of neurotoxic
chemicals (Bondy, 1986; Mailman,
1987; Morell and Mailman, 1987; Costa,
1988). Normal functioning of the
nervous system depends on the
synthesis and release of specific
neurotransmitters and activation of their
receptors at specific presynaptic and
postsynaptic sites. Chemicals can
interfere with the ionic balance of a,
neuron, act as a cytotoxicant after
transport into a nerve terminal, block
reuptake of neurotransmitters and their
precursors, act as a metabolic poison J
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 Wrm KNOWN NEUROCHEMICAL MECHANISMS
Site of action
Example*
1. Neurotoxicants Acting on Ionic Balance:
A. Inhibit sodium entry
B. Block dosing of sodhjm channel ....
C. Increase permeability to sodium ....
0. Increase intracelluiar calcium
2. Cytotoxicanta—Depend on uptake into nerve terminal.
3. Uptake blockers .._.__. „..._.._„_
4. Metabolic poisons „„ ._.._..
5. Hyperactrvation of receptors
6. Blocks transmitter release (AcetytehoKne [AChJ) ,
7. Inhibition of transmitter degradation (ACn)
8. Blocks axonai transport
TeliodotoxirL
pjj'-OOT, pyrettiroids.
Bafrachotaort
Chtofdecone.
MPTP.
Henvcfaofiniunx
Cyanide..
Demote acid.
BoftJnun toxin.
Pesticides of the organopnosphate and carbamate ctasse*.
Acrytamide.
As stated previously, any
neurochemical change is potentially
neurotoxic. but each determination
requires professional judgment.
Persistent or irreversible chemically
induced neurochemical changes are
indicative of neurotoxicity. Because the
ultimate functional significance of some
biochemical changes is not known at
this time, neurochemical studies should
be interpreted with reference to the
presumed neurotoxic consequence(s) of
the neurochemical changes. For
example, many neuroactive agents can
increase or decrease neurotransmitter
levels, but such changes are not
necessarily indicative of a neurotoxic
effect. If, however, these neurochemical
changes may be expected to have
neurophysiological, neuropathological.
or neurobehavioral correlates, then the
neurochemical changes could be
classified as neurotoxic effects.
Some neurotoxicants, such as the
orgmnophosphate and carbamate
pesticides, are known to inhibit the
activity of a specific enzyme,
acetylcholinesterase (for a review sea
Costa. 1988), which hydrolyzest the
neurorransmitter acetylcholine.
Inhibition of the enzyme prolongs the
action of the acetylcholine at the
neuron's synaptic receptors and is
responsible for the autonomic
stimulation and death that them agents
cause.
Within EPA and elsewhere, questions
have arisen as to whether inhibition of
cholinestense activity constitutes an
adverse effect for defining hazard
potential and evaluating risk. There is
agreement among scientists that
statistically significant inhibition of
cholinesterase activity in multiple
organs and tissues accompanied by
clinical effects constitutes a hazard.
However, there is scientific uncertainty
and related controversy aboutthe risk
assessment implications of data
describing inhibition of cholinesterase
enzyme activity in the absence of
observable clinical effects. While there
is agreement that such inhibition is a
biomarker of exposure, there is
continued disagreement over whether •
cholinesterase inhibition^especially in
blood, constitutes an adverse effect.
At this point, it can be stated that
there is general agreement among
scientists that objective clinical
measures of dysfunction/impairment
can be overt manifestations of inhibition
of cholinesterase in the nervous system.
On the basis of Hiniml manifestations.
e.g.. muscle weakness, tremor, blurred
vision, one should be able to evaluate
dose-response and dose-effect
relationships and define the presence
and absence of given effects. A
relationship between the effect and
cholinesterase inhibition should be
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Federal Register / Vol. 60. No.- 192 / Wednesday. October 4. 1995 / .Notices
confirmed by biochemical measures of
reduced cholinesterase activity.
In addition, a reduction in brain
cholinesterase activity may or may not
be accompanied by clinical
manifestations. Most experts in the field
acknowledge that when significant
reductions in brain cholinesterase
activity alone occur, reduced
cholinesterase levels either are
themselves toxic or would lead to a
neurotoxic effect if exposure were to
persist over time or increase in
magnitude. Therefore, statistically
significant decreases in brain
cholinesterase could be considered to be
a biologically significant effect. •
A reduction in RBG and/or plasma
cholinesterase activity also may or may
not be accompanied by clinical
manifestations. At this time, there is
general agreement that the observation
of inhibition of RBC and/or plasma
cholinesterasa contributes to the overall
hazard identification of cholinesterasa
inhibiting agents by serving as
biomarkers. As such, these enzyme
parameters can provide information that
will help scientists evaluate whether
reported clinical effects are associated
with cholinesterase inhibition. There
remains, however, a lack of consensus
as to whether RBC and/or plasma
cholinesterase represent biologically
significant events. Discussions on this
topic are continuing within the Agency.
A subset of organophosphate agents
also produces organophosphate-induced
delayed neuropathy (OPIDN) after acute
or repeated exposure. Prolonged
inhibition (i.e.. aging) of neurotoxic
esterase (or neuropathy target enzyme)
has been associated with agents that
produce OPIDN (Johnson, 1990), a clear
neurotoxic effect
d. Behavioral'End Points of
Neurotoxicity. EPA's testing guidelines
developed for the Toxic Substances
Control Act and the Federal Insecticide,
Fungicide and Rodentitide Act describe
the use of functional observational
batteries (FOB), motor activity, and
schedule-controlled behavior for
assessing neurotoxic potential (U.S.
EPA, 1991a). There are many other
measures of behavior, including
specialized tests of motor and sensory
function and of learning and memory
(Tilson. 1987; Anger. 1984). Examples of
behavioral end points that have been
used to detect neurotoxicity are
included in Table 1. The risk assessor
should know that the literature is'clear
that a number of other behaviors besides
those listed in Tables 1 and 5 could be
affected fay chemical exposure. For
example, alterations in food and water ^
intake, reproduction, sleep, temperature'
regulation, and circadian rhythmicity *
are controlled by specific regions of the
brain and chemical-induced alterations
in these behaviors could be indicative of
neurotoxicity. It is reasonable to assume
that a NOAEL or LOAEL could be based
on one or more of these end points.
TABLE 5.—SUMMARY OF MEASURES IN A REPRESENTATIVE FUNCTIONAL OBSERVATIONAL BATTERY, AND THE TYPE OF
DATA PRODUCED BY EACH
Home cage and open field
Posture (D)
Convulsions, tremors (D)
Palpetxal closure (R)
LacrtmaHon (R)
PiloerectJon (Q)
Salivation (R)
Vocalizations (Q)
Rearing (C)
Urination (Q
Defecation (C)
Gait (D, R)
Arous* (R)
Mobility (R).
Sterectypy (D).
Biza/re behavior (D)
Manipulative
Ease of removal (R)
HandBng reactivity (R)
Palpetoral closure (R).
Approach response (R).
CBck response (R).
Touch response (R).
Tail pinch response (R).
Higjrang renex (n).
Landing foot splay (1).
ForeKmb grip strength (1).
HindUmb grip strength (1).
Pupil response- (Q).
Physiologic
Body temperature (1).
Body weight (1).
£>
D—-descriptive data; R—rank order data; Q—quanta! data; I—interval data; C—court data.
Behavior is an indication of the
overall well-being of the organism.
Changes in behavior can arise from a
direct effect of a toxicant on the nervous
system or indirectly from its effects on
other physiological systems.
Understanding the interrelationship
between systemic toxicity and
behavioral changes is extremely
important (e.g.. the relationship between
Uver damage and motor activity). The
presence of systemic toxicity may
complicate, but does-not necessarily
preclude, interpretation of behavioral
changes as evidence of neurotoxicity. In
addition, a number of behaviors (e.g..
schedule-controlled behavior) may
require a motivational component for
successful completion of the task. In '
such cases, experimental paradigms
designed to assess the motivation of an
animal during behavior might be
necessary to interpret tha meaning of
some chemical-induced changes in
behavior.
The following sections describe in
general behavioral tests and their uses
and offer guidance on interpreting data.
(1) Functional observational battery.
A functional observational battery is
designed to detect and quantify major
overt behavioral, physiological, and
neurological signs (Gad. 1982;
OTJonoghue, 1989; Moser, 1989). A
number of batteries have been
developed, each consisting of tests
generally intended to evaluate various
aspects of sensorimotor function (Tilson
and Moser. 1992). Many FOB tests are
essentially clinical neurological
examinations that rate the presence or
absence, and in many cases the severity,
of specific neurological signs. Some
FOBs in animals are similar to clinical
neurological examinations used with
human patients. Most FOBs have
several components or tests. A typical
FOB is summarized in Table 5 and
evaluates several functional domains,
including neuromuscular (i.e.,
weakness, incoordination. gait, and
tremor), sensory (i.e., audition, vision.
and somatosensory), and autonomic
(i.e., pupil response and salivation)
function. FOB data may be in the form
of interval, ordinal, or continuous
measurements.
The relevance of statistically
significant test results from an FOB is
judged according to the number of signs
affected, the dose(s) at which effects ar»
observed, and the nature, severity, and
persistence of the effects and their
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52045
incidence in relation to control animals.
If only a few unrelated measures in the
FOB are affected, or the effects are
unrelated to dose, the results are not
considered evidence of a neurotoxic
effect. If several neurological signs are •
affected but only at the high dose and
in conjunction with other overt signs of
toxicity, including systemic toxicity,
large decreases in body weight,
decreases in body temperature, or
debilitation, there is ncr conclusive
evidence of a direct neurotoxic effect. In
cases where several related measures in
a battery of tests are affected and the
effects appear to bo dose dependent, the
data are considered to be evidence of a
neurotoxic effect, especially in the
absence of systemic toxicity. Recently, it.
was proposed that data from FOB
studies be grouped into several .
neurobiological domains, including
neuromuscular (i.e., weakness,
^coordination, abnormal movements,
gait), sensory (i.e. auditory, visual,. '
somatosensory), and autonomic
functions (Tilson andMoser. 1992).
This statistical technique is useful when
separating changes that occur on the
basis of chance or in conjunction with
systemic toxicity from those treatment-
related changes indicative of neurotoxic
effects. In the case of the developing
organism, chemicals may alter the
maturation or appearance of
sensorimotor reflexes. Significant
alterations in or delay of such reflexes
is evidence of a neurotoxic effect.
Examples of chemicals that affect
neuromuscular function are 3-
acetylpyridine, acrylamide, and
triethyltin. Organophosphate and
carbamate insecticides produce
autonomic dysfunction, while
organochlorine and pyrethroid
insecticides increase sensorimotor
sensitivity, produce tremors, and in
some cases, cause seizures and
convulsions (Spencer and Schaumberg.
1980).
(2) Motor activity. Motor activity
represents a broad class of behaviors
involving coordinated participation of
sensory, motor, and integntive
processes. Assessment of motor activity
is noninvasive and has been used to
evaluate the effects, of acute and
repeated exposure to neurotoxicants
(MaePhail et aL, 1989). An organism's
level of activity can, however, be
affected by many different types of
environmental agents-, including
nonneurotoxic agents. Motor activity
measurements also have been used in
humans to evaluate disease states.
including disorders of the nervous
system (Goldstein and Stein, 1985).
Motor activity is usually quantified as
the frequency of movements over a
period of time. The'-total counts
generated during a test period will
depend on the recording mechanism
and size and configuration of the testing
apparatus. Effects of agents on motor
activity can be expressed as absolute
activity counts or as a percentage of
control values. In some cases, a
transformation (e.g.. square root) may be
used to achieve a normal distribution of
the data. The frequency of-motor
activity within a session usually
decreases and is reported as the average
number of counts occurring in each
successive block of time. The EPA's
Office of Prevention, Pesticides and
Toxic Substances guidelines (U.S. EPA,
1991a), for example, call for test-
sessions of sufficient duration to allow
motor activity to approach steady-state
levels during the last 20 percent of the
session for control animals. A sum of
the counts in each epoch will add up to
the total number of counts per session.
In the adult, nourotoxic agents
generally decrease motor activity
(MaePhail et aL, 1989). Examples
include many pesticides (e.g.,
carfaamates. chlorinated hydrocarbons,
organophosphates, and pyrethroids).
heavy metals (lead, tin, and mercury),
and other agents (3*acatylpyridine,-
acrylamide, and 2,4-dithiobiuret). Some
neurotoxicants (e.g., toluene, xylene.
triadimefon) produce transient increases
in activity by. presumably stimulating
neurotransmitter release, while others
(e.g., trimethyltin) produce persistent .
increases in motor activity by destroying
specific regions of the brain (e.g.,
hippocampus).
Following developmental exposures,
neurotoxic effects are often observed as
a change in the developmental profile or
maturation of motor activity patterns.
Frequently, developmental exposure to
neurotoxic agents will produce an
increase.in motor activity that persists
into adulthood or that results in changes
in other behaviors. This type of effect is
evidence of a neurotoxic effect. Like
other organ systems, the nervous system
may be differentially sensitive to
toxicants in groups such as the young.
For example, toxicants introduced to the
developing nervous system may kill
stem cells and thus cause profound •
effects on adult structure and function.
Moreover, toxicants may have greater
access to the developing nervous system
before the blood-brain barrier is
completely formed or before metabolic
detoxifying systems are functional.
Motor activity measurements are
typically used with other tests (e.g.,
FOB)-to help detect neurotoxic effects.
Agent-induced changes in motor
activity associated with other overt
signs of toxicity (e.g., loss of body
weight, systemic toxicity) or occurring
in non-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)-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 7
performance of a learned behavior (e.g.,
lever press or key peck) and involves
training and motivational variables that
must be considered in-'evaluating the
data. Agents may interact with sensory
processing, motor output, motivational
variables (i.e., related to reinforcement),
training history, and baseline
characteristics (Bice, 1988: Cory-
Slechta, 1989). Rates and patterns of
SCOB display remarkable species and
experimental generality.
In laboratory animals. SCOB has been
used to study a wide range of
neurotoxicants. including
methylmercury, many pesticides,
carbon disulfide, organic and inorganic
lead, and triethyl and trimethyltin
(MaePhail. 1985; Tilson, 1987; Rice,
1988). The primary SCOB end points for
evaluation are response rate and the
temporal pattern of responding. These
end points may vary as a function of the
contingency between responding and
reinforcement presentation (i.e.,
schedule of reinforcement). While most
chemicals decrease the efficiency of
responding at some dose, some agents
may increase, response efficiency on
schedules requiring high response rates
due to a stimulant effect or an increase
in central nervous system excitability.
Agent-induced changes in responding.
between reinforcements (i.e., the
temporal pattern of responding) may
occur independently of changes in the
overall rate of responding. Chemicals
may also affect the reaction time to
respond following presentation of a
stimulus. Agent-induced changes in
response rate or temporal patterning
associated with other overt signs of
toxicity (e.g.. body weight loss, systemic
toxicity. or occurring in a non-dose-
related fashion) are of less concern than
changes that are dose dependent, related
to structural or other functional changes
in the nervous system, or occur in the
absence of life-threatening toxicity.
(4) Convulsions. Observable
convulsions in animals are indicative of
an adverse effect. These events can
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52046
Federal Register / Vol. so. No. 192 / Wednesday, October 4. 1995 / Notices
reflect central nervous system activity
comparable to that of epilepsy in
humans and could be defined as
aeurotoxicity. Occasionally, other toxic
actions of compounds, such as direct
effects on muscle, might mimic some
convulsion-like behaviors. In some
cases, convulsions or convulsion-like
behaviors may be observed in animals
that are otherwise severely
compromised, moribund, OF near death.
In such cases, convulsions might reflect
an indirect effect of systemic toxicity
and are less dearly indicative of
neurotoxicity. As discussed in the
section on neurophysiological measures,
electrical recordings of brain activity
could be used to determine specificity
of effects on the nervous system.
(5) Specialized tests for neurotoxicity.
Several procedures have been
developed to measure agent-induced
changes in specific neurobehavioral
functions such as motor, sensory, or
cognitive function (Tilson, 1987; Cory-
Slechta. 1989). Table 6 lists several
well-known behavioral tests, the
neurobehavioral functions they were
designed to assess, and agents known to
affect the response. Many of these tests
in animals have been designed to assess
neural functions in humane using
similar testing procedures.
TABLE 6.—EXAMPLES OF SPEOAUZED BEHAVIORAL TESTS TO MEASURE NEUROTOXICITY
Function
Procedure
Representative agents
incooFcSnsDsn •
Tremor —
Myodooia spasms
Soraory:
AuoTtory
Vouai .
Grip sliefiejlte iwwwnjng dftdurance;
sion rod! dacnrrinalive motor function.
Rotorod. ga* maasuemana; nghting reflex
Rating 3caiet specksl anafystt
Rating scale, spectral analysis
n-Hexane. mathJ n-outy*eton«, carbaryt.
3-Acetylpyriolne, •thanei.
CHofoacone. Type I pyreflvortt, DOT.
DDT. Type H |
/
Dtacriminalion coneVoning Reflex modHlcafion
i. WnethyUta.
Pain s*ns£vity
conoMoning (trtration); func-
Aaytamide.
ParBttion.
9/Menxxy:
tional otaarvationeJ battery.
consWoning ._
OBseoropyM
Oa
xrtng
latal meeiyknereuy.
Ahmnum.
CaiberyL
TnineHiyNki, IOPN.
Neonatal MnettiyMn.
ophospnate (OFP) Pre/
Delayed matching to sample
RepexHd acQuiafcn
visual decranras«n
PnVheoaatat lead.
HypetvitHTinois A.
Styrena.
DFP.
TrimostyiBfL
OFP.
A statistically significant chemically
induced chang* in any measure in Tab)*
6 is presumptive evidence of advers*
effect Judgments of neurotoxicity may
involve not only tha analysis of change*
seen but the structurs and class of the
chemical and othar available
neurochsmical, neurophysiological, and
neuropathological evidenc*. In general.
behavioral changes seen across broader
dos« ranges indicate mom specific
actions on the systems underlying those
changes, i.e.. the nervous system.
Changes that an not dose dependent or
that are confounded with body weight
changes and/or other systemic toxicity
may be more difficult to interpret as ,
neurotoxic effects.
(a) Motor function: Neurotoxicants
commonly affect motor function. These
effects can be categorized generally into
(1) weakness or decreased strength, (2)
tremor; (3) Lncootdination, and (4)
spasms, myoclonia, or abnormal motor
movements (Tilson. 1987; Cory-Slechta.
1989). Specialized tests used to assess
weakness include- measures of grip
strength, swimming endurance.
suspension from a hanging rod. and
discriminative motor function. Rotarod
and gait assessments are used to
measure incoordination. while rating
scales and spectral analysis techniques
can be used to quantify tremor and other
abnormal movements.
(b) Sensory function: Gross
perturbations of sensory function can be
observed in simple neurological
assessments such as the FOB. However.
these tests may not be sufficiently
sensitive to detect subtle sensory
changes. Psychophysical procedures
that study the relationship between a
physical dimension (e.g.. intensity,
frequency) of a stimulus and behavior
may be necessary to quantify agent-
induced alterations in sensory function.
Examples of psychophysical procedures
include discriminated conditioning and
startle reflex modification.
(c) Cognitive function: Alterations in
teaming and memory in experimental
anitnaU must be inferred from changes
in behavior following exposure when
compared with that either seen prior to
exposure or with a nonexposed control
group. Learning is defined as a
relatively lasting change in behavior due
to experience, and memory is defined as
the persistence of a teamed behavior
over time. Table 8 lists several examples
of learning and memory tests and
representative neurotoxicants known to
affect these tests.. Measurement of
changes in learning and memory must
be separated from other changes in
behavior that do not involve cognitive
or associative processes (i.e., motor
function, sensory capabilities.
motivational factors). In addition, any
apparent toxicant-induced change in
learning or memory should ideally be
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Federal Register-/ VoL. 60. N'o. 192 f Wednesday. October 4, -\.gg&j Notices
5204?
demonstrated avara range, of stimulus
and response conditions and testing
conditions. In developmental exposures.
it should be shown that the animals
have matured enough to perform the
specified task. Developmental
neurotoxicants can accelerate or delay
the ability to learn a response or
interfere with cognitive function at the
time of testing. Older animals frequently
perform- poorly on some types of tests,
and it must be demonstrated that
control animals in this population are
capable of performing the procedure.
Neurotoxicants might accelerate age-
related dysfunction or alter motivational
variables that are important for learning
to occur. Further, it is not necessarily
the case that a decrease in responding
on a learning task is adverse while an
increase in performance on a learning
task is not. It is well known that lesions
in certain regions of the brain can
facilitate the acquisition of certain types
of behaviors by removing preexisting
response tendencies (e.g., inhibitory
responses due to stress) that moderate
the rate of learning under normal
circumstances. Examples of learning
and memory procedures include simple
habituation, classical conditioning, and
operant (or instrumental) conditioning,
including tests for spatial learning and
memory.
e. Developmental Neurotoxicity.
Although the previous discussion of
various neurotoxicity end points and
tests applies to studies in which
developmental exposures are used.
there are particular issues, of importance
in the evaluation of developmental
neurotoxicity studies. Exposure to
chemicals during development can
result in a spectrum of effects, including
death, structural abnormalities, altered
growth, and functional deficits (U.S.
EPA, 1991b). Children are often
differentially sensitive to chemical
exposure. A number of agents have been
shown to cause developmental
neurotoxicity when exposure occurred
during the period between conception
and sexual maturity (e.g., Riley and
Vorhees, 1988; 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)
(Kimmei et al.. 1990; Needleman. 1990:
Jacobson et al.. 1985; Needleman, 1988).
In many of these cases, functional
deficits are observed at dose levels
beiownthosa:at.which other-indicators-of
developmental toxicity are evident or at
minimally toxic doses in adults. Such
effects may be transient, but generally
are considered to Be adverse effects.
TABLE 7.—EXAMPLES OF
DEVELOPMENTAL NEUROTOXICANTS
Alcohols .....
Antirmtotics-.
Insecticides
Metals
Poryhatogenated hy-
drocarbons.
Solvents
Memanot, ethanof •
X-fadatioo, :
azacytioine.
DDT.Jsepone.
LeaoV-methylmercury,
cadmium.
PCSs, PBBs.
Carbon disurflde. tolu-
ene^ ' ..•'
Testing for developmental"^ »"••: • •
neurotoxicity has not been required-"'
routinely by regulatory agencies in the
United States, but is required by. the
EPA when other information indicates
the potential for developmental .
neurotoxicity (U.S. EPA, 1988,1988a,
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 that in studies with shorter
term exposure.
Important design issues to be -
evaluated for developmental
neurotoxicity studies are similar to
those for standard developmental
toxicity studies (e.g., a dose-response
approach with the highest dosa
producing minimal overt maternal or
perinatal toxicity. number of litters large
enough for adequate statistical power.
randomization of animals to dosa
groups and test groups, litter generally
considered as the statistical unit). In
addition, the us* of a replicate study
design provide* added confidence in the
interpretation of data. A
pharmacological/physiological
challenge may also be valuable in
evaluating neurologic function and
"unmasking" effects not otherwise
detectable. For example, a challenge
with a psychomotor stimulant such as
d-amphetamina may unmask latent
developmental neurotoxicity (Hughes
and Sparber. 1978; Adams and Buelke-
Sam. 1981; Buelke-Sam et al.. 1985).
Direct extrapolation of developmental
neurotoxicity to humans is limited in
the same way as for other end points of
toxicity. i.e., by the lack of knowledge
about underlying lexicological
mechanisms and their significance (U.S.
EPA, 1991b). However, comparisons of
human and animal data for several
agsnts known-to-cause developmental"'
neurotoxicity in-humans showed many
similarities in effects (Kimmei et al..
1990). Comparisons at the level of
functional category (sensory,
motivational, cognitive, and motor
function and social behavior) showed
close agreement across species for the
agents evaluated, even though the
specific end-points used to assess these
functions varied considerably across
species (Stanton and Spear, 1990). Thus,
. it can be assumed that developmental
neurotoxicity effects in animal studies
indicate the potential for altered
neurobehavioral development, in"n
humans, although the specific types of .
developmental effects seen in ''" f
experimental animal studies will nof"
necessarily be the same as those that
may be produced in humans. Therefore,
when data suggesting adverse effects in
developmental neurotoxicity studies are
encountered for particular agents, they
should be considered in the risk
assessment process.
Functional tests with a moderate
degree of background variability, (e.g.. a
coefficient of variability of 20 percent or
less) may be more sensitive to the effects
of an agent on behavioral end points
than are tests with low variability that
may be impossible to disrupt without
using life^thraatening doses. A battery of
functional tests, in contrast to a single
test, is usually needed to evaluate the
full complement of nervous system
functions in an animal. Likewise, a
series of tests conducted in animals in
several age groups may provide more
information about maturational changes
and their persistence than tests
conducted at a single age.
It is a well-established principle that
there are critical developmental periods
for the disruption of functional
competence, which include both the
prenatal and postnatal periods to the
time of sexual maturation, and the effect
of a toxicant is likely to vary depending
on the time and degree of exposure
(Rodier. 1978.1990). It is also important
to consider the data from studies in
which postnatal exposure is included,
as there may be an interaction of the
agent with maternal behavior, milk.
composition, pup suckling behavior, as
well as possible direct exposure of pups
via dosed food or water (Kimmei et al..
1992).
Agents that produce developmental
neurotoxicity at a dose that is not toxic
to the maternal animal are of special
concern. However, adverse
developmental effects are often
produced at doses that cause maternal
toxicity (e.g., <20 percent reduction in
weight gain during gestation and
lactation). In these cases, the
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52048
Federal Register Vol. 60. N'o. 192 / Wednesday-, October 4. 1995 / Notices
Developmental effects are still
Considered to represent neurotoxicity
and should not be discounted as being .
secondary to maternal toxicity. At doses
causing moderate maternal toxicity (i.e.,
>20 percent reduction in weight gain
during gestation and lactation),
interpretation of developmental effects
may be confounded. Current
information is inadequate to assume
that developmental effects at doses
causing minimal maternal toxicity result
only from maternal toxicity; rather, it
may bt that the mother and developing
organism are equally sensitive to that
doss level. Moreover, whether
developmental effects are secondary to •
maternal toxicity or not. the maternal
effects may be reversible while the
effects on the offspring may be
permanent. These are important
considerations for agents to which
humans may be exposed at minimally
toxic levels either voluntarily or
involuntarily, because several agents are
known to produce adverse
developmental effects at minimally
toxic doses in adult humans (e.g.,
alcohol) (Coles et al., 1991).
Although interpretation of
developmental neurotoxicity data may
be limited, it is dear that functional
effects must be evaluated in light of
other toxicity data, including other
forms of developmental toxicity (e.g.,
structural abnormalities, perinatal
death, and growth retardation). For
example, alterations in motor
performance may be due to a skeletal
malformation rather than nervous
system change. Changes in learning
tasks that require a visual cue might be
influenced by structural abnormalities
in the eye. The level of confidence that
an agent produces an adverse effect may.
be as important as the type of change
seen, and confidence may be increased
by such factors as reproducibility of the
effect either in. another study of the
same function or by convergence of data.
from tests that purport to measure
similar functions-. A dose-response
relationship is an extremely important
measure of a chemical's effect; in the
case of developmental neurotoxicity
both monotonic and biphasic dose-
response curves are likely, depending
on the function being tested. The EPA
Guidelines for Developmental Toxicity
Risk Assessment (U.S. EPA, 1991b) may
be consulted for more information on
interpreting developmental toxicity
studies. The endpoints frequently used
to assess developmental neurotoxicity
in exposed children was recently
reviewed by Winneke (1995).
3. Other Considerations
a. Pharmacokinetics. Extrapolation of
test results between species can be
aided considerably by data on the
pharnracokinetics of a particular agent
in the species tested and. if possible, in
humans. Information on a toxicant's
half-life, metabolism, absorption.
excretion, and distribution to the
peripheral and central nervous system
may be useful in predicting risk. Of
particular importance for the
pharmacokinetics of neurotoxicants is
the blood-brain barrier, which
ordinarily excludes ionic and nonlipid
soluble chemicals from the central
nervous system. The brain contains
tircumventricular organs whose
purpose seems to be to sense the
chemical composition of the peripheral
circulation and activate mechanisms to
bring the composition of the blood back
to equilibrium if disturbed. These areas
are technically inside the brain, but they
lie outside of the blood-brain-barrier.
Therefore, chemicals from the periphery
can pass directly into the brain at these
sites. The majority of these structures
are located within or near the
hypothalamus, an area that is crucial for
maintenance of neuroendocrine
function. Phannacokinetic data may be
helpful in defining the dose-response
curve, developing a more accurate basis
for comparing species sensitivity
(including that of humans), determining
dosimetry at target sites, and comparing
phannacokinetic profiles for various
dosing regimens or routes of
administration. The correlation of
phannacokinetic parameters and
neurotoxicity data may be useful in
determining the contribution of specific
phannacokinetic processes to the effects
observed.
b. Comparisons of Molecular
Structure. Comparisons of the chemical
or physical properties of an agent with
those of known neurotoxicants may
provide some indication of the potential
for neurotoxicity. Such information may
be helpful for evaluating potential
toxicity when only minimal data are
available. The structure-activity
relationships (SAR) of some chemical •
classes have been studied, including
hexacarbons, organophosphates,
caroaraates, and pyrethroids. Therefore,
class relationships or SAR may help
predict neurotqxicity or interpret data
from neurotoxicological studies. Under
certain circumstances (e.g.. in the case
of new chemicals), this procedure is one
of the primary methods used to evaluate
the potential for toxicity when little or
no empirical toxicity data are available.
It should be recognized, however, that
effects of chemicals in the same class
can vary widely. Moser (1994), for
example, reported that the behavioral
effects of prototypic cholinesterase-
inhibiting pesticides differed
qualitatively in a battery of behavioral
tests.
c. Statistical Considerations. Properly
designed, studies on the neurotoxic
effects of compounds will include
appropriate statistical tests of
significance. In general, the likelihood
of obtaining a significant effect will
depend jointly on the magnitude of the
effect and the variability obtained in
control and treated groups. A number of
texts are available on standard statistical
tests (e.g.. Siegel, 1956; Winer, 1971;
Sokal and Rohlf, 1969; Salsburg. 1986:
Gad and Weil. 1988).
Neurotoxicity data present some
unique features that must be considered
in selecting statistical tests for analysis.
Data may involve several different
measurement scales, including
categorical (affected or not), rank (more
or less affected), and interval and ratio
scales of measurement (affected by some
percentage). For example, convulsions
are usually recorded as being present or
absent (categorical), whereas
neuropathological changes are
frequently described in terms of the
degree of damage (rank). Many tests of
neurotoxicity involve interval or ratio
measurements (e.g., frequency of
photocell interruptions or amplitude of
an evoked potential), which are the
most powerful and sensitive scales of
measurement. In addition.
measurements are frequently made
repeatedly in control and treated
subjects, especially in the case of
behavioral and neurophysiological end
points. For example, OPPTS guidelines
for FOB assessment call for evaluations
before exposure and at several times
during exposure in a subchronic study
(U.S. EPA. 1991a).
Descriptive data (categorical) and rank
order data can be analyzed using
standard nonparametric techniques
(Siegel. 1956). In some cases, if it is
determined that the data fit the linear
model, the categorical modeling
procedure can be used for weighted
least;squares estimation of parameters
for a wide range of general linear
models, including repeated-measures
analyses. The weighted least-squares
approach to categorical and rank data
allows computation of statistics for
testing the significance of sources of
variation as reflected by the model. In
the case of studies assessing effects in
the same animals at several time points.
univariate analyses can Be carried out at
each time point when the overall dose
effect or the dose-by-time interaction is
significant.
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Federal Register / Vol. 60. No. 192 / Wednesday. October 4. 1995 ' Nouces 52049
Continuous data (e.g.. magnitude,
rate, amplitude), if found to be normally
distributed, can be analyzed with
general linear models using a grouping
factor of dose and. if necessary; repeated
measures across time (Winer, 1971).
Univariate analyses of dose, comparing •
dose groups to the control group at each
time point, are performed when there is
a significant overall dose effect or a
dose-by-time interaction. Post hoc
comparisons between control and
treatment groups can be made following
tests for overall significance. In the case
of multiple end points within a series of
evaluations, some type of correction' for
multiple observations is warranted
(Winer, 1971).
d. In Vitro Data in Neurotoxicology.
Methods and procedures that fall under
the general heading of short-term tests-
include an array of in vitro tests that
have been proposed as alternatives to
whole-animal tests (Goldberg and
Frazier, 1989). In vitro approaches use
animal or human cells, tissues, or
organs and maintain them in a nutritive
medium. Various typea of in vitro
techniques produce data for evaluating
potential and known neurotoxic
substances, including primary cell
cultures, cell Lines, and cloned cells.
While such procedures are important in
studying the mechanism of action of
toxic agents, their use in hazard
identification in human health risk .
• assessment has not been explored to any
great extent
Data from in vitro procedures are
generally based on simplified
approaches that require less time to
yield information than do many in vivo
techniques. However, in vitro methods
generally do not take into account thft
distribution of the toxicant in the body.
die route of administration, or the
metabolism of the substance. It also is
difficult to extrapolate in vitro data to
animal or human neurotoxicity end
points, which include behavioral
changes, motor disorders, sensory and
perceptual disorders, lack of
coordination, and learning deficits. In
addition, data from in vitro tests cannot
duplicate the complex neuronal
circuitry characteristic of the intact
animal.
Many in vitro systems are now being
evaluated for their ability to predict the
neurotoxicity of various agents seen, in
intact animals. This validation process
requires'considerations in study design.
including defined end points of toxicity
and an understanding of how a test
agent would bo 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 thVreliabiiity';pf the in
vivo results. -""S.y/ /-'^r*"'
B. -Dose-Response Evaluation
Dose-response evaluation is a critical
part of hazard characterization and
involves the description of the dose
response relationship in the available
data. Human, studies covering a range of
exposures are rarely available and
therefore animal data are typically used:
for estimating exposure levels likely to
produce adverse effects in humans.
Evidence for a dose-response
relationship is an important criterion in
establishing a neurotoxic effect.
although this analysis may be limited
when based on standard studies using
three dose groups or fewer. The
evaluation of dose-response
relationships includes identifying
effective dose levels as well as doses
associated with no increase in incidence
of adverse effects when compared with
controls. Much of the focus is on
identifying die critical effect(s) observed
at the lowest-observed-adverse-effect-
level and die-no-observed-adverse-
effect-level associated with that effect.
The NOAEL is defined as the highest' '
dose at which there is no statistically or
biologically significant increase in the
frequency of an adverse neurotoxic
effect when compared with the
appropriate control group in a data base-
characterized as having sufficient
evidence for use in a risk assessment
(see section C). Although a threshold is
assumed for neurotoxic effects, the •
existence of a NOAEL in an animal
study does not prove or disprove the1
existence or level of a biological
threshold. Alternatively, mathematical
modeling of the dose-response
relationship may be performed to
determine a quantitative estimate of
responses in the experimental range.
This approach can be used to determine
a BMD, which may be used in place of
the NOAEL (Crump, 1994) (see Dose-
Response Analysis. Section IV).
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 die
impairments less obvious. At levels of
exposure near the NOAEL and LOAEL,
the effects will often be mild, possibly
reversible, and inconsistently found. In
addition, the end points showing / .
responses may be at levels of .f
organization below the whole organism
(e.g., neurochemical or
electrophysiological end points). The •
adversity of such effects can be
contentious (e.g., choiinesterase
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.
C. Characterization of the Health-
Related Data Base
. This section describes a scheme for
characterizing the sufficiency of
evidence for neurotoxic effects. This -
scheme defines two broad'categories:
sufficient and insufficient (Table 8).
Categorization is aimed at providing
certain criteria for the Agency to use to
define the minimum evidence necessary
to define hazards and to conduct 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 to estimate
die risks of those hazards from
anticipated or estimated exposures.
Table 8:—Characterization of the Health-
Rtlattd Deiabue
Sufficient Evidence
.. The sufficient evidence category includes
data that collectively provide enough
information to judge whether or not a human
neurotoxic hazard could exist This category
may include both human and experimental
animal evidence. .
Sufficient Human Evidence
This category includes agents for which
there is sufficient evidence from
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52050 Federal Register / Vol. 60, No. 192 / Wednesday, October 4. 1995 / Notices
studies, s g.. case control and
toaort studies, to :udge that some aeurotoxic
affect is associated with exposure. A case
ienes in conjunction with other supporting
evidence may also be judged "sufficient
evidence." Epideraiologic and clinical case
studies should discuss whether the observed
affects can be considered biologically
plausible in relation to chemical exposure.
.Historically, much often has been made of
the notion of causality in epidemiologic
studies. Causality is a more stringent
criterion than association and has become a
topic of scientific and philosophical debate.
See Susser [1986], for example, for a
discussion of inference in epidemiology.)
Sufficient Experimental Animal Evidence/
Limited or No Human Data
This category includes agents for which
there is sufficient evidence from
experimental animal studies and/or limited
human data to judge whether a potential
ueurotoxic hazard may exist Generally.
agents that have been tested according to
current test guidelines would be included in
this category. The minimum evidence
necessary to Judge that a potential hazard
exists would be data demonstrating an effect
in a single appropriate, well-executed study
In a single experimental animal species.
whereas the minimum evidence needed to
judge that a potential hazard does not exist
would include data from appropriate, well-
executed laboratory •"'"'•I studies that
evaluated a variety of the potential
manifestations of neuroxtoxicity and showed
no effects at.doses that were at least
minimally toxic. Information on
pharmacokinetics. mechanisms, or known
properties of the chemical class may also
strengthen the evidence.,
Insufficient Evidence
This category includes agents for which
there is less than the minimum evidence
sufficient for identifying whether or not a
nourotoxlc hazard exists, such as agents for
which there are no data on neurotoxicity oe
agents with data bases from studies in
animals or humans that are limited by study
design or conduct (e.g., inadequate conduct
or report of clinical signs )TMany general
toxicity studies, for example, are considered
insufficient in terms of the conduct of
clinical neurobehAvioral observations or the
number of samples taken for histopathology
of the nervous system. Thus, a battery of
negative toxicity studies with the**
•shortcomings would be regarded as providing
insufficient evidence of the lack of a
neurotoxic effect of the test matariaL Further.
most screening studies based on simple
observations involving autonomic and motor
function provide insufficient evaluation of
many sensory or cognitive functions. Data,
which by itself would likely fall in this
category, would also include information on
structure-activity relationships or data from
in vitro tests. While such information would
be insufficient by itself to proceed further in
the assessment it could be used to support
the need for additional testing.
Data from all potentially relevant studies.
whether indicative of potential hazard or not
should be included in this characterization.
The primary sources of data are human
studies and case reports, experimental
animal studies, other supporting data, and in
vitro and/or structure-activity relationship
data. Because a complex interrelationship
exists among study design, statistical
analysis, and biological significance of the
data, a great deal of scientific judgment.
based on experience with neurotoxicity data
and with the principles of study design and
statistical analysis, is required to adequately
evaluate the data base on neurotoxicity. In
many cases, interaction with scientists in
specific disciplines either within or outside.
the field of neurotoxicology (e.g.,
epidemiology, statistics) may be appropriate.
The adverse nature of different
neurotoxicity end points may be a complex
judgment In general, most neuropathological
and many neurooehavioral changes are
regarded as adverse. However, there are
adverse behavioral effects that may not
reflect a direct action on the nervous system.
Neurochemical and electrophysiological
changes may be regarded as adverse as a
function of their known or presumed relation
to neuropathological and/or neurooehavioral
consequences. In the absence of supportive
information, a professional judgment must be
made regarding the adversity of such
outcomes, considering factors such as the
nature, magnitude; and duration of the effects
reported. Thus, correlated measures of
neurotoxicity strengthen the evidence fix 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, an the most
common and striking example of this form of
validity. Correlations support a coherent and
logical link between behavioral effects and
biochemical mechanisms. Replication of &
finding also strengthens the evidence for a
hazard. Some neurotoxicants cause similar
effects across most spades. Many chemicals
shows to produce neurotoxicity in laboratory
anim«l« have similar effects in humans.
Some neurologic effects may be considered
adverse even if they are small in magnitude.
reversible, or the result of indirect
Because of the inherent difficulty in
"proving any negative." it is more difficult to
. document a finding of no apparent adverse
effect than a finding of an adverse effect.
Neurotoxic effects (and most kinds of
toxicity) can be observed at many different
levels, so that only a single end point needs
to be found to demonstrate a hazard, but-
many end points need to be examined to
demonstrate no effect For example, to judge
that a hazard for neurotoxicity could exist for
a given agent, the minimum evidence
sufficient would be data on a single adverse
end point from a well-conducted study. In
contrast, to judge that an agent is unlikely to
pose a hazard for neurotoxicity, the
minimum evidence would include data from
a host of end points that revealed no
neurotoxic effects. This may include human
data from appropriate studies that could
support a. conclusion of no evidence of a
neurotoxic effect. With respect to clinical
signs aad symptoms, human exposures can
reveal rat 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, as well as
other factors may affect the strength of the
evidence in these situations. For example,
given that gamma diketones are known to
cause motor system neurotoxicity, a marginal
data.set on a candidate gamrna diketone, e.g.,
Vio animals affected, might be more likely to
be judged sufficient than equivalent data
from a member of a chemical class about
which nothing is known. ;
A judgment that the toxicology data base
is sufficient to indicate a potential neurotoxic
hazard is not the end of analysis. The
circumstances of expression of hazard are *
essential to describing human hazard ,/
potential Thus, reporting should contain tpe
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 rag/kg have been
associated with ataxia and peripheral nerve
damage."
IV. Dose-Response Analysis
This section describes several
approaches (including the LOAEL/
NOAEL and BMD) for determining tha
reference dose or reference
concentration. The NOAEL or BMD/
uncertainty factor approach results in a
RfD or RfC. which is an estimate (with
uncertainty spanning perhaps an order
of magnitude) of a daily exposure to the
human population (including sensitive
subgroups) that is likely to be without
an appreciable risk of deleterious effects
during a lifetime.
The dose-response analysis
characterization should:
a. Describe how the RfD/RfC was
calculated:
b. Discuss the confidence in the
estimates;
c. Describe the assumptions or
uncertainty factors used; and
d. Discuss the route and level of
exposure observed, as compared to
expected human exposures.
(Specifically, are the available data from
the same route of exposure as the
expected human exposures? How many
orders of magnitude do you need to
extrapolate from the observed data to
environmental exposures?)
A. LOAEUNOAEL and Benchmark Dose
(BMD) Determination
As indicated earlier, the LOAEL and
NOAEL are determined for endpoints
that are seen at the lowest dose level
(so-called critical effect). Several
Limitations in the use of the NOAEL
have been identified and described (e.g.,
Barnes and Dourson. 1986; Crump,
1984). For example, the NOAEL is
derived from a single end point from a
single study (the critical study) and
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/ Notices
52051
ignores both the slope of the dose-
response function and baseline
variability in the end point of concern.
Because the baseline variability is not
taken into account, the NOAEL from a
study using small group sizes may be
higher than the NOAEL from a similar-
study in the same species that uses
larger group sizes. The NOAEL is also
directly dependent on the dose spacing
used in the study. Finally, and perhaps
most importantly, use of the NOAEL
does not allow estimates of risk or
extrapolation of risk to lower dose
levels.
Because of these and other limitations
in the NOAEL approach, mathematical
curve-fitting techniques (Crump, 1984:
Gaylor and Slikker, 1990; Glowa. 1991;
U.S. EPA, 1995a) are beginning to be
used with, or as an alternative to, the
NOAEL in calculating the RfD or RfC.
The Agency is in the process of
implementing these newer techniques •
and strongly encourages the calculation
of BMDs for neurotoxicity and other
health effect end points. These-
techniques typically apply a
mathematical function that describes
the dose-response relationship and then
interpolate to a level of exposure
associated with a small increase in
effect over that occurring in the control
group or under baseline conditions. The
BMD has been defined as a lower
confidence limit on the effective dose
associated with some defined level of
effect, e.g., a 5 percent or 10 percent
increase in response (i.e., a BMDos or
BMDio for a particular effect). Because
the model is only used to interpolate
within the dose range of the study, no
assumptions about the existence (or
nonexistence) of a threshold are needed.
Thus, any model that fits the data well
is likely to provide a reasonable
estimate-of the BMD.
Many neurotoxic end points provide
continuous measures of response, such
as response speed, nerve conduction
velocity, IQ score, degree of enzyme
inhibition, or the accuracy of task
performance. Although it is possible to
impose a dichotomy on a continuous
effects distribution and to classify some
level of response as "affected" and the
remainder as "unaffected," it may be
very difficult and inappropriate to
establish such dear distinctions,
' because such a dichotomy would
misrepresent the true nature of the
neurotoxic response. Alternatively.
quantitative models designed to analyze
continuous effect variables may be
preferable. Other techniques that allow
this approach, with transformation of
the information into estimates of the
incidence or frequency of affected
individuals in a population, have been
proposed (Cnup|,, 1984;,'G^,ylor and
Slikker. 1990).'Ga'tegorical*regression
analysis has been;pr6posed since it can
evaluate different types of data and
derive estimates for short-term
exposures (Rees and Hattis, 1994).
Decisions about the most appropriate
approach require professional judgment,
taking into account the biological nature
of the continuous effect variable and its
distribution in the population under
study.
Although dose-response functions in
neurotoxicology are generally linear or
monotonic. curvilinear functions,
especially U-shaped or inverted U-
shaped curves, have been reported as
noted earlier (Section ffl B). 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.
B. Determination of the Reference Dose
or Reference Concentration
Since the availability of dose-response
data in humans is limited, extrapolation
of data from «nimq|§ to humans usually
involves the application of uncertainty
factors to the NOAEL/LOAEL or BMD.
The NOAEL or BMD/uncertainty factor
approach results in a RfD or RfC, which
is an estimate (with uncertainty
spanning perhaps an order of
magnitude) of a daily exposure to the
human population (including sensitive
subgroups) that is likely to be without
an appreciable risk of deleterious effects
during a lifetime. The oral RfD and
inhalation RfC are applicable to chronic
exposure situations and are based on an
evaluation of all the noncancor health
effects, including neurotoxicity data.
RfDs and RfC« in the Integrated Risk
Information System (IRIS-2) data base
for several agents are based on
neurotoxicity end points and include a
few cases in which the RfD or RfC is
calculated using the BMD approach
(e.g., methylmercury, carbon disulfide).
The size of the final uncertainty factor
used will vary from agent to agent and
will require the exercise of scientific
judgment, taking into account
intersperies differences, the shape of the
dose-response curve, and the
neurotoxicity end points observed.
Default uncertainty factors are typically
multiples of 10 and ate used to
compensate for human variability in
sensitivity, the need to extrapolate from
animals to humans, and the need to
extrapolate from less than lifetime (e.g.,
subchronic) to lifetime exposures. An
additional factor of up to 10 may be
included when only a LOAEL (and not
a NOAEL) is available from a study, or
depending on the completeness of the
data base, a modifying factor of up to 10
may be applied, depending on the
confidence one has in the data base. -.
Barnes and Dourson (1988) provide a
more complete description of the
calculation, use. and significance of
RfDs in-setting exposure limits to toxic
agents by the oral route. Jarabek et al.
(1990) provide a more complete
description of the calculation, use, and
significance of RfCs in setting exposure
limits to toxic agents in air.
Neurotoxicity can result from acutefc
shorter term exposures, and it may/be
appropriate in some cases, e.g., for air
pollutants or water contaminants, to set
shorter term exposure limits for
neurotoxicity as well as for other
noncancer health effects.
V. Exposure Assessment
Exposure assessment describes the
magnitude, duration, frequency, and
routes of exposure to the agent of '•
interest. This information may come
from hypothetical values, models, or
actual experimental values, including
ambient environmental sampling
results. Guidelines for exposure
assessment have been published
sepa-ately (U.S. EPA. 1992) and will.
therefore, be discussed only briefly here.
The exposure assessment should
include an exposure characterization '
that:
a. Provides a statement of the
purpose, scope, level of detail, and.
approach used in the exposure
assessment:
b. Presents the estimates of exposure
and dose by pathway and route for
individuals, population segments, and
populations in a manner appropriate for
the intended risk characterization;
c. Provides an evaluation of the
overall level of confidence in the
estimate of exposure and dose and the
conclusions drawn; and
d. Communicates the results of the
exposure assessment to the risk
assessor, who can then use the exposure
characterization, along with the
characterization of the other risk
assessment elements, to develop a risk
characterization.
A number of considerations are
relevant to exposure assessment for
neurotoxicants. An appropriate
evaluation of exposure should consider
the potential for exposure via ingestion,
inhalation, and dermal penetration from
relevant sources of exposures, including
multiple avenues of intake from the
same source. On-going Agency activities
that support neurotoxicity exposure
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Federal Register Vol. 60. Mo. 192 / Wednesday. October 4. 1995 / N'otices
assessment include characterizing
cumulative nsk and revising the
Guidelines for the Health Risk
Assessment of Chemical Mixtures.
In addition, neurotoxic effects may
result from short-term (acute), high-
concentration exposures as well as from
longer term (subchronic), lower level
exposures. Neurotoxic effects may-occur
after a period of time following initial
exposure or be obfuscated by repair
mechanisms or apparent tolerance. The
type and severity of effect may depend
significantly on the pattern of exposure
rather t.h«n 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.
VI. Risk Characterization
A. Overview
Risk characterization, the culmination
of the risk assessment process, consists
of an integrative analysis and a risk
characterization summary. The
integrative analysis (a) involves
integration of the toxicity information
from the hazard characterization and
dose-response analysis with the human
exposure estimates, (b) provides an
evaluation of the overall quality of the
assessment and the degree of confidence
in the estimates of risk and conclusions
drawn, and (c) describes risk in terms of
the nature and extent of harm. The risk
characterization summary
communicates the results of the risk
assessment to the risk manager.
This summary should include but is
not limited to a discussion of the
following elements:
a. Quality of and confidence in the
available data;
b. Uncertainty analysis;
c. Justification of defaults or
assumptions;
d. Related research recommendations:
e. Contentious issues and extent of
scientific consensus;
f. Effect of reasonable alternative
assumptions on conclusions and
estimates:
g. Highlight reasonable plausible
ranges;
h. Reasonable alternative models; and
i. Perspective through analogy.
The risk manager can then use the
risk assessment, along with other risk
management elements, to make public
health decisions.
An effective risk characterization
must fully, openly, and clearly
characterize risks and disclose the
scientific analyses, uncertainties.
assumptions, and science policies that
underlie decisions throughout the risk
assessment and risk management
processes. The risk characterization
must feature values such as
transparency in the decision-making
process; clarity in communicating with
each other and the public regarding
environmental risk and the '
uncertainties associated with
assessments of environmental risk; and
consistency across program offices in
core assumptions and science policies.
which are well grounded in science and
reasonable.
The following sections describe these
four aspects of die risk characterization
in more detail.
B. Integration of Hazard
Characterization, Dose-Response
Analysis and Exposure Assessment
In developing the hazard
characterization, dose-response analysis
and exposure portions of the risk
assessment, the assessor must take into
account many judgments concerning
human relevance of the toxicity data.
including the appropriateness of the
various animal models for which data
are available and the route, timing, and
duration of exposure relative to
expected human exposure. These
judgments should be summarized at
each stage of the risk assessment process
(e.g., the biological relevance of
anatomical variations may be
established in the hazard
characterization process, or the
influence of species differences in
metabolic patterns in the dose-response
analysis). In integrating the information
from the assessment the risk assessor
must determine if some of these
judgments have implications for other
portions of the assessment and whether
the various components of the
assessment are compatible.
The risk characterization should not
only examine the judgments but also
explain the constraints of available data
and the state of knowledge about the
phenomena studied in making them.
including (1} the qualitative conclusions
about the likelihood that the chemical
may pose a specific hazard to human
health, the nature of the observed
effects, under what conditions (route,
dose levels, time, and duration} of
.exposure these effects occur, and
whether the health-related data are
sufficient to use in a risk assessment; (2)
a discussion of the dose-response
characteristics of the critical effects(s),
data such as the shapes and slopes of
the dose-response curves for the various
end points, the rationale behind the
determination of the NOAEL and
LOAEL and calculation of the
benchmark dose, and the assumptions
underlying the estimation of the RfD or
RfC; and (3) the estimates of the
magnitude of human exposure; the
route, duration, and pattern of the
exposure; relevant pharmacokinetics:
and the number and characteristics of
the population(s) exposed.
If data to be used in a risk
characterization are from a route of
exposure other >b«" 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, 4
the Agency makes certain assumptions/
concerning, the amount of absorption y
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 bo stated to the
extent possible, including the
appropriate category regarding
sufficiency of the health-related data. A
comprehensive risk assessment ideally
includes information on a variety of end
points that provide insight into the full
spectrum of potential neurotoxicological
responses. A profile that integrates both
human and test species data and
incorporates a broad range of potential
adverse neurotoxic effects provides
more confidence in a risk assessment for
a given agent
The ability to describe the nature of
the potential human exposure is
important to predict when certain
outcomes can be anticipated and the •
likelihood of permanence or
reversibility of the effect An important
part of this effort is a description of the
nature of the exposed population and
the potential for sensitive, highly
susceptible, or highly exposed
populations. For example, the
consequences of exposure to the
developing individual versus the adult
can differ markedly and can influence
whether the effects are transient or
permanent Other considerations
relative to human exposures might
include the likelihood -of exposures to
other agents, concurrent disease, and
nutritional status.
The presentation of the integrated
results of the assessment should* draw
from and highlight key points of the
individual characterizations of
component analyses performed under
these Guidelines. The overall risk
characterization represents the
integration of these component
characterizations. If relevant risk
assessments on the agent or an
analogous agent have been done by EPA
or other Federal agencies, these should
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be described and the similarities and
differences discussed.
C. Quality of the Data Base and Degree
of Confidence in the Assessment
The risk characterization should
summarize the kinds of data brought
together in the analysis and the
reasoning on. which the assessment is
based. The description should convey
the major strengths and weaknesses of
the assessment that arise from
availability of data and the current
limits of our understanding of the
mechanisms of toxicity.
Health risk is a function of the hazard
characterization, 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 dear picture of consensus or
lack of consensus that exists about -
significant aspects of the assessment.
Whenever more »h»" one view is •
supported by the data and choosing
between them is difficult, all views
should be presented. If one has been
selected over the others, the rationale
should be given; if not. then all should
be presented as plausible alternative
results.
D. Descriptors of Neurotoxicity Risk
There are a number of ways to
. describe risks. Several ways that are
relevant to describing risks for
neurotoxicity are as follows:
1. Estimation of the Number of
Individuals
The RfD or RfC is taken to be a
chronic exposure level at or below
which no significant risk occurs.
Therefore, presentation of th»
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.
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 limit, what would be
the resulting risk for neurotoxicity
above the RfD or RfC?
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 tor exposure' at still higher
levels.
If population data are absent, it will
often be possible to describe a scenario
representingjiigh-end exposures using
upper percentile or judgment-based
values for exposure variables. In these
instances caution should be used not to
compound a substantial number of high-
end values for variables if a
"reasonable** exposure estimate is to be
achieved.
4. Risk Characterization for Highly
Sensitive or Susceptible Individuals
This measure identifies populations
sensitive or susceptible to the effect of
concern. Sensitive or susceptible
individuals are those within the
exposed population at increased risk of
expressing the toxic effect All stages of
nervous system maturation might be
considered highly sensitive or
susceptible, but certain subpopulations
can sometimes be identified because of
critical periods for exposure, for
example, pregnant or lactatmg women,
infants, children.
In general, not enough is understood
about the mechanisms of tbxicity to
identify sensitive subgroups for all
agents, although factors such as
nutrition, personal habits (e.g.. smoking.
alcohol consumption, illicit drug abuse),
or preexisting disease (e.g.. diabetes,
sexually transmitted diseases) may
predispose some individuals to be more
sensitive to the neurotoxic effects of
various agents.
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^s
not available, a LOAEL may be used in •
calculating the MOE Alternative! jTa
benchmark dose may be compared with
the estimated human exposure level to
obtain the MOE. Considerations for the
evaluation of the MOE are similar to
those for the uncertainty factor applied
to the LOAEL/NOAEL or the benchmark
dose. The MOE is presented along with
a discussion of the adequacy of the data
base, including the nature and quality of
the hazard and exposure data, the
number of species affected, and the
dose-response information.
The RfD or RfC comparison with the
human exposure estimate and the
calculation of the MOE are conceptually
similar but are used in different
regulatory situations. The choice of
approach depends on several factors.
including the statute involved, the
situation being addressed, the data base
used, and the needs of the decision
maker. The RfD or RfC and the MOE are
considered along with other risk
assessment and risk management issues
in making risk management decisions.
but the scientific issues that must be
taken into account in establishing them
have been addressed here.
If the MOE is equal to or more than
the uncertainty factor x any modifying
factor used as a basis for an RfD or RfC.
then the need for regulatory concern is
likely to be small. Although these
methods of describing risk do not
actually estimate risks per se, they give
the risk manager some sense of how
close the exposures are to levels of
concern.
E. Communicating Results
Once the risk characterization is
completed, the focus turns to
communicating results to the risk
manager. The risk manager uses the
results of the risk characterization along
with other technological, social, and
economic considerations in reaching a
regulatory decision. Because of the way
in which these risk management factors
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Federal Register < Vol. 60, No. 192 / Wednesday. October 4, 1995 / Notices
may affect different cases, consistent but
not necessarily identical risk
management decisions must be made on
a case-by-case basis. These Guidelines
are not intended to give guidance on the
nonscientific aspects of risk
management decisions.
F. Summary and Research Needs
These Guidelines summarize the-
procadures 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 toxitity, provide insight
into the concept of threshold, develop
approaches for improved mathematical
modeling of neurotoxic effects, improve
animal models for examining the effects
of agents given by various routes of
exposure, and address the synergistic or
antagonistic effects of mixtures of
chemicals and neurotoxic response.
Such research will aid in the evaluation
and interpretation of data on
neurotoxicity and should provide
methods to assess risk more precisely.
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(FR Doc, 95-246S2 Filed 10-3-95; 8:45 ami
BaiMQCOM tMO-tO-*>
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