[Federal Register Volume 63, Number 93 (Thursday, May 14, 1998)]
[Notices]
[Pages 26926-26954]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 98-12303]



[[Page 26925]]

_______________________________________________________________________

Part III





Environmental Protection Agency





_______________________________________________________________________



Guidelines For Neurotoxicity Risk Assessment; Notice

Federal Register / Vol. 63, No. 93 / Thursday, May 14, 1998 / 
Notices

[[Page 26926]]



ENVIRONMENTAL PROTECTION AGENCY

[FRL-6011-3]
RIN 2080-AA08


Guidelines for Neurotoxicity Risk Assessment

AGENCY: Environmental Protection Agency.

ACTION: Notice of availability of final Guidelines for Neurotoxicity 
Risk Assessment.

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

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

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

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

FOR FURTHER INFORMATION CONTACT: Dr. Hugh A. Tilson, Neurotoxicology 
Division, National Health and Environmental Effects Research 
Laboratory, U.S. Environmental Protection Agency, Research Triangle 
Park, NC 27711, Tel: 919-541-2671; Fax: 919-541-4849; E-mail: 
<a href="/cdn-cgi/l/email-protection#f4809d98879b9ada9c81939cb491849599959d98da918495da939b82"><span class="__cf_email__" data-cfemail="f7839e9b849899d99f82909fb79287969a969e9bd9928796d9909881">[email protected]</span></a>.

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

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

Contents

Part A: Guidelines for Neurotoxicity Risk Assessment

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

[[Page 26927]]

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

Part B: Response to Science Advisory Board and Public Comments

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

List of Tables

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

Part A: Guidelines for Neurotoxicity Risk Assessment

1. Introduction

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

[[Page 26928]]

more quantitative dose-response evaluation when sufficient data are 
available. The benchmark dose approach takes into account the 
variability in the data and the slope of the dose-response curve, and 
provides a more consistent basis for calculation of the RfD or RfC. If 
data are considered sufficient for risk assessment, and if 
neurotoxicity is the effect occurring at the lowest dose level (i.e., 
the critical effect), an oral or dermal RfD or an inhalation RfC, based 
on neurotoxic effects, is then derived. This RfD or RfC is derived 
using the NOAEL or benchmark dose divided by uncertainty factors to 
account for interspecies differences in response, intraspecies 
variability, and other factors of study design or the database. A 
statement of the potential for human risk and the consequences of 
exposure can come only from integrating the hazard characterization and 
dose-response analysis with the human exposure estimates in the final 
risk characterization.
    The section on exposure assessment (section 5) identifies human 
populations exposed or potentially exposed to an agent, describes their 
composition and size, and presents the types, magnitudes, frequencies, 
and durations of exposure to the agent. The exposure assessment 
provides an estimate of human exposure levels for particular 
populations from all potential sources.
    In risk characterization (section 6), the hazard characterization, 
dose-response analysis, and exposure assessment for given populations 
are combined to estimate some measure of the risk for neurotoxicity. As 
part of risk characterization, a summary of the strengths and 
weaknesses of each component of the risk assessment is given, along 
with major assumptions, scientific judgments and, to the extent 
possible, qualitative and quantitative estimates of the uncertainties. 
This characterization of the health-related database is always 
presented in conjunction with information on the dose, route, duration, 
and timing of exposure as well as the dose-response analysis including 
the RfD or RfC. If human exposure estimates are available, the exposure 
basis used for the risk assessment is clearly described, e.g., highly 
exposed individuals or highly sensitive or susceptible individuals. The 
NOAEL may be compared to the various estimates of human exposure to 
calculate the margin(s) of exposure (MOE). The considerations for 
judging the acceptability of the MOE are similar to those for 
determining the appropriate size of the uncertainty factor for 
calculating the RfD or RfC.
    The Agency recently issued a policy statement and associated 
guidance for risk characterization (U.S. EPA, 1995b, 1995c), which is 
currently being implemented throughout EPA. This statement is designed 
to ensure that critical information from each stage of a risk 
assessment is used in forming conclusions about risk and that this 
information is communicated from risk assessors to risk managers 
(policy makers), from middle to upper management, and from the Agency 
to the public. Additionally, the policy provides a basis for greater 
clarity, transparency, reasonableness, and consistency in risk 
assessments across Agency programs.
    Final neurotoxicity risk assessment guidelines may reflect 
additional changes in risk characterization practices resulting from 
implementation activities. Risk assessment is just one component of the 
regulatory process and defines the potential adverse health 
consequences of exposure to a toxic agent. The other component, risk 
management, combines risk assessment with statutory directives 
regarding socioeconomic, technical, political, and other considerations 
in order to decide whether to control future exposure to the suspected 
toxic agent and, if so, the nature and level of control. One major 
objective of these Guidelines is to help the risk assessor determine 
whether the experimental animal or human data indicate the potential 
for a neurotoxic effect. Such information can then be used to 
categorize evidence that will identify and characterize neurotoxic 
hazards, as described in section 3.3, Characterization of the Health-
Related Database, and Table 8 of these Guidelines. Risk management is 
not dealt with directly in these Guidelines because the basis for 
decision making goes beyond scientific considerations alone, but the 
use of scientific information in this process is discussed. For 
example, the acceptability of the MOE is a risk management decision, 
but the scientific bases for establishing this value are discussed 
here.
1.2. The Role of Environmental Agents in Neurotoxicity
    Chemicals are an integral part of life, with the capacity to 
improve as well as endanger health. The general population is exposed 
to chemicals in air, water, foods, cosmetics, household products, and 
drugs used therapeutically or illicitly. During daily life, a person 
experiences a multitude of exposures to potentially neuroactive 
substances, singly and in combination, both synthetic and natural. 
Levels of exposure vary and may or may not pose a hazard, depending on 
dose, route, and duration of exposure.
    A link between human exposure to some chemical substances and 
neurotoxicity has been firmly established (Anger, 1986; OTA, 1990). 
Because many natural and synthetic chemicals are present in today's 
environment, there is growing scientific and regulatory interest in the 
potential for risks to humans from exposure to neurotoxic agents. If 
sufficient exposure occurs, the effects resulting from such exposures 
can have a significant adverse impact on human health. It is not known 
how many chemicals may be neurotoxic in humans (Reiter, 1987). EPA's 
TSCA inventory of chemical substances manufactured, imported, or 
processed in the United States includes more than 65,000 substances and 
is increasing yearly. An overwhelming majority of the materials in 
commercial use have not been tested for neurotoxic potential (NRC, 
1984).
    Estimates of the number of chemicals with neurotoxic properties 
have been made for subsets of substances. For instance, a large 
percentage of the more than 500 registered active pesticide ingredients 
affect the nervous system of the target species to varying degrees. Of 
588 chemicals listed by the American Conference of Governmental 
Industrial Hygienists, 167 affected the nervous system or behavior at 
some exposure level (Anger, 1984). Anger (1990) estimated that of the 
approximately 200 chemicals to which 1 million or more American workers 
are exposed, more than one-third may have adverse effects on the 
nervous system if sufficient exposure occurs. Anger (1984) also 
recognized neurotoxic effects as one of the 10 leading workplace 
disorders. A number of therapeutic substances, including some 
anticancer and antiviral agents and abused drugs, can cause adverse or 
neurotoxicological side effects at therapeutic levels (OTA, 1990). The 
number of chemicals with neurotoxic potential has been estimated to 
range from 3% to 28% of all chemicals (OTA, 1990). Thus, estimating the 
risks of exposure to chemicals with neurotoxic potential is of concern 
with regard to their overall impact on human health.
1.3. Neurotoxicity Risk Assessment
    In addition to its primary role in psychological functions, the 
nervous system controls most, if not all, other bodily processes. It is 
sensitive to perturbation from various sources and has limited ability 
to regenerate. There is evidence that even small anatomical, 
biochemical, or physiological insults to the nervous system may result 
in

[[Page 26929]]

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

2. Definitions and Critical Concepts

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

[[Page 26930]]

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

[[Page 26931]]

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

3. Hazard Characterization

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

[[Page 26932]]

Peripheral neuropathies (with sensory and motor disturbances), 
encephalopathies, organic brain syndromes, extrapyramidal syndromes, 
demyelination, autonomic changes, and dementia are well-characterized 
consequences of acute and chronic exposure to chemical agents. The 
range of exposure conditions that produce clinical signs of 
neurotoxicity also has been defined by these clinical methods. It is 
very important to make external/internal dose measurements in humans to 
determine the actual dose(s) that can cause unwanted effects.
    Aspects of the neurological examination approach limit its 
usefulness for neurotoxicological risk assessment. Information obtained 
from the neurological exam is mostly qualitative and descriptive rather 
than quantitative. Estimates of the severity of functional impairment 
can be reliably placed into only three or four categories (for example, 
mild, moderate, severe). Much of the assessment depends on the 
subjective judgment of the examiner. For example, the magnitude and 
symmetry of muscle strength are often judged by having the patient push 
against the resistance of the examiner's hands. The endpoints are 
therefore the absolute and relative amount of muscle load sensed by the 
examiner in his or her arms.
    Compared with other methods, the neurological exam may be less 
sensitive in detecting early neurotoxicity in peripheral sensory and 
motor nerves. While clinicians' judgments are equal in sensitivity to 
quantitative methods in assessing the amplitude of tremor, tremor 
frequency is poorly quantified by clinicians. Thus, important aspects 
of the clinical neurologic exam may be insufficiently quantified and 
lack sufficient sensitivity for detecting early neurobehavioral 
toxicity produced by environmental or workplace exposure conditions. 
However, a neurological evaluation of persons with documented 
neurobehavioral impairment would be helpful for identifying nonchemical 
causes of neurotoxicity, such as diabetes and cardiovascular 
insufficiency.
    Administration of a neuropsychological battery also requires a 
trained technician, and interpretation requires a trained and 
experienced neuropsychologist. Depending on the capabilities of the 
patient, 2 to 4 hours may be needed to administer a full battery; 1 
hour may be needed for the shorter screening versions. These practical 
considerations may limit the usefulness of neuropsychological 
assessment in large field studies of suspected neurotoxicity.
    In addition to logistical problems in administration and 
interpretation, neuropsychological batteries and neurological exams 
share two disadvantages with respect to neurotoxicity risk assessment. 
First, neurological exams and neuropsychological test batteries are 
designed to confirm and classify functional problems in individuals 
selected on the basis of signs and symptoms identified by the patient, 
family, or other health professionals. Their usefulness in detecting 
low base-rate impairment in workers or the general population is 
generally thought to be limited, decreasing the usefulness of clinical 
assessment approaches for epidemiologic risk assessment.
    Second, neurological exams and neuropsychological test batteries 
were developed to assess the functional correlates of the most common 
forms of nervous system dysfunction: brain trauma, focal lesions, and 
degenerative conditions. The clinical tests were validated against 
these neurological disease states. With a few notable exceptions, 
chemicals are not believed to produce impairment similar to that from 
trauma or lesions; neurotoxic effects are more similar to the effects 
of degenerative disease. There has been insufficient research to 
demonstrate which tests designed to assess functional expression of 
neurologic disease are useful in characterizing the modes of central 
nervous system impairment produced by chemical agents and drugs.
    It should be noted that alternative approaches are available that 
avoid many of the limitations of clinical and neurological and 
traditional neuropsychological methods. Computerized behavioral 
assessment systems designed for field testing of populations exposed to 
chemicals in the community or workplace have been developed during the 
past decade. The most widely used system is the Neurobehavioral 
Evaluation System (NES) developed by Baker et al. (1985). Advantages of 
computerized tests include (1) standardized administration to eliminate 
intertester variability and minimize subject-experimenter interaction; 
(2) automated data collection and scoring, which is faster, easier, and 
less error-prone than traditional methods; and (3) test administration 
requires minimal training and experience. NES tests have proven 
sensitive to a variety of solvents, metals, and pesticides (Otto, 
1992). Computerized systems available for human neurotoxicity testing 
are critically reviewed in Anger et al. (1996).
3.1.1.2. Case Reports
    The first type of human data available is often the case report or 
case series, which can identify cases of a disease and are reported by 
clinicians or discerned through active or passive surveillance, usually 
in the workplace. However, case reports involving a single neurotoxic 
agent, although informative, are rare in the literature; for example, 
farmers are likely to be exposed to a wide variety of potentially 
neurotoxic pesticides. Careful case histories assist in identifying 
common risk factors, especially when the association between the 
exposure and disease is strong, the mode of action of the agent is 
biologically plausible, and clusters occur in a limited period of time.
    Case reports can be obtained more quickly than more complex 
studies. Case reports of acute high-level exposure to a toxicant can be 
useful for identifying signs and symptoms that may also apply to lower 
exposure. Case reports can also be useful when corroborating 
epidemiological data are available.
3.1.1.3. Epidemiologic Studies
    Epidemiology has been defined as ``the study of the distributions 
and determinants of disease and injuries in human populations'' 
(Mausner and Kramer, 1985). Knowing the frequency of illness in groups 
and the factors that influence the distribution is the tool of 
epidemiology that allows the evaluation of causal inference with the 
goal of prevention and cure of disease (Friedlander and Hearn, 1980). 
Epidemiologic studies are a useful means of evaluating the effects of 
neurotoxic substances on human populations, particularly if effects of 
exposure are cumulative or exposures are repeated. Such studies are 
less useful in cases of acute exposure, where the effects are short-
term. Frequently, determining the precise dose or exposure 
concentration in epidemiological studies can be difficult.
3.1.1.3.1. Cross-Sectional Studies.
    In cross-sectional studies or surveys, both the disease and 
suspected risk factors are ascertained at the same time, and the 
findings are useful in generating hypotheses. A group of people are 
interviewed, examined, and tested at a single point in time to 
ascertain a relationship between a disease and a neurotoxic exposure. 
This study design does not allow the investigator to determine whether 
the disease or the exposure came first, rendering it less useful in 
estimating risk. These studies are intermediate in cost and time

[[Page 26933]]

required to complete compared with case reports and more complex 
analytical studies, but should be augmented with additional data.
3.1.1.3.2. Case-Control (Retrospective) Studies.
    Last (1986) defines a case-control study as one that ``starts with 
the identification of persons with the disease (or other outcome 
variable) of interest, and a suitable control population (comparison, 
reference group) of persons without the disease.'' He states that the 
relationship of an ``attribute'' to the disease is measured by 
comparing the diseased with the nondiseased with regard to how 
frequently the attribute is present in each of the groups. The cases 
are assembled from a population of persons with and without exposure, 
and the comparison group is selected from the same population; the 
relative distribution of the potential risk factor (exposure) in both 
groups is evaluated by computing an odds ratio that serves as an 
estimate of the strength of the association between the disease and the 
potential risk factor. The statistical significance of the ratio is 
determined by calculating a p-value and is used to approximate relative 
risk.
    The case-control approach to the study of potential neurotoxicants 
in the environment provides a great deal of useful information for the 
risk assessor. In his textbook, Valciukas (1991) notes that the case-
control approach is the strategy of choice when no other environmental 
or biological indicator of neurotoxic exposure is available. He further 
states: ``Considering the fact that for the vast majority of neurotoxic 
chemical compounds, no objective biological indicators of exposure are 
available (or if they are, their half-life is too short to be of any 
practical value), the case-control paradigm is a widely accepted 
strategy for the assessment of toxic causation.'' The case-control 
study design, however, can be very susceptible to bias. The potential 
sources of bias are numerous and can be specific to a particular study. 
Many of these biases also can be present in cross-sectional studies. 
For example, recall bias or faulty recall of information by study 
subjects in a questionnaire-based study can distort the results. 
Analysis of the case-comparison study design assumes that the selected 
cases are representative persons with the disease--either all cases 
with the disease or a representative sample of them have been 
ascertained. It further assumes that the control or comparison group is 
representative of the nonexposed population (or that the prevalence of 
the characteristic under study is the same in the control group as in 
the general population). Failure to satisfy these assumptions may 
result in selection bias that may invalidate study results.
    An additional source of bias in case-control studies is the 
presence of confounding variables, i.e., factors known to be associated 
with the exposure and causally related to the disease under study. 
These should be controlled, either in the design of the study by 
matching cases to controls on the basis of the confounding factor, or 
in the analysis of the data by using statistical techniques such as 
stratification or regression. Matching requires time to identify an 
adequate number of potential controls to distinguish those with the 
proper characteristics, while statistical control of confounding 
factors requires a larger study.
    The definition of exposure is critical in epidemiologic studies. In 
occupational settings, exposure assessment often is based on the job 
assignment of the study subjects, but can be more precise if detailed 
company records allow the development of exposure profiles. Positive 
results from a properly controlled retrospective study should weigh 
heavily in the risk assessment process.
3.1.1.3.3. Cohort (Prospective, Follow-Up) Studies.
    In a prospective study design, a healthy group of people is 
assembled and followed forward in time and observed for the development 
of dysfunction. Such studies are invaluable for determining the time 
course for development of dysfunction (e.g., follow-up studies 
performed in various cities on the effects of lead on child 
development). This approach allows the direct estimate of risks 
attributed to a particular exposure, since toxic incidence rates in the 
cohort can be determined. Prospective study designs also allow the 
study of chronic effects of exposure. One major strength of the cohort 
design is that it allows the calculation of rates to determine the 
excess risk associated with an exposure. Also, biases are reduced by 
obtaining information before the disease develops. This approach, 
however, can be very time-consuming and costly.
    In cohort studies information bias can be introduced when 
individuals provide distorted information about their health because 
they know their exposure status and may have been told of the expected 
health effects of the exposure under study. More credence should be 
given to those studies in which both observer and subject bias are 
carefully controlled (e.g., double-blind studies).
    A special type of cohort study is the retrospective cohort study, 
in which the investigator goes back in time to select the study groups 
and traces them over time, often to the present. The studies usually 
involve specially exposed groups and have provided much assistance in 
estimating risks due to occupational exposures. Occupational 
retrospective cohort studies rely on company records of past and 
current employees that include information on the dates of employment, 
age at employment, date of departure, and whether diseased (or dead in 
the case of mortality studies). Workers can then be classified by 
duration and degree of exposure. Positive or negative results from a 
properly controlled prospective study should weigh heavily in the risk 
assessment process.
3.1.1.4. Human Laboratory Exposure Studies
    Neurotoxicity assessment has an advantage not afforded to the 
evaluation of other toxic endpoints, such as cancer or reproductive 
toxicity, in that the effects of some chemicals are short in duration 
and reversible. This makes it ethically possible to perform human 
laboratory exposure studies and obtain data relevant to the risk 
assessment process. Information from experimental human exposure 
studies has been used to set occupational exposure limits, mostly for 
organic solvents that can be inhaled. Laboratory exposure studies have 
contributed to risk assessment and the setting of exposure limits for 
several solvents and other chemicals with acute reversible effects.
    Human exposure studies sometimes offer advantages over 
epidemiologic field studies. Combined with appropriate sampling of 
biological fluids (urine or blood), it is possible to calculate body 
concentrations, examine toxicokinetics, and identify metabolites. 
Bioavailability, elimination, dose-related changes in metabolic 
pathways, individual variability, time course of effects, interactions 
between chemicals, and interactions between chemical and environmental/
biobehavioral processes (stressors, workload/respiratory rate) are 
factors that are generally easier to collect under controlled 
conditions.
    Other goals of laboratory studies include the in-depth 
characterization of effects, the development of new assessment methods, 
and the examination of the sensitivity, specificity, and reliability of 
neurobehavioral assessment methods across chemical classes. The 
laboratory is the most appropriate setting for the

[[Page 26934]]

study of environmental and biobehavioral variables that affect the 
action of chemical agents. The effects of ambient temperature, task 
difficulty, rate of ongoing behavior, conditioning variables, 
tolerance/sensitization, sleep deprivation, motivation, and so forth 
are sometimes studied.
    From a methodological standpoint, human laboratory studies can be 
divided into two categories: between-subjects and within-subjects 
designs. In the former, the neurobehavioral performance of exposed 
volunteers is compared with that of nonexposed participants. In the 
latter, preexposure performance is compared with neurobehavioral 
function under the influence of the chemical or drug. Within-subjects 
designs have the advantage of requiring fewer participants, eliminating 
individual differences as a source of variability, and controlling for 
chronic mediating variables, such as caffeine use and educational 
achievement. A disadvantage of the within-subjects design is that 
neurobehavioral tests must be administered more than once. Practice on 
many neurobehavioral tests often leads to improved performance that may 
confound the effect of the chemical/drug. There should be a sufficient 
number of test sessions in the pre-exposure phase to allow performance 
on all tests to achieve a relatively stable baseline level.
    Participants in laboratory exposure studies may have been recruited 
from populations of persons already exposed to the chemical/drug or 
from chemical-naive populations. Although the use of exposed volunteers 
has ethical advantages, can mitigate against novelty effects, and 
allows evaluation of tolerance/sensitization, finding an accessible 
exposed population in reasonable proximity to the laboratory can be 
difficult. Chemical-naive participants are more easily recruited but 
may differ significantly in important characteristics from a 
representative sample of exposed persons. Chemical-naive volunteers are 
often younger, healthier, and better educated than the populations 
exposed environmentally, in the workplace, or pharmacotherapeutically.
    Compared with workplace and environmental exposures, laboratory 
exposure conditions can be controlled more precisely, but exposure 
periods are much shorter. Generally only one or two relatively pure 
chemicals are studied for several hours, whereas the population of 
interest may be exposed to multiple chemicals containing impurities for 
months or years. Laboratory studies are therefore better at identifying 
and characterizing effects with acute onset and the selective effects 
of pure agents. In all cases, the potential for participant bias should 
be as carefully controlled for as possible. Even the consent form can 
lead to participant bias, as toxic effects have been reported in some 
individuals who were warned of such effects in an informed consent 
form. In addition, double-blind studies have been shown to provide some 
control for observer bias that may occur in single-blind studies. More 
credence should be given to those studies in which both observer and 
subject bias are carefully controlled (Benignus, 1993).
    A test battery that examines multiple neurobehavioral functions may 
be more useful for screening and the initial characterization of acute 
effects. Selected neurobehavioral tests that measure a limited number 
of functions in multiple ways may be more useful for elucidating 
mechanisms or validating specific effects.
    Both chemical and behavioral control procedures are valuable for 
examining the specificity of the effects. A concordant effect among 
different measures of the same neurobehavioral function (e.g., reaction 
time) and a lack of effect on some other measures of psychomotor 
function (e.g., untimed manual dexterity) would increase the confidence 
in a selective effect on motor speed and not on attention or another 
nonspecific motor function. Likewise, finding concordant effects among 
similar chemical or drug classes along with different effects from 
dissimilar classes would support the specificity of chemical effect. 
For example, finding that the effects of a solvent were similar to 
those of ethanol but not caffeine would support the specificity of 
solvent effects on a given measure of neurotoxicity.
3.1.2. Animal Studies
    This section provides an overview of the major types of endpoints 
that may be evaluated in animal neurotoxicity studies, describes the 
kinds of effects that may be observed and some of the tests used to 
detect and quantify these effects, and provides guidance for 
interpreting data. Compared with human studies, animal studies are more 
often available for specific chemicals, provide more precise exposure 
information, and control environmental factors better (Anger, 1984). 
For these reasons, risk assessments tend to rely heavily on animal 
studies.
    Many tests that can measure some aspect of neurotoxicity have been 
used in the field of neurobiology in the past 50 years. The Office of 
Prevention, Pesticides and Toxic Substances (OPPTS) has published 
animal testing guidelines that were developed in cooperation with the 
Office of Research and Development (U.S. EPA, 1991a). While the test 
endpoints included in the 1991 document serve as a convenient focus for 
this section, there are many other endpoints for which there are no 
current EPA guidelines. The goal of the current document is to provide 
a framework for interpreting data collected in tests frequently used by 
neurotoxicologists.
    Five categories of endpoints will be described: structural or 
neuropathological, neurophysiological, neurochemical, behavioral, and 
developmental. Table 1 lists a number of endpoints in each of these 
categories. It is imperative for the risk assessor to understand that 
the interpretation of the indicators listed in Table 1 as neurotoxic 
effects is dependent on the dose at which such changes occur and the 
possibility that damage to other organ systems may contribute to or 
cause such changes indirectly.

    Table 1.--Examples of Possible Indicators of a Neurotoxic Effect    
------------------------------------------------------------------------
                                                                        
-------------------------------------------------------------------------
Structural or neuropathological endpoints:                              
    Gross changes in morphology, including brain weight.                
    Histologic changes in neurons or glia (neuronopathy, axonopathy,    
     myelinopathy).                                                     
Neurochemical endpoints:                                                
    Alterations in synthesis, release, uptake, degradation of           
     neurotransmitters.                                                 
    Alterations in second-messenger-associated signal transduction.     
    Alterations in membrane-bound enzymes regulating neuronal activity. 
    Inhibition and aging of neuropathy enzyme.                          
    Increases in glial fibrillary acidic protein in adults.             
Neurophysiological endpoints:                                           
    Change in velocity, amplitude, or refractory period of nerve        
     conduction.                                                        

[[Page 26935]]

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

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

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

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

[[Page 26936]]

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

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

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

[[Page 26937]]

refractory periods in a well-controlled study indicate a neurotoxic 
effect.
    In summary, alterations in peripheral nerve response amplitude and 
refractory period in studies that are well controlled for temperature 
are indicative of a neurotoxic effect. Alterations in peripheral nerve 
function are frequently associated with clinical signs such as 
numbness, tingling, or burning sensations or with motor impairments 
such as weakness. Examples of compounds that alter peripheral nerve 
function in humans or experimental animals include acrylamide, carbon 
disulfide, n-hexane, lead, and some organophosphates.
    3.1.2.2.2. Sensory, Motor, and Other Evoked Potentials. Evoked 
potential studies are electrophysiological procedures that measure the 
response elicited from a defined stimulus such as a tone, a light, or a 
brief electrical pulse. Evoked potentials reflect the function of the 
system under study, including visual, auditory, or somatosensory; 
motor, involving motor nerves and innervated muscles; or other neural 
pathways in the central or peripheral nervous system (Rebert, 1983; 
Dyer, 1985; Mattsson and Albee, 1988; Mattsson et al., 1992; Boyes, 
1992, 1993). Evoked potential studies should be interpreted with 
respect to the known or presumed neural generators of the responses, 
and their likely relationships with behavioral outcomes, when such 
information is available. Such correlative information strengthens the 
confidence in electrophysiological outcomes. In the absence of such 
supportive information, the extent to which evoked potential studies 
provide convincing evidence of neurotoxicity is a matter of 
professional judgment on a case-by-case basis. Judgments should 
consider the nature, magnitude, and duration of such effects, along 
with other factors discussed elsewhere in this document.
    Data are in the form of a voltage record collected over time and 
can be quantified in several ways. Commonly, the latency (time from 
stimulus onset) and amplitude (voltage) of the positive and negative 
voltage peaks are identified and measured. Alternative measurement 
schemes may involve substitution of spectral phase or template shifts 
for peak latency and spectral power, spectral amplitude, root-mean-
square, or integrated area under the curve for peak amplitude. Latency 
measurements are dependent on both the velocity of nerve conduction and 
the time of synaptic transmission. Both of these factors depend on 
temperature, as discussed in regard to nerve conduction, and similar 
caveats apply for sensory evoked potential studies. In studies that are 
well controlled for temperature, increases in latencies or related 
measures can reflect deficits in nerve conduction, including 
demyelination or delayed synaptic transmission, and are indicators of a 
neurotoxic effect.
    Decreases in peak latencies, like increases in nerve conduction 
velocity, are unusual, but the neural systems under study in sensory 
evoked potentials are complex, and situations that might cause a peak 
measurement to occur earlier are conceivable. Two such situations are a 
reduced threshold for spatial or temporal summation of afferent neural 
transmission and a selective loss of cells responding late in the peak, 
thus making the measured peak occur earlier. Decreases in peak latency 
should not be dismissed outright as experimental or statistical error, 
but should be examined carefully and perhaps replicated to assess 
possible neurotoxicity. A decrease in latency is not conclusive 
evidence of a neurotoxic effect.
    Changes in peak amplitudes or equivalent measures reflect changes 
in the magnitude of the neural population responsive to stimulation. 
Both increases and decreases in amplitude are possible following 
exposure to chemicals. Whether excitatory or inhibitory neural activity 
is translated into a positive or negative deflection in the sensory 
evoked potential is dependent on the physical orientation of the 
electrode with respect to the tissue generating the response, which is 
frequently unknown. Comparisons should be based on the absolute change 
in amplitude. Therefore, either increases or decreases in amplitude may 
be indicative of a neurotoxic effect.
    Within any given sensory system, the neural circuits that generate 
various evoked potential peaks differ as a function of peak latency. In 
general, early latency peaks reflect the transmission of afferent 
sensory information. Changes in either the latency or amplitude of 
these peaks are considered convincing evidence of a neurotoxic effect 
that is likely to be reflected in deficits in sensory perception. The 
later-latency peaks, in general, reflect not only the sensory input but 
also the more nonspecific factors such as the behavioral state of the 
subject, including such factors as arousal level, habituation, or 
sensitization (Dyer, 1987). Thus, changes in later-latency evoked 
potential peaks should be interpreted in light of the behavioral status 
of the subject and would generally be considered evidence of a 
neurotoxic effect.
    3.1.2.2.3. Seizures/Convulsions. Some neurotoxicants (e.g., 
lindane, pyrethroids, trimethyltin, dichlorodiphenyltrichloroethane 
[DDT]) produce observable convulsions. When convulsionlike behaviors 
are observed, as described in the behavioral section on convulsions, 
neurophysiological recordings can provide additional information to 
help interpret the results. Recordings of brain electrical activity 
that demonstrate seizurelike activity are indicative of a neurotoxic 
effect.
    In addition to producing seizures directly, chemicals may also 
alter the frequency, severity, duration, or threshold for eliciting 
seizures through other means by a phenomenon known as ``kindling.'' 
Such alterations can occur after acute exposure or after repeated 
exposure to dose levels below the acute threshold. In experiments 
demonstrating changes in sensitivity following repeated exposures to 
the test compound, information regarding possible changes in the 
pharmacokinetic distribution of the compound is required before the 
seizure susceptibility changes can be interpreted as evidence of 
neurotoxicity. Increases in susceptibility to seizures are considered 
adverse.
    3.1.2.2.4. Electroencephalography (EEG). EEG analysis is used 
widely in clinical settings for the diagnosis of neurological 
disorders, and less often for the detection of subtle toxicant-induced 
dysfunction (WHO, 1986; Eccles, 1988). The basis for using EEG in 
either setting is the relationship between specific patterns of EEG 
waveforms and specific behavioral states. Because states of alertness 
and stages of sleep are associated with distinct patterns of electrical 
activity in the brain, it is generally thought that arousal level can 
be evaluated by monitoring the EEG. Dissociation of EEG activity and 
behavior can, however, occur after exposure to certain chemicals. 
Normal patterns of transition between sleep stages or between sleeping 
and waking states are known to remain disturbed for prolonged periods 
of time after exposure to some chemicals. Changes in the pattern of the 
EEG can be elicited by anesthetic drugs and stimuli producing arousal 
(e.g., lights, sounds). In studies with toxicants, changes in EEG 
pattern can sometimes precede alterations in other objective signs of 
neurotoxicity (Dyer, 1987).
    EEG studies should be done under highly controlled conditions, and 
the data should be considered on a case-by-case basis. Chemically 
induced seizure activity detected in the EEG pattern is evidence of a 
neurotoxic effect.

[[Page 26938]]

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

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

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

[[Page 26939]]

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

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


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

[[Page 26940]]

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

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

[[Page 26941]]

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

[[Page 26942]]

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

  Table 7.--Examples of Compounds or Treatments Producing Developmental 
                              Neurotoxicity                             
------------------------------------------------------------------------
                                                                        
------------------------------------------------------------------------
Alcohols..................................  Methanol, ethanol.          
Antimitotics..............................  X-radiation, azacytidine.   
Insecticides..............................  DDT, chlordecone.           
Metals....................................  Lead, methylmercury,        
                                             cadmium.                   
Polyhalogenated hydrocarbons..............  PCBs, PBBs.                 
------------------------------------------------------------------------

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

[[Page 26943]]

evaluating neurological function and ``unmasking'' effects not 
otherwise detectable. For example, a challenge with a psychomotor 
stimulant such as d-amphetamine may unmask latent developmental 
neurotoxicity (Hughes and Sparber, 1978; Adams and Buelke-Sam, 1981; 
Buelke-Sam et al., 1985).
    Direct extrapolation of developmental neurotoxicity to humans is 
limited in the same way as for other endpoints of toxicity, i.e., by 
the lack of knowledge about underlying toxicological mechanisms and 
their significance (U.S. EPA, 1991b). However, comparisons of human and 
animal data for several agents known to cause developmental 
neurotoxicity in humans showed many similarities in effects (Kimmel et 
al., 1990). As evidenced primarily by observations in laboratory 
animals, comparisons at the level of functional category (sensory, 
motivational, cognitive, motor function, and social behavior) showed 
close agreement across species for the agents evaluated, even though 
the specific endpoints used to assess these functions varied 
considerably across species (Stanton and Spear, 1990). Thus, it can be 
assumed that developmental neurotoxicity effects in animal studies 
indicate the potential for altered neurobehavioral development in 
humans, although the specific types of developmental effects seen in 
experimental animal studies will not be the same as those that may be 
produced in humans. Therefore, when data suggesting adverse effects in 
developmental neurotoxicity studies are encountered for particular 
agents, they should be considered in the risk assessment process.
    Functional tests with a moderate degree of background variability 
(e.g., a coefficient of variability of 20% or less) may be more 
sensitive to the effects of an agent on behavioral endpoints than are 
tests with low variability that may be impossible to disrupt without 
using life-threatening doses. A battery of functional tests, in 
contrast to a single test, is usually needed to evaluate the full 
complement of nervous system functions in an animal. Likewise, a series 
of tests conducted in animals in several age groups may provide more 
information about maturational changes and their persistence than tests 
conducted at a single age.
    It is a well-established principle that there are critical 
developmental periods for the disruption of functional competence, 
which include both the prenatal and postnatal periods to the time of 
sexual maturation, and the effect of a toxicant is likely to vary 
depending on the time and degree of exposure (Rodier, 1978, 1990). It 
is also important to consider the data from studies in which postnatal 
exposure is included, as there may be an interaction of the agent with 
maternal behavior, milk composition, or pup suckling behavior, as well 
as possible direct exposure of pups via dosed food or water (Kimmel et 
al., 1992).
    Agents that produce developmental neurotoxicity at a dose that is 
not toxic to the maternal animal are of special concern. However, 
adverse developmental effects are often produced at doses that cause 
mild maternal toxicity (e.g., 10%-20% reduction in weight gain during 
gestation and lactation). At doses causing moderate maternal toxicity 
(i.e., 20% or more reduction in weight gain during gestation and 
lactation), interpretation of developmental effects may be confounded. 
Current information is inadequate to assume that developmental effects 
at doses causing minimal maternal toxicity result only from maternal 
toxicity; rather, it may be that the mother and developing organism are 
equally sensitive to that dose level. Moreover, whether developmental 
effects are secondary to maternal toxicity or not, the maternal effects 
may be reversible while the effects on the offspring may be permanent. 
These are important considerations for agents to which humans may be 
exposed at minimally toxic levels either voluntarily or involuntarily, 
because several agents (e.g., alcohol) are known to produce adverse 
developmental effects at minimally toxic doses in adult humans (Coles 
et al., 1991).
    Although interpretation of developmental neurotoxicity data may be 
limited, it is clear that functional effects should be evaluated in 
light of other toxicity data, including other forms of developmental 
toxicity (e.g., structural abnormalities, perinatal death, and growth 
retardation). For example, alterations in motor performance may be due 
to a skeletal malformation rather than nervous system change. Changes 
in learning tasks that require a visual cue might be influenced by 
structural abnormalities in the eye. The level of confidence that an 
agent produces an adverse effect may be as important as the type of 
change seen, and confidence may be increased by such factors as 
reproducibility of the effect, either in another study of the same 
function or by convergence of data from tests that purport to measure 
similar functions. A dose-response relationship is an extremely 
important measure of a chemical's effect; in the case of developmental 
neurotoxicity both monotonic and biphasic dose-response curves are 
likely, depending on the function being tested. The EPA Guidelines for 
Developmental Toxicity Risk Assessment (U.S. EPA, 1991b) may be 
consulted for more information on interpreting developmental toxicity 
studies. The endpoints frequently used to assess developmental 
neurotoxicity in exposed children have been reviewed by Winneke (1995).
3.1.3. Other Considerations
3.1.3.1. Pharmacokinetics
    Extrapolation of test results between species can be aided 
considerably by data on the pharmacokinetics of a particular agent in 
the species tested and, if possible, in humans. Information on a 
toxicant's half-life, metabolism, absorption, excretion, and 
distribution to the peripheral and central nervous system may be useful 
in predicting risk. Of particular importance for the pharmacokinetics 
of neurotoxicants is the blood-brain barrier. The vast majority of the 
central nervous system is served by blood vessels with blood-brain 
barrier properties, which exclude most ionic and nonlipid-soluble 
chemicals from the brain and spinal cord. The brain contains several 
structures called circumventricular organs (CVOs) that are served by 
blood vessels lacking blood-brain barrier properties. Brain regions 
adjacent to these CVOs are thus exposed to relatively high levels of 
many neurotoxicants. Pharmacokinetic data may be helpful in defining 
the dose-response curve, developing a more accurate basis for comparing 
species sensitivity (including that of humans), determining dosimetry 
at sites, and comparing pharmacokinetic profiles for various dosing 
regimens or routes of administration. The correlation of 
pharmacokinetic parameters and neurotoxicity data may be useful in 
determining the contribution of specific pharmacokinetic processes to 
the effects observed.
3.1.3.2. Comparisons of Molecular Structure
    Comparisons of the chemical or physical properties of an agent with 
those of known neurotoxicants may provide some indication of the 
potential for neurotoxicity. Such information may be helpful for 
evaluating potential toxicity when only minimal data are available. The 
structure-activity relationships (SAR) of some chemical classes have 
been studied, including hexacarbons, organophosphates, carbamates, and 
pyrethroids. Therefore, class relationships or SAR may help

[[Page 26944]]

predict neurotoxicity or interpret data from neurotoxicological 
studies. Under certain circumstances (e.g., in the case of new 
chemicals), this procedure is one of the primary methods used to 
evaluate the potential for toxicity when little or no empirical 
toxicity data are available. It should be recognized, however, that 
effects of chemicals in the same class can vary widely. Moser (1995), 
for example, reported that the behavioral effects of prototypic 
cholinesterase-inhibiting pesticides differed qualitatively in a 
battery of behavioral tests.
3.1.3.3. Statistical Considerations
    Properly designed studies on the neurotoxic effects of compounds 
will include appropriate statistical tests of significance. In general, 
the likelihood of obtaining a significant effect will depend jointly on 
the magnitude of the effect and the variability obtained in control and 
treated groups. The risk assessor should be aware that some 
neurotoxicants may induce a greater variability in biologic response, 
rather than a clear shift in mean or other parameters (Laties and 
Evans, 1980; Glowa and MacPhail, 1995). A number of texts are available 
on standard statistical tests (e.g., Siegel, 1956; Winer, 1971; Sokal 
and Rohlf, 1969; Salsburg, 1986; Gad and Weil, 1988).
    Neurotoxicity data present some unique features that should be 
considered in selecting statistical tests for analysis. Data may 
involve several different measurement scales, including categorical 
(affected or not), rank (more or less affected), and interval and ratio 
scales of measurement (affected by some percentage). For example, 
convulsions are usually recorded as being present or absent 
(categorical), whereas neuropathological changes are frequently 
described in terms of the degree of damage (rank). Many tests of 
neurotoxicity involve interval or ratio measurements (e.g., frequency 
of photocell interruptions or amplitude of an evoked potential), which 
are the most powerful and sensitive scales of measurement. In addition, 
measurements are frequently made repeatedly in control and treated 
subjects, especially in the case of behavioral and neurophysiological 
endpoints. For example, OPPTS guidelines for FOB assessment call for 
evaluations before exposure and at several times during exposure in a 
subchronic study (U.S. EPA, 1991a).
    Descriptive data (categorical) and rank order data can be analyzed 
using standard nonparametric techniques (Siegel, 1956). In some cases, 
if it is determined that the data fit the linear model, the categorical 
modeling procedure can be used for weighted least-squares estimation of 
parameters for a wide range of general linear models, including 
repeated-measures analyses. The weighted least-squares approach to 
categorical and rank data allows computation of statistics for testing 
the significance of sources of variation as reflected by the model. In 
the case of studies assessing effects in the same animals at several 
time points, univariate analyses can be carried out at each time point 
when the overall dose effect or the dose-by-time interaction is 
significant.
    Continuous data (e.g., magnitude, rate, amplitude), if found to be 
normally distributed, can be analyzed with general linear models using 
a grouping factor of dose and, if necessary, repeated measures across 
time (Winer, 1971). Univariate analyses of dose, comparing dose groups 
to the control group at each time point, can be performed when there is 
a significant overall dose effect or a dose-by-time interaction. Post 
hoc comparisons between control and treatment groups can be made 
following tests for overall significance. In the case of multiple 
endpoints within a series of evaluations, some type of correction for 
multiple observations is warranted (Winer, 1971).
3.1.3.4. In Vitro Data in Neurotoxicology
    Methods and procedures that fall under the general heading of 
short-term tests include an array of in vitro tests that have been 
proposed as alternatives to whole-animal tests (Goldberg and Frazier, 
1989). In vitro approaches use animal or human cells, tissues, or 
organs and maintain them in a nutritive medium. Various types of in 
vitro techniques, including primary cell cultures, cell lines, and 
cloned cells, produce data for evaluating potential and known 
neurotoxic substances. While such procedures are important in studying 
the mechanism of action of toxic agents, their use in hazard 
identification in human health risk assessment has not been explored to 
any great extent.
    Data from in vitro procedures are generally based on simplified 
approaches that require less time to yield information than do many in 
vivo techniques. However, in vitro methods generally do not take into 
account the distribution of the toxicant in the body, the route of 
administration, or the metabolism of the substance. It also is 
difficult to extrapolate in vitro data to animal or human neurotoxicity 
endpoints, which include behavioral changes, motor disorders, sensory 
and perceptual disorders, lack of coordination, and learning deficits. 
In addition, data from in vitro tests cannot duplicate the complex 
neuronal circuitry characteristic of the intact animal.
    Many in vitro systems are now being evaluated for their ability to 
predict the neurotoxicity of various agents seen in intact animals. 
This validation process requires considerations in study design, 
including defined endpoints of toxicity and an understanding of how a 
test agent would be handled in vitro as compared to the intact 
organism. Demonstrated neurotoxicity in vitro in the absence of in vivo 
data is suggestive but inadequate evidence of a neurotoxic effect. In 
vivo data supported by in vitro data enhance the reliability of the in 
vivo results.
3.1.3.5. Neuroendocrine Effects
    Neuroendocrine dysfunction may occur because of a disturbance in 
the regulation and modulation of neuroendocrine feedback systems. One 
major indicator of neuroendocrine function is secretion of hormones 
from the pituitary. Hypothalamic control of anterior pituitary 
secretions is also involved in a number of important bodily functions. 
Many types of behaviors (e.g., reproductive behaviors, sexually 
dimorphic behaviors in animals) are dependent on the integrity of the 
hypothalamic-pituitary system, which could represent a potential site 
of neurotoxicity. Pituitary secretions arise from a number of different 
cell types in this gland, and neurotoxicants could affect these cells 
directly or indirectly. Morphological changes in cells mediating 
neuroendocrine secretions could be associated with adverse effects on 
the pituitary or hypothalamus and could ultimately affect behavior and 
the functioning of the nervous system. Biochemical changes in the 
hypothalamus may also be used as indicators of potential adverse 
effects on neuroendocrine function. Finally, the development of the 
nervous system is intimately associated with the presence of 
circulating hormones such as thyroid hormone (Porterfield, 1994). The 
nature of the nervous system deficit, which could include cognitive 
dysfunction, altered neurological development, or visual deficits, 
depends on the severity of the thyroid disturbance and the specific 
developmental period when exposure to the chemical occurred.
3.2. Dose-Response Evaluation
    Dose-response evaluation is a critical part of the qualitative 
characterization of a chemical's potential to produce neurotoxicity and 
involves the description of the dose-response

[[Page 26945]]

relationship in the available data. Human studies covering a range of 
exposures are rarely available, and therefore animal data are typically 
used for estimating exposure levels likely to produce adverse effects 
in humans. Evidence for a dose-response relationship is an important 
criterion in establishing a neurotoxic effect, although this analysis 
may be limited when based on standard studies using three dose groups 
or fewer. The evaluation of dose-response relationships includes 
identifying effective dose levels as well as doses associated with no 
increase in adverse effects when compared with controls. The lack of a 
dose-response relationship in the data may suggest that the effect is 
not related to the putative neurotoxic effect or that the study was not 
appropriately controlled. Much of the focus is on identifying the 
critical effect(s) observed at the LOAEL and the NOAEL associated with 
that effect. The NOAEL is defined as the highest dose at which there is 
no statistically or biologically significant increase in the frequency 
of an adverse neurotoxic effect when compared with the appropriate 
control group in a database characterized as having sufficient evidence 
for use in a risk assessment (see section 3.3). The risk assessor 
should be aware of possible problems associated with estimating a NOAEL 
in studies involving a small number of test subjects and that have a 
poor dose-response relationship.
    In addition to identifying the NOAEL/LOAEL or BMD, the dose-
response evaluation defines the range of doses that are neurotoxic for 
a given agent, species, route of exposure, and duration of exposure. In 
addition to these considerations, pharmacokinetic factors and other 
aspects that might influence comparisons with human exposure scenarios 
should be taken into account. For example, dose-response curves may 
exhibit not only monotonic but also U-shaped or inverted U-shaped 
functions (Davis and Svendsgaard, 1990). Such curves are hypothesized 
to reflect multiple mechanisms of action, the presence of homeostatic 
mechanisms, and/or activation of compensatory or protective mechanisms. 
In addition to considering the shape of the dose-response curve, it 
should also be recognized that neurotoxic effects vary in terms of 
nature and severity across dose or exposure level. At high levels of 
exposure, frank lesions accompanied by severe functional impairment may 
be observed. Such effects are widely accepted as adverse. At 
progressively lower levels of exposure, however, the lesions may become 
less severe and the impairments less obvious. At levels of exposure 
near the NOAEL and LOAEL, the effects will often be mild, possibly 
reversible, and inconsistently found. In addition, the endpoints 
showing responses may be at levels of organization below the whole 
organism (e.g., neurochemical or electrophysiological endpoints). The 
adversity of such effects can be disputed (e.g., cholinesterase 
inhibition), yet it is such effects that are likely to be the focus of 
risk assessment decisions. To the extent possible, this document 
provides guidance on determining the adversity of neurotoxic effects. 
However, the identification of a critical adverse effect often requires 
considerable professional judgment and should consider factors such as 
the biological plausibility of the effect, the evidence of a dose-
effect continuum, and the likelihood for progression of the effect with 
continued exposure.
3.3. Characterization of the Health-Related Database
    This section describes a scheme for characterizing the sufficiency 
of evidence for neurotoxic effects. This scheme defines two broad 
categories: sufficient and insufficient (Table 8). Categorization is 
aimed at providing certain criteria for the Agency to use to define the 
minimum evidence necessary to define hazards and to conduct dose-
response analyses. It does not address the issues related to 
characterization of risk, which requires analysis of potential human 
exposures and their relation to potential hazards in order to estimate 
the risks of those hazards from anticipated or estimated exposures. 
Several examples using a weight-of-evidence approach similar to that 
described in these Guidelines have been described elsewhere (Tilson et 
al., 1995; Tilson et al., 1996).

                            Table 8.--Characterization of the Health-Related Database                           
----------------------------------------------------------------------------------------------------------------
                                                                                                                
----------------------------------------------------------------------------------------------------------------
Sufficient evidence.................................  The sufficient evidence category includes data that       
                                                       collectively provide enough information to judge whether 
                                                       or not a human neurotoxic hazard could exist. This       
                                                       category may include both human and experimental animal  
                                                       evidence.                                                
Sufficient human evidence...........................  This category includes agents for which there is          
                                                       sufficient evidence from epidemiologic studies, e.g.,    
                                                       case control and cohort studies, to judge that some      
                                                       neurotoxic effect is associated with exposure. A case    
                                                       series in conjunction with other supporting evidence may 
                                                       also be judged ``sufficient evidence.'' Epidemiologic and
                                                       clinical case studies should discuss whether the observed
                                                       effects can be considered biologically plausible in      
                                                       relation to chemical exposure. (Historically, often much 
                                                       has been made of the notion of causality in epidemiologic
                                                       studies. Causality is a more stringent criterion than    
                                                       association and has become a topic of scientific and     
                                                       philosophical debate. See Susser [1986], for example, for
                                                       a discussion of inference in epidemiology.)              
Sufficient experimental animal evidence/limited       This category includes agents for which there is          
 human data.                                           sufficient evidence from experimental animal studies and/
                                                       or limited human data to judge whether a potential       
                                                       neurotoxic hazard may exist. Generally, agents that have 
                                                       been tested according to current test guidelines would be
                                                       included in this category. The minimum evidence necessary
                                                       to judge that a potential hazard exists would be data    
                                                       demonstrating an adverse neurotoxic effect in a single   
                                                       appropriate, well-executed study in a single experimental
                                                       animal species. The minimum evidence needed to judge that
                                                       a potential hazard does not exist would include data from
                                                       an appropriate number of endpoints from more than one    
                                                       study and two species showing no adverse neurotoxic      
                                                       effects at doses that were minimally toxic in terms of   
                                                       producing an adverse effect. Information on              
                                                       pharmacokinetics, mechanisms, or known properties of the 
                                                       chemical class may also strengthen the evidence.         

[[Page 26946]]

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

4. Quantitative Dose-Response Analysis

    This section describes several approaches (including the LOAEL/
NOAEL and BMD) for determining the reference dose (RfD) or reference 
concentration (RfC). The NOAEL or BMD/uncertainty factor approach 
results in an RfD or RfC, which is an estimate (with uncertainty 
spanning perhaps an order of magnitude) of a daily exposure to the 
human population (including sensitive subgroups) that is likely to be 
without an appreciable risk of deleterious effects during a lifetime.
    The dose-response analysis characterization should:
    <bullet> Describe how the RfD/RfC was calculated;
    <bullet> Discuss the confidence in the estimates;
    <bullet> Describe the assumptions or uncertainty factors used; and
    <bullet> Discuss the route and level of exposure observed, as 
compared to expected human exposures.
4.1. LOAEL/NOAEL and BMD Determination
    As indicated earlier, the LOAEL and NOAEL are determined for 
endpoints that are seen at the lowest dose level (so-called critical 
effect). Several limitations in the use of the NOAEL have been 
identified and described (e.g., Barnes and Dourson, 1988; Crump, 1984). 
For example, the NOAEL is derived from a single endpoint from a single 
study (the critical study) and

[[Page 26947]]

ignores both the slope of the dose-response function and baseline 
variability in the endpoint of concern. Because the baseline 
variability is not taken into account, the NOAEL from a study using 
small group sizes may be higher than the NOAEL from a similar study in 
the same species that uses larger group sizes. The NOAEL is also 
directly dependent on the dose spacing used in the study. Finally, and 
perhaps most importantly, use of the NOAEL does not allow estimates of 
risk or extrapolation of risk to lower dose levels. Because of these 
and other limitations in the NOAEL approach, it has been proposed that 
mathematical curve-fitting techniques (Crump, 1984; Gaylor and Slikker, 
1990; Glowa, 1991; Glowa and MacPhail, 1995; U.S. EPA, 1995a) be 
compared with the NOAEL procedure in calculating the RfD or RfC. These 
techniques typically apply a mathematical function that describes the 
dose-response relationship and then interpolate to a level of exposure 
associated with a small increase in effect over that occurring in the 
control group or under baseline conditions. The BMD has been defined as 
a lower confidence limit on the effective dose associated with some 
defined level of effect, e.g., a 5% or 10% increase in response. These 
guidelines suggest that the use of the BMD should be explored in 
specific situations. The Agency is currently developing guidelines for 
the use of the BMD in risk assessment.
    Many neurotoxic endpoints provide continuous measures of response, 
such as response speed, nerve conduction velocity, IQ score, degree of 
enzyme inhibition, or the accuracy of task performance. Although it is 
possible to impose a dichotomy on a continuous effects distribution and 
to classify some level of response as ``affected'' and the remainder as 
``unaffected,'' it may be very difficult and inappropriate to establish 
such clear distinctions, because such a dichotomy would misrepresent 
the true nature of the neurotoxic response. The risk assessor should be 
aware of the importance of trying to reconcile findings from several 
studies that seem to report widely divergent results. Alternatively, 
quantitative models designed to analyze continuous effect variables may 
be preferable. Other techniques that allow this approach, with 
transformation of the information into estimates of the incidence or 
frequency of affected individuals in a population, have been proposed 
(Crump, 1984; Gaylor and Slikker, 1990; Glowa and MacPhail, 1995). 
Categorical regression analysis has been proposed because it can 
evaluate different types of data and derive estimates for short-term 
exposures (Rees and Hattis, 1994). Decisions about the most appropriate 
approach require professional judgment, taking into account the 
biological nature of the continuous effect variable and its 
distribution in the population under study.
    Although dose-response functions in neurotoxicology are generally 
linear or monotonic, curvilinear functions, especially U-shaped or 
inverted U-shaped curves, have been reported as noted earlier (section 
3.2). Dose-response analyses should consider the uncertainty that U-
shaped dose-response functions might contribute to the estimate of the 
NOAEL/LOAEL or BMD. Typically, estimates of the NOAEL/LOAEL are taken 
from the lowest part of the dose-response curve associated with 
impaired function or adverse effect.
4.2. Determination of the Reference Dose or Reference Concentration
    Since the availability of dose-response data in humans is limited, 
extrapolation of data from animals to humans usually involves the 
application of uncertainty factors to the NOAEL/LOAEL or BMD. The NOAEL 
or BMD/uncertainty factor approach results in an RfD or RfC, which is 
an estimate (with uncertainty spanning perhaps an order of magnitude) 
of a daily exposure to the human population (including sensitive 
subgroups) that is likely to be without an appreciable risk of 
deleterious effects during a lifetime. The oral RfD and inhalation RfC 
are applicable to chronic exposure situations and are based on an 
evaluation of all the noncancer health effects, including neurotoxicity 
data. RfDs and RfCs in the Integrated Risk Information System (IRIS-2) 
database for several agents are based on neurotoxicity endpoints and 
include a few cases in which the RfD or RfC is calculated using the BMD 
approach (e.g., methylmercury, carbon disulfide). The size of the final 
uncertainty factor used will vary from agent to agent and will require 
the exercise of scientific judgment, taking into account interspecies 
differences, the shape of the dose-response curve, and the 
neurotoxicity endpoints observed. Uncertainty factors are typically 
multiples of 10 and are used to compensate for human variability in 
sensitivity, the need to extrapolate from animals to humans, and the 
need to extrapolate from less than lifetime (e.g., subchronic) to 
lifetime exposures. An additional factor of up to 10 may be included 
when only a LOAEL (and not a NOAEL) is available from a study, or 
depending on the completeness of the database, a modifying factor of up 
to 10 may be applied, depending on the confidence one has in the 
database. Uncertainty factors of less than 10 can be used, depending 
upon the availability of relevant information. Barnes and Dourson 
(1988) provide a more complete description of the calculation, use, and 
significance of RfDs in setting exposure limits to toxic agents by the 
oral route. Jarabek et al. (1990) provide a more complete description 
of the calculation, use, and significance of RfCs in setting exposure 
limits to toxic agents in air. Neurotoxicity can result from acute, 
shorter term exposures, and it may be appropriate in some cases, e.g., 
for air pollutants or water contaminants, to set shorter term exposure 
limits for neurotoxicity as well as for other noncancer health effects.

5. Exposure Assessment

    Exposure assessment describes the magnitude, duration, frequency, 
and routes of exposure to the agent of interest. This information may 
come from hypothetical values, models, or actual experimental values, 
including ambient environmental sampling results. Guidelines for 
exposure assessment have been published separately (U.S. EPA, 1992) and 
will, therefore, be discussed only briefly here.
    The exposure assessment should include an exposure characterization 
that:
    <bullet> Provides a statement of the purpose, scope, level of 
detail, and approach used in the exposure assessment;
    <bullet> 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;
    <bullet> Provides an evaluation of the overall level of confidence 
in the estimate of exposure and dose and the conclusions drawn; and
    <bullet> Communicates the results of the exposure assessment to the 
risk assessor, who can then use the exposure characterization, along 
with the hazard and dose/response characterizations, to develop a risk 
characterization.
    A number of considerations are relevant to exposure assessment for 
neurotoxicants. An appropriate evaluation of exposure should consider 
the potential for exposure via ingestion, inhalation, and dermal 
penetration from relevant sources of exposure, including multiple 
avenues of intake from the same source.
    In addition, neurotoxic effects may result from short-term (acute), 
high-concentration exposures as well as from

[[Page 26948]]

longer term (subchronic), lower level exposures. Neurotoxic effects may 
occur after a period of time following initial exposure or be 
obfuscated by repair mechanisms or apparent tolerance. The type and 
severity of effect may depend significantly on the pattern of exposure 
rather than on the average dose over a long period of time. For this 
reason, exposure assessments for neurotoxicants may be much more 
complicated than those for long-latency effects such as 
carcinogenicity. It is rare for sufficient data to be available to 
construct such patterns of exposure or dose, and professional judgment 
may be necessary to evaluate exposure to neurotoxic agents.

6. Risk Characterization

6.1. Overview
    Risk characterization is the summarization step of the risk 
assessment process and consists of an integrative analysis and a 
summary. The integrative analysis (a) involves integration of the 
toxicity information from the hazard characterization and dose-response 
analysis with the human exposure estimates, (b) provides an evaluation 
of the overall quality of the assessment and the degree of confidence 
in the estimates of risk and conclusions drawn, and <Copyright> 
describes risk in terms of the nature and extent of harm. The risk 
characterization summary communicates the results of the risk 
assessment to the risk manager in a complete, informative, and useful 
format.
    This summary should include, but is not limited to, a discussion of 
the following elements:
    <bullet> Quality of and confidence in the available data;
    <bullet> Uncertainty analysis;
    <bullet> Justification of defaults or assumptions;
    <bullet> Related research recommendations;
    <bullet> Contentious issues and extent of scientific consensus;
    <bullet> Effect of reasonable alternative assumptions on 
conclusions and estimates;
    <bullet> Highlights of reasonable plausible ranges;
    <bullet> Reasonable alternative models; and
    <bullet> Perspectives through analogy.
    The risk manager can then use the derived risk to make public 
health decisions.
    An effective risk characterization should fully, openly, and 
clearly characterize risks and disclose the scientific analyses, 
uncertainties, assumptions, and science policies that underlie 
decisions throughout the risk assessment and risk management processes. 
The risk characterization should feature values such as transparency in 
the decision-making process; clarity in communicating with the 
scientific community and the public regarding environmental risk and 
the uncertainties associated with assessments of environmental risk; 
and consistency across program offices in core assumptions and science 
policies, which are well grounded in science and reasonable. The 
following sections describe these four aspects of the risk 
characterization in more detail.
6.2. Integration of Hazard Characterization, Dose-Response Analysis, 
and Exposure Assessment
    In developing the hazard characterization, dose-response analysis, 
and exposure portions of the risk assessment, the risk assessor should 
take into account many judgments concerning human relevance of the 
toxicity data, including the appropriateness of the various animal 
models for which data are available and the route, timing, and duration 
of exposure relative to expected human exposure. These judgments should 
be summarized at each stage of the risk assessment process (e.g., the 
biological relevance of anatomical variations may be established in the 
hazard characterization process, or the influence of species 
differences in metabolic patterns in the dose-response analysis). In 
integrating the information from the assessment, the risk assessor 
should determine if some of these judgments have implications for other 
portions of the assessment and whether the various components of the 
assessment are compatible.
    The risk characterization should not only examine the judgments but 
also explain the constraints of available data and the state of 
knowledge about the phenomena studied in making them, including (1) the 
qualitative conclusions about the likelihood that the chemical may pose 
a specific hazard to human health, the nature of the observed effects, 
under what conditions (route, dose levels, time, and duration) of 
exposure these effects occur, and whether the health-related data are 
sufficient to use in a risk assessment; (2) a discussion of the dose-
response characteristics of the critical effects, data such as the 
shapes and slopes of the dose-response curves for the various 
endpoints, the rationale behind the determination of the NOAEL and 
LOAEL and calculation of the benchmark dose, and the assumptions 
underlying the estimation of the RfD or RfC; and (3) the estimates of 
the magnitude of human exposure; the route, duration, and pattern of 
the exposure; relevant pharmacokinetics; and the number and 
characteristics of the population(s) exposed.
    If data to be used in a risk characterization are from a route of 
exposure other than the expected human exposure, then pharmacokinetic 
data should be used, if available, to make extrapolations across routes 
of exposure. If such data are not available, the Agency makes certain 
assumptions concerning the amount of absorption likely or the 
applicability of the data from one route to another (U.S. EPA, 1992).
    The level of confidence in the hazard characterization should be 
stated to the extent possible, including the appropriate category 
regarding sufficiency of the health-related data. A comprehensive risk 
assessment ideally includes information on a variety of endpoints that 
provide insight into the full spectrum of potential neurotoxicological 
responses. A profile that integrates both human and test species data 
and incorporates a broad range of potential adverse neurotoxic effects 
provides more confidence in a risk assessment for a given agent.
    The ability to describe the nature of the potential human exposure 
is important in order to predict when certain outcomes can be 
anticipated and the likelihood of permanence or reversibility of the 
effect. An important part of this effort is a description of the nature 
of the exposed population and the potential for sensitive, highly 
susceptible, or highly exposed populations. For example, the 
consequences of exposure to the developing individual versus the adult 
can differ markedly and can influence whether the effects are transient 
or permanent. Other considerations relative to human exposures might 
include the likelihood of exposures to other agents, concurrent 
disease, and nutritional status.
    The presentation of the integrated results of the assessment should 
draw from and highlight key points of the individual characterizations 
of component analyses performed under these Guidelines. The overall 
risk characterization represents the integration of these component 
characterizations. If relevant risk assessments on the agent or an 
analogous agent have been done by EPA or other Federal agencies, these 
should be described and the similarities and differences discussed.

[[Page 26949]]

6.3. Quality of the Database and Degree of Confidence in the Assessment
    The risk characterization should summarize the kinds of data 
brought together in the analysis and the reasoning on which the 
assessment is based. The description should convey the major strengths 
and weaknesses of the assessment that arise from availability of data 
and the current limits of our understanding of the mechanisms of 
toxicity.
    A health risk assessment is only as good as its component parts, 
i.e., hazard characterization, dose-response analysis, and exposure 
assessment. Confidence in the results of a risk assessment is thus a 
function of confidence in the results of the analysis of these 
elements. Each of these elements should have its own characterization 
as a part of the assessment. Within each characterization, the 
important uncertainties of the analysis and interpretation of data 
should be explained, and the risk manager should be given a clear 
picture of consensus or lack of consensus that exists about significant 
aspects of the assessment. Whenever more than one view is supported by 
the data and choosing between them is difficult, all views should be 
presented. If one has been selected over the others, the rationale 
should be given; if not, then all should be presented as plausible 
alternative results.
6.4. Descriptors of Neurotoxicity Risk
    There are a number of ways to describe risks. Several relevant ways 
for neurotoxicity are as follows:
6.4.1. Estimation of the Number of Individuals
    The RfD or RfC is taken to be a chronic exposure level at or below 
which no significant risk occurs. Therefore, presentation of the 
population in terms of those at or below the RfD or RfC (``not at 
risk'') and above the RfD or RfC (``may be at risk'') may be useful 
information for risk managers. This method is particularly useful to a 
risk manager considering possible actions to ameliorate risk for a 
population. If the number of persons in the at-risk category can be 
estimated, then the number of persons removed from the at-risk category 
after a contemplated action is taken can be used as an indication of 
the efficacy of the action.
6.4.2. Presentation of Specific Scenarios
    Presenting specific scenarios in the form of ``what if?'' questions 
is particularly useful to give perspective to the risk manager, 
especially where criteria, tolerance limits, or media quality limits 
are being set. The question being asked in these cases is, at this 
proposed exposure limit, what would be the resulting risk for 
neurotoxicity above the RfD or RfC?
6.4.3. Risk Characterization for Highly Exposed Individuals
    This measure is one example of the just-discussed descriptor. This 
measure describes the magnitude of concern at the upper end of the 
exposure distribution. This allows risk managers to evaluate whether 
certain individuals are at disproportionately high or unacceptably high 
risk.
    The objective of looking at the upper end of the exposure 
distribution is to derive a realistic estimate of a relatively highly 
exposed individual or individuals. This measure could be addressed by 
identifying a specified upper percentile of exposure in the population 
and/or by estimating the exposure of the highest exposed individual(s). 
Whenever possible, it is important to express the number of individuals 
who comprise the selected highly exposed group and discuss the 
potential for exposure at still higher levels.
    If population data are absent, it will often be possible to 
describe a scenario representing high-end exposures using upper 
percentile or judgment-based values for exposure variables. In these 
instances caution should be used in order not to compound a substantial 
number of high-end values for variables if a ``reasonable'' exposure 
estimate is to be achieved.
6.4.4. Risk Characterization for Highly Sensitive or Susceptible 
Individuals
    This measure identifies populations sensitive or susceptible to the 
effect of concern. Sensitive or susceptible individuals are those 
within the exposed population at increased risk of expressing the toxic 
effect. All stages of nervous system maturation might be considered 
highly sensitive or susceptible, but certain subpopulations can 
sometimes be identified because of critical periods for exposure, for 
example, pregnant or lactating women, infants, or children. The aged 
population is considered to be at particular risk because of the 
limited ability of the nervous system to regenerate or compensate to 
neurotoxic insult.
    In general, not enough is understood about the mechanisms of 
toxicity to identify sensitive subgroups for all agents, although 
factors such as nutrition (e.g., vitamin B), personal habits (e.g., 
smoking, alcohol consumption, illicit drug abuse), or preexisting 
disease (e.g., diabetes, neurological diseases, sexually transmitted 
diseases, polymorphisms for certain metabolic enzymes) may predispose 
some individuals to be more sensitive to the neurotoxic effects of 
specific agents. Gender-related differences in response to 
neurotoxicants have been noted, but these appear to be related to 
gender-dependent toxicodynamic or toxicokinetic factors.
    In general, it is assumed that an uncertainty factor of 10 for 
intrapopulation variability will be able to accommodate differences in 
sensitivity among various subpopulations, including children and the 
elderly. However, in cases where it can be demonstrated that a factor 
of 10 does not afford adequate protection, another uncertainty factor 
may be considered in conducting the risk assessment.
6.4.5. Other Risk Descriptors
    In risk characterization, dose-response information and the human 
exposure estimates may be combined either by comparing the RfD or RfC 
and the human exposure estimate or by calculating the margin of 
exposure (MOE). The MOE is the ratio of the NOAEL from the most 
appropriate or sensitive species to the estimated human exposure level. 
If a NOAEL is not available, a LOAEL may be used in calculating the 
MOE. Alternatively, a benchmark dose may be compared with the estimated 
human exposure level to obtain the MOE. Considerations for the 
evaluation of the MOE are similar to those for the uncertainty factor 
applied to the LOAEL/NOAEL or the benchmark dose. The MOE is presented 
along with a discussion of the adequacy of the database, including the 
nature and quality of the hazard and exposure data, the number of 
species affected, and the dose-response information.
    The RfD or RfC comparison with the human exposure estimate and the 
calculation of the MOE are conceptually similar but are used in 
different regulatory situations. The choice of approach depends on 
several factors, including the statute involved, the situation being 
addressed, the database used, and the needs of the decision maker. The 
RfD or RfC and the MOE are considered along with other risk assessment 
and risk management issues in making risk management decisions, but the 
scientific issues that should be taken into account in establishing 
them have been addressed here.

[[Page 26950]]

    If the MOE is equal to or more than the uncertainty factor 
multiplied by 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.
6.5. Communicating Results
    Once the risk characterization is completed, the focus turns to 
communicating results to the risk manager. The risk manager uses the 
results of the risk characterization along with other technological, 
social, and economic considerations in reaching a regulatory decision. 
Because of the way in which these risk management factors may affect 
different cases, consistent but not necessarily identical risk 
management decisions should be made on a case-by-case basis. These 
Guidelines are not intended to give guidance on the nonscientific 
aspects of risk management decisions.
6.6. Summary and Research Needs
    These Guidelines summarize the procedures that the U.S. 
Environmental Protection Agency would use in evaluating the potential 
for agents to cause neurotoxicity. These Guidelines discuss the general 
default assumptions that should be made in risk assessment for 
neurotoxicity because of gaps in our knowledge about underlying 
biological processes and how these compare across species. Research to 
improve the risk assessment process is needed in a number of areas. For 
example, research is needed to delineate the mechanisms of 
neurotoxicity and pathogenesis, provide comparative pharmacokinetic 
data, examine the validity of short-term in vivo and in vitro tests, 
elucidate the functional modalities that may be altered, develop 
improved animal models to examine the neurotoxic effects of exposure 
during the premating and early postmating periods and in neonates, 
further evaluate the relationship between maternal and developmental 
toxicity, provide insight into the concept of threshold, develop 
approaches for improved mathematical modeling of neurotoxic effects, 
improve animal models for examining the effects of agents given by 
various routes of exposure, determine the effects of recurrent 
exposures over prolonged periods of time, and address the synergistic 
or antagonistic effects of mixed exposures and neurotoxic response. 
Such research will aid in the evaluation and interpretation of data on 
neurotoxicity and should provide methods to assess risk more precisely. 
Additional research is needed to determine the most appropriate dose-
response approach to be used in neurotoxicity risk assessments.

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Organization.

Part B: Response to Science Advisory Board and Public Comments

1. Introduction

    A notice of availability for public comments of these Guidelines 
was published in the Federal Register in October 1995. Twenty-five 
responses were received. These Guidelines were presented to the 
Environmental Health Committee of the Science Advisory Board (SAB) on 
July 18, 1996. The report of the SAB was provided to the Agency in 
April 1997. The SAB and public comments were diverse and represented 
varying perspectives. Many of the comments were favorable and expressed 
agreement with positions taken in the proposed Guidelines. Some 
comments addressed items that were more pertinent to testing guidance 
than risk assessment guidance or were otherwise beyond the scope of 
these Guidelines. Some of the comments concerned generic points that 
were not specific to neurotoxicity issues. Others

[[Page 26953]]

addressed topics that have not been developed sufficiently and should 
be viewed as research issues. There were conflicting views about the 
need to provide additional detailed guidance about decision making in 
the evaluation process as opposed to promoting extensive use of 
scientific judgment. Many public comments provided specific suggestions 
for clarification of details and corrections of factual material in the 
Guidelines.

2. Response to Science Advisory Board Comments

    The SAB found the Guidelines ``* * * to be quite successful, and, 
all things considered, well suited to its intended task.'' However, 
recommendations were made to improve specific areas.
    The SAB recommended that EPA keep hazard identification as an 
identifiable qualitative step in the risk assessment process and that 
steps should be taken to decouple the qualitative step of hazard 
identification from the more quantitatively rigorous steps of exposure 
evaluation and dose-response assessment. These Guidelines now include a 
hazard characterization step that clearly describes a qualitative 
evaluation of hazard within the context of the dose, route, timing and 
duration of exposure. This step is clearly differentiated from the 
quantitative dose-response analysis, which describes approaches for 
determining an RfD or RfC.
    The SAB supported the presumption that what appears to be 
reversible neurotoxicity, especially when arising from gestational or 
neonatal exposure and observed before adulthood, should not be 
dismissed as of little practical consequence. They may be indices of 
silent toxicity that emerge later in life or may suggest more robust 
and enduring responses in aged individuals. These Guidelines explain 
the concept of functional reserve and advise caution in instances where 
reversibility is seen and in cases where exposure to a chemical may 
result in delayed-onset neurotoxicity. These Guidelines also indicate 
that reversibility may vary with the region of the nervous system 
damaged, the neurotoxic agent involved, and organismic factors such as 
age.
    The SAB restated previous positions concerning cholinesterase-
inhibiting chemicals. Agent-induced clinical signs of cholinergic 
dysfunction could be used to evaluate dose-response and dose-effect 
relationships and define the presence and absence of given effects in 
risk assessment. The SAB also indicated that inhibition of RBC and 
plasma cholinesterase activity could serve as a biomarker of exposure 
to cholinesterase-inhibiting agents and thereby corroborate 
observations concerning the presence of clinical effects associated 
with cholinesterase inhibition. The SAB also indicated that reduced 
brain cholinesterase activity should be assessed in the context of the 
biological consequences of the reduction. These Guidelines indicate 
that inhibition of cholinesterase in the nervous system reduces the 
organism's level of ``reserve'' cholinesterase and, therefore, limits 
the subsequent ability to respond successfully to additional exposures 
and that prolonged inhibition could lead to adverse functional changes 
associated with compensatory neurochemical mechanisms. In general, an 
attempt was made to coordinate these Guidelines with the views of a 
recently convened Scientific Advisory Panel regarding the risk 
assessment of cholinesterase-inhibiting pesticides (Office of Pesticide 
Programs, Science Policy on the Use of Cholinesterase Inhibition for 
Risk Assessments of Organophosphate and Carbamate Pesticides, 1997).
    The SAB indicated that the Guidelines were inclusive of the major 
neurotoxicity endpoints of concern. No additional neurochemical, 
neurophysiological, or structural endpoints were suggested. Comments 
indicated that there was no need to consider endocrine disruptors 
differently from other potential neurotoxic agents.
    The SAB found that the descriptions of the endpoints used in human 
and animal neurotoxicological assessments were thorough and well 
documented. Several sections, particularly concerning some of the 
neurochemical and neurobehavioral measures, were corrected for factual 
errors or supported with more detailed descriptions.
    The SAB recommended that the use of the threshold assumption should 
occur after an evaluation of likely biological mechanisms and available 
data to provide evidence that linear responses would be expected. A 
strict threshold is not always clear in the human population because of 
the wide variation in background levels for some functions. Cumulative 
neurotoxicological effects might also alter the response of some 
individuals within a special population, which might allow the Agency 
to characterize the risk to the sensitive population. Although the SAB 
did not disagree with the Guidelines' assumption of a threshold as a 
default for neurotoxic effects, it was suggested that the term 
``nonlinear dose-response curve for most neurotoxicants'' be 
substituted for the term ``threshold.'' The Neurotoxicity Risk 
Assessment Guidelines have been amended to harmonize their treatment of 
the issue of threshold with the presentation and position taken with 
other guidelines.
    The SAB also recommended that the topic of susceptible populations 
be expanded to include the elderly and other groups. The elderly could 
be at increased risk of toxic effects for a number of reasons, 
including a decline in the reserve capacity with aging, changes in the 
ability to detoxify or excrete xenobiotics with age, and the potential 
to interact with medicines or other compounds that could synergize 
interactions with toxic chemicals. The SAB also indicated that other 
populations should be considered, including those with chronic and 
debilitating conditions, groups of workers with potential exposure to 
chemicals that may be neurotoxic, individuals with genetic 
polymorphisms that could affect responsiveness to certain 
neurotoxicants, and individuals that may experience differential 
exposure because of their proximity to chemicals in the environment or 
diet. The Guidelines have been modified to emphasize the possible 
presence of all of these susceptible populations. When specific 
information on differential risk is not available, the Agency will 
continue to apply a default uncertainty factor to account for potential 
differences in susceptibility.
    The SAB recommended that the benchmark dose (BMD) was not ready for 
immediate incorporation into adjustment-factor-based safety assessment 
or to serve as a substitute or replacement for the more familiar NOAEL 
or LOAEL. The SAB also recommended that research and development on the 
BMD should be aggressively encouraged and actively supported. The BMD 
could be a replacement for the NOAEL or LOAEL after the appropriate 
research has been conducted.

3. Response to Public Comments

    In addition to numerous supportive statements, several issues were 
indicated, although each issue was raised by only a few commentators. 
The public comment supported the SAB recommendation that there was no 
clear consensus concerning replacing the NOAEL approach with the BMD to 
calculate RfDs and RfCs for neurotoxicity endpoints. There was also 
support for ensuring that dose-response and other experimental design 
information be considered in interpreting the results of hazard 
identification studies before proceeding

[[Page 26954]]

to quantitative dose-response analysis. Public comment also supported 
the position that reversibility cannot be ignored in neurotoxicity risk 
assessment and that the risk assessor should exert caution in 
interpreting reversible effects, especially where an apparent transient 
effect is cited to support evidence for relatively benign effects. The 
public comment also supported the use of clinical signs in the risk 
assessment of cholinesterase-inhibiting compounds and the finding that 
inhibition of brain cholinesterase was an adverse effect. The 
Guidelines emphasize the importance of brain cholinesterase inhibition, 
particularly in cases of repeated exposure. The public comment agreed 
with the SAB that RBC and plasma cholinesterase activity are biomarkers 
of exposure. It was recommended that the Guidelines incorporate 
additional information addressing the neuroendocrine system as a 
potential target site, and a section has been added that defines the 
vulnerable components of the neuroendocrine system and the behavioral, 
hormonal, and physiological endpoints that may be indicative of a 
direct or indirect effect on the neuroendocrine system.
    Public comment strongly endorsed the default assumption that there 
is a threshold for neurotoxic effects. The Guidelines, however, reflect 
the argument of the SAB that the term ``nonlinear dose-response curve 
for most neurotoxicants'' be substituted for ``threshold'' in order to 
be consistent with the presentation and positions taken by other risk 
assessment guidelines.
    The public comments made a number of recommendations to improve the 
Guidelines with regard to consistency of language between text and 
tables, improve the clarity of some of the tables, and improve the 
description of some of the endpoints used in animal studies. A number 
of factual errors were corrected, including the description of the 
blood-brain barrier and the degree of inhibition of neurotoxic esterase 
associated with organophosphate-induced delayed-onset neuropathy. 
Therefore, a number of changes have been made in the Guidelines to 
clarify and correct specific passages, but every effort was made to 
maintain the original intent concerning the use and interpretation of 
results from various neurotoxicological endpoints. Finally, the public 
comment agreed with the SAB that factors such as nutrition, personal 
habits, age, or preexisting disease may predispose some individuals to 
be differentially sensitive to neurotoxic chemicals. The risk 
characterization section has been expanded to reflect these potentially 
sensitive subpopulations.

[FR Doc. 98-12303 Filed 5-13-98; 8:45 am]
BILLING CODE 6560-50-P


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