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:

[email protected].

SUPPLEMENTARY INFORMATION: In its 1983 book Risk Assessment in the

Federal Government: Managing the Process, the National Academy of

Sciences recommended that Federal regulatory agencies establish

``inference guidelines'' to promote consistency and technical quality

in risk assessment, and to ensure that the risk assessment process is

maintained as a scientific effort separate from risk management. A task

force within EPA accepted that recommendation and requested that Agency

scientists begin to develop such guidelines. In 1984, EPA scientists

began work on risk assessment guidelines for carcinogenicity,

mutagenicity, suspect developmental toxicants, chemical mixtures, and

exposure assessment. Following extensive scientific and public review,

these first five guidelines were issued on September 24, 1986 (51 FR

33992-34054). Since 1986, additional risk assessment guidelines have

been proposed, revised, reproposed, and finalized. These guidelines

continue the process initiated in 1984. As with other EPA guidelines

(e.g., developmental toxicity, 56 FR 63798-63826; exposure assessment,

57 FR 22888-22938; and carcinogenicity, 61 FR 17960-18011), EPA will

revisit these guidelines as experience and scientific consensus evolve.

These Guidelines set forth principles and procedures to guide EPA

scientists in the conduct of Agency risk assessments and to inform

Agency decision makers and the public about these procedures. Policies

in this document are intended as internal guidance for EPA. Risk

assessors and risk managers at EPA are the primary audience, although

these Guidelines may be useful to others outside the Agency. In

particular, the Guidelines emphasize that risk assessments will be

conducted on a case-by-case basis, giving full consideration to all

relevant scientific information. This approach means that Agency

experts study scientific information on each chemical under review and

use the most scientifically appropriate interpretation to assess risk.

The Guidelines also stress that this information will be fully

presented in Agency risk assessment documents, and that Agency

scientists will identify the strengths and weaknesses of each

assessment by describing uncertainties, assumptions, and limitations,

as well as the scientific basis and rationale for each assessment. The

Guidelines are formulated in part to bridge gaps in risk assessment

methodology and data. By identifying these gaps and the importance of

the missing information to the risk assessment process, EPA wishes to

encourage research and analysis that will lead to new risk assessment

methods and data.

Dated: April 30, 1998.

Carol M. Browner,

Administrator.

Contents

Part A: Guidelines for Neurotoxicity Risk Assessment

List of Tables

1. Introduction

1.1. Organization of These Guidelines

1.2. The Role of Environmental Agents in Neurotoxicity

1.3. Neurotoxicity Risk Assessment

1.4. Assumptions

2. Definitions and Critical Concepts

3. Hazard Characterization

3.1. Neurotoxicological Studies: Endpoints and Their

Interpretation

3.1.1. Human Studies

3.1.1.1. Clinical Evaluations

3.1.1.2. Case Reports

3.1.1.3. Epidemiologic Studies

3.1.1.4. Human Laboratory Exposure Studies

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3.1.2. Animal Studies

3.1.2.1. Structural Endpoints of Neurotoxicity

3.1.2.2. Neurophysiological Endpoints of Neurotoxicity

3.1.2.3. Neurochemical Endpoints of Neurotoxicity

3.1.2.4. Behavioral Endpoints of Neurotoxicity

3.1.3. Other Considerations

3.1.3.1. Pharmacokinetics

3.1.3.2. Comparisons of Molecular Structure

3.1.3.3. Statistical Considerations

3.1.3.4. In Vitro Data in Neurotoxicology

3.1.3.5. Neuroendocrine Effects

3.2. Dose-Response Evaluation

3.3. Characterization of the Health-Related Database

4. Quantitative Dose-Response Analysis

4.1. LOAEL/NOAEL and BMD Determination

4.2. Determination of the Reference Dose or Reference

Concentration

5. Exposure Assessment

6. Risk Characterization

6.1. Overview

6.2. Integration of Hazard Characterization, Dose-Response

Analysis, and Exposure Assessment

6.3. Quality of the Database and Degree of Confidence in the

Assessment

6.4. Descriptors of Neurotoxicity Risk

6.4.1. Estimation of the Number of Individuals

6.4.2. Presentation of Specific Scenarios

6.4.3. Risk Characterization for Highly Exposed Individuals

6.4.4. Risk Characterization for Highly Sensitive or Susceptible

Individuals

6.5.5. Other Risk Descriptors

6.5. Communicating Results

6.6. Summary and Research Needs

References

Part B: Response to Science Advisory Board and Public Comments

1. Introduction

2. Response to Science Advisory Board Comments

3. Response to Public Comments

List of Tables

Table 1. Examples of possible indicators of a neurotoxic effect

Table 2. Neurotoxicants and disorders with specific neurological

targets

Table 3. Examples of neurophysiological measures of neurotoxicity

Table 4. Examples of neurotoxicants with known neurochemical

mechanisms

Table 5. Examples of measures in a representative functional

observational battery

Table 6. Examples of specialized behavioral tests to measure

neurotoxicity

Table 7. Examples of compounds or treatments producing developmental

neurotoxicity

Table 8. Characterization of the health-related database

Part A: Guidelines for Neurotoxicity Risk Assessment

1. Introduction

These Guidelines describe the principles, concepts, and procedures

that the U.S. Environmental Protection Agency (EPA) will follow in

evaluating data on potential neurotoxicity associated with exposure to

environmental toxicants. The Agency's authority to regulate substances

that have the potential to interfere with human health is derived from

a number of statutes that are implemented through multiple offices

within EPA. The procedures outlined here are intended to help develop a

sound scientific basis for neurotoxicity risk assessment, promote

consistency in the Agency's assessment of toxic effects on the nervous

system, and inform others of the approaches used by the Agency in those

assessments. This document is not a regulation and is not intended for

EPA regulations. The Guidelines set forth current scientific thinking

and approaches for conducting and evaluating neurotoxic risk

assessments. They are not intended, nor can they be relied upon, to

create any rights enforceable by any party in litigation with the

United States.

1.1. Organization of These Guidelines

This introduction (section 1) summarizes the purpose of these

Guidelines within the overall framework of risk assessment at EPA. It

also outlines the organization of the guidance and describes several

default assumptions to be used in the risk assessment process, as

discussed in the recent National Research Council report ``Science and

Judgment in Risk Assessment'' (NRC, 1994).

Section 2 sets forth definitions of particular terms widely used in

the field of neurotoxicology. These include ``neurotoxicity'' and

``behavioral alterations.'' Also included in this section are

discussions concerning reversible and irreversible effects and direct

versus indirect effects.

Risk assessment is the process by which scientific judgments are

made concerning the potential for toxicity in humans. The National

Research Council (NRC, 1983) has defined risk assessment as including

some or all of the following components (paradigm): hazard

identification, dose-response assessment, exposure assessment, and risk

characterization. In its 1994 report ``Science and Judgment in Risk

Assessment'' the NRC extended its view of the paradigm to include

characterization of each component (NRC, 1994). In addition, it noted

the importance of an approach that is less fragmented and more

holistic, less linear and more interactive, and that deals with

recurring conceptual issues that cut across all stages of risk

assessment. These Guidelines describe a more interactive approach by

organizing the process around the qualitative evaluation of the

toxicity data (hazard characterization), the quantitative dose-response

analysis, the exposure assessment, and the risk characterization. In

these Guidelines, hazard characterization includes deciding whether a

chemical has an effect by means of qualitative consideration of dose-

response relationships, route, and duration of exposure. Determining a

hazard often depends on whether a dose-response relationship is present

(Kimmel et al., 1990). This approach combines the information important

in comparing the toxicity of a chemical with potential human exposure

scenarios (section 3). In addition, it avoids the potential for

labeling chemicals as ``neurotoxicants'' on a purely qualitative basis.

This organization of the risk assessment process is similar to that

discussed in the Guidelines for Developmental Toxicity Risk Assessment

(56 FR 63798), the main difference being that the quantitative dose-

response analysis is discussed under a separate section in these

Guidelines.

Hazard characterization involves examining all available

experimental animal and human data and the associated doses, routes,

timing, and durations of exposure to determine qualitatively if an

agent causes neurotoxicity in that species and under what conditions.

From the hazard characterization and criteria provided in these

Guidelines, the health-related database can be characterized as

sufficient or insufficient for use in risk assessment (section 3.3).

Combining hazard identification and some aspects of dose-response

evaluation into hazard characterization does not preclude the

evaluation and use of data for other purposes when quantitative

information for setting reference doses (RfDs) and reference

concentrations (RfCs) is not available.

The next step in the dose-response analysis (section 4) is the

quantitative analysis, which includes determining the no-observed-

adverse-effect-level (NOAEL) and/or the lowest-observed-adverse-effect-

level (LOAEL) for each study and type of effect. Because of the

limitations associated with the use of the NOAEL, the Agency is

beginning to use an additional approach, the benchmark dose approach

(BMD) (Crump, 1984; U.S. EPA, 1995a), for

[[Page 26928]]

more quantitative dose-response evaluation when sufficient data are

available. The benchmark dose approach takes into account the

variability in the data and the slope of the dose-response curve, and

provides a more consistent basis for calculation of the RfD or RfC. If

data are considered sufficient for risk assessment, and if

neurotoxicity is the effect occurring at the lowest dose level (i.e.,

the critical effect), an oral or dermal RfD or an inhalation RfC, based

on neurotoxic effects, is then derived. This RfD or RfC is derived

using the NOAEL or benchmark dose divided by uncertainty factors to

account for interspecies differences in response, intraspecies

variability, and other factors of study design or the database. A

statement of the potential for human risk and the consequences of

exposure can come only from integrating the hazard characterization and

dose-response analysis with the human exposure estimates in the final

risk characterization.

The section on exposure assessment (section 5) identifies human

populations exposed or potentially exposed to an agent, describes their

composition and size, and presents the types, magnitudes, frequencies,

and durations of exposure to the agent. The exposure assessment

provides an estimate of human exposure levels for particular

populations from all potential sources.

In risk characterization (section 6), the hazard characterization,

dose-response analysis, and exposure assessment for given populations

are combined to estimate some measure of the risk for neurotoxicity. As

part of risk characterization, a summary of the strengths and

weaknesses of each component of the risk assessment is given, along

with major assumptions, scientific judgments and, to the extent

possible, qualitative and quantitative estimates of the uncertainties.

This characterization of the health-related database is always

presented in conjunction with information on the dose, route, duration,

and timing of exposure as well as the dose-response analysis including

the RfD or RfC. If human exposure estimates are available, the exposure

basis used for the risk assessment is clearly described, e.g., highly

exposed individuals or highly sensitive or susceptible individuals. The

NOAEL may be compared to the various estimates of human exposure to

calculate the margin(s) of exposure (MOE). The considerations for

judging the acceptability of the MOE are similar to those for

determining the appropriate size of the uncertainty factor for

calculating the RfD or RfC.

The Agency recently issued a policy statement and associated

guidance for risk characterization (U.S. EPA, 1995b, 1995c), which is

currently being implemented throughout EPA. This statement is designed

to ensure that critical information from each stage of a risk

assessment is used in forming conclusions about risk and that this

information is communicated from risk assessors to risk managers

(policy makers), from middle to upper management, and from the Agency

to the public. Additionally, the policy provides a basis for greater

clarity, transparency, reasonableness, and consistency in risk

assessments across Agency programs.

Final neurotoxicity risk assessment guidelines may reflect

additional changes in risk characterization practices resulting from

implementation activities. Risk assessment is just one component of the

regulatory process and defines the potential adverse health

consequences of exposure to a toxic agent. The other component, risk

management, combines risk assessment with statutory directives

regarding socioeconomic, technical, political, and other considerations

in order to decide whether to control future exposure to the suspected

toxic agent and, if so, the nature and level of control. One major

objective of these Guidelines is to help the risk assessor determine

whether the experimental animal or human data indicate the potential

for a neurotoxic effect. Such information can then be used to

categorize evidence that will identify and characterize neurotoxic

hazards, as described in section 3.3, Characterization of the Health-

Related Database, and Table 8 of these Guidelines. Risk management is

not dealt with directly in these Guidelines because the basis for

decision making goes beyond scientific considerations alone, but the

use of scientific information in this process is discussed. For

example, the acceptability of the MOE is a risk management decision,

but the scientific bases for establishing this value are discussed

here.

1.2. The Role of Environmental Agents in Neurotoxicity

Chemicals are an integral part of life, with the capacity to

improve as well as endanger health. The general population is exposed

to chemicals in air, water, foods, cosmetics, household products, and

drugs used therapeutically or illicitly. During daily life, a person

experiences a multitude of exposures to potentially neuroactive

substances, singly and in combination, both synthetic and natural.

Levels of exposure vary and may or may not pose a hazard, depending on

dose, route, and duration of exposure.

A link between human exposure to some chemical substances and

neurotoxicity has been firmly established (Anger, 1986; OTA, 1990).

Because many natural and synthetic chemicals are present in today's

environment, there is growing scientific and regulatory interest in the

potential for risks to humans from exposure to neurotoxic agents. If

sufficient exposure occurs, the effects resulting from such exposures

can have a significant adverse impact on human health. It is not known

how many chemicals may be neurotoxic in humans (Reiter, 1987). EPA's

TSCA inventory of chemical substances manufactured, imported, or

processed in the United States includes more than 65,000 substances and

is increasing yearly. An overwhelming majority of the materials in

commercial use have not been tested for neurotoxic potential (NRC,

1984).

Estimates of the number of chemicals with neurotoxic properties

have been made for subsets of substances. For instance, a large

percentage of the more than 500 registered active pesticide ingredients

affect the nervous system of the target species to varying degrees. Of

588 chemicals listed by the American Conference of Governmental

Industrial Hygienists, 167 affected the nervous system or behavior at

some exposure level (Anger, 1984). Anger (1990) estimated that of the

approximately 200 chemicals to which 1 million or more American workers

are exposed, more than one-third may have adverse effects on the

nervous system if sufficient exposure occurs. Anger (1984) also

recognized neurotoxic effects as one of the 10 leading workplace

disorders. A number of therapeutic substances, including some

anticancer and antiviral agents and abused drugs, can cause adverse or

neurotoxicological side effects at therapeutic levels (OTA, 1990). The

number of chemicals with neurotoxic potential has been estimated to

range from 3% to 28% of all chemicals (OTA, 1990). Thus, estimating the

risks of exposure to chemicals with neurotoxic potential is of concern

with regard to their overall impact on human health.

1.3. Neurotoxicity Risk Assessment

In addition to its primary role in psychological functions, the

nervous system controls most, if not all, other bodily processes. It is

sensitive to perturbation from various sources and has limited ability

to regenerate. There is evidence that even small anatomical,

biochemical, or physiological insults to the nervous system may result

in

[[Page 26929]]

adverse effects on human health. Therefore, there is a need for

consistent guidance on how to evaluate data on neurotoxic substances

and assess their potential to cause transient or persistent and direct

or indirect effects on human health.

These Guidelines develop principles and concepts in several areas.

They outline the scientific basis for evaluating effects due to

exposure to neurotoxicants and discuss principles and methods for

evaluating data from human and animal studies on behavior,

neurochemistry, neurophysiology, and neuropathology. They also discuss

adverse effects on neurological development and function in infants and

children following prenatal and perinatal exposure to chemical agents.

They outline the methods for calculating reference doses or reference

concentrations when neurotoxicity is the critical effect, discuss the

availability of alternative mathematical approaches to dose-response

analyses, characterize the health-related database for neurotoxicity

risk assessment, and discuss the integration of exposure information

with results of the dose-response assessment to characterize risks.

These Guidelines do not advocate developing reference doses specific

for neurotoxicity, but rather support the use of neurotoxicity as one

possible endpoint to develop reference doses. EPA offices have

published guidelines for neurotoxicity testing in animals (U.S. EPA,

1986, 1987, 1988a, 1991a). The testing guidelines address the

development of new data for use in risk assessment.

These neurotoxicity risk assessment guidelines provide the Agency's

first comprehensive guidance on the use and interpretation of

neurotoxicity data, and are part of the Agency's risk assessment

guidelines development process, which was initiated in 1984. As part of

its neurotoxicity guidelines development program, EPA has sponsored or

participated in several conferences on relevant issues (Tilson, 1990);

these and other sources (see references) provide the scientific basis

for these Guidelines.

This guidance is intended for use by Agency risk assessors and is

separate and distinct from the recently published document on

principles of neurotoxicity risk assessment (U.S. EPA, 1994). The

document on principles was prepared under the auspices of the

Subcommittee on Risk Assessment of the Federal Coordinating Council for

Science, Engineering, and Technology and was not intended to provide

specific directives for how neurotoxicity risk assessment should be

performed. It is expected that, like other EPA risk assessment

guidelines for noncancer endpoints (U.S. EPA, 1991b), this document

will encourage research and analysis leading to new risk assessment

methods and data, which in turn would be used to revise and improve the

Guidelines and better guide Agency risk assessors.

1.4. Assumptions

There are a number of unknowns in the extrapolation of data from

animal studies to humans. Therefore, a number of default assumptions

are made that are generally applied in the absence of data on the

relevance of effects to potential human risk. Default assumptions

should not be applied indiscriminately. First, all available

mechanistic and pharmacokinetic data should be considered. If these

data indicate that an alternative assumption is appropriate or if they

obviate the need for applying an assumption, such information should be

used in risk assessment. For example, research in rats may determine

that the neurotoxicity of a chemical is caused by a metabolite. If

subsequent research finds that the chemical is metabolized to a lesser

degree or not at all in humans, then this information should be used in

formulating the default assumptions. The following default assumptions

form the basis of the approaches taken in these Guidelines:

(1) It is assumed that an agent that produces detectable adverse

neurotoxic effects in experimental animal studies will pose a potential

hazard to humans. This assumption is based on the comparisons of data

for known human neurotoxicants (Anger, 1990; Kimmel et al., 1990;

Spencer and Schaumburg, 1980), which indicate that experimental animal

data are frequently predictive of a neurotoxic effect in humans.

(2) It is assumed that behavioral, neurophysiological,

neurochemical, and neuroanatomical manifestations are of concern. In

the past, the tendency has been to consider only neuropathological

changes as endpoints of concern. Based on data on agents that are known

human neurotoxicants (Anger, 1990; Kimmel et al., 1990; Spencer and

Schaumberg, 1980), there is usually at least one experimental species

that mimics the types of effects seen in humans, but in other species

tested, the neurotoxic effect may be different or absent. For example,

certain organophosphate compounds produce a delayed-onset neuropathy in

hens similar to that seen in humans, whereas rodents are

characteristically insensitive to these compounds. A biologically

significant increase in any of the manifestations is considered

indicative of an agent's potential for disrupting the structure or

function of the human nervous system.

(3) It is assumed that the neurotoxic effects seen in animal

studies may not always be the same as those produced in humans.

Therefore, it may be difficult to determine the most appropriate

species in terms of predicting specific effects in humans. The fact

that every species may not react in the same way is probably due to

species-specific differences in maturation of the nervous system,

differences in timing of exposure, metabolism, or mechanisms of action.

(4) It is also assumed that, in the absence of data to the

contrary, the most sensitive species is used to estimate human risk.

This is based on the assumption that humans are as sensitive as the

most sensitive animal species tested. This provides a conservative

estimate of sensitivity for added protection to the public. As with

other noncancer endpoints, it is assumed that there is a nonlinear

dose-response relationship for neurotoxicants. Although there may be a

threshold for neurotoxic effects, these are often difficult to

determine empirically. Therefore, a nonlinear relationship is assumed

to exist for neurotoxicants.

These assumptions are ``plausibly conservative'' (NRC, 1994) in

that they are protective of public health and are also well founded in

scientific knowledge about the effects of concern.

2. Definitions and Critical Concepts

This section defines the key terms and concepts that EPA will use

in the identification and evaluation of neurotoxicity. The various

health effects that fall within the broad classification of

neurotoxicity are described and examples are provided. Adverse effects

include alterations from baseline or normal conditions that diminish an

organism's ability to survive, reproduce, or adapt to the environment.

Neurotoxicity is an adverse change in the structure or function of the

central and/or peripheral nervous system following exposure to a

chemical, physical, or biological agent (Tilson, 1990). Functional

neurotoxic effects include adverse changes in somatic/autonomic,

sensory, motor, and/or cognitive function. Structural neurotoxic

effects are defined as neuroanatomical changes occurring at any level

of nervous system organization; functional changes are defined as

neurochemical, neurophysiological, or behavioral effects. Chemicals can

also be categorized into four classes: Those that act on the central

nervous system, the peripheral nerve fibers, the peripheral

[[Page 26930]]

nerve endings, or muscles or other tissues (Albert, 1973). Changes in

function can result from toxicity to other specific organ systems, and

these indirect changes may be considered adverse. For example, exposure

to a high dose of a chemical may cause damage to the liver, resulting

in general sickness and a decrease in a functional endpoint such as

motor activity. In this case, the change in motor activity could be

considered as adverse, but not necessarily neurotoxic. A discussion

concerning problems associated with risk assessment of high doses of

chemicals in the context of drinking water and health was published by

the National Research Council (1986).

The risk assessor should also know that there are different levels

of concern based on the magnitude of effect, duration of exposure, and

reversibility of some neurotoxic effects. Neurotoxic effects may be

irreversible (the organism cannot return to the state prior to

exposure, resulting in a permanent change) or reversible (the organism

can return to the pre-exposure condition). Clear or demonstrable

irreversible change in either the structure or function of the nervous

system causes greater concern than do reversible changes. If neurotoxic

effects are observed at some time during the lifespan of the organism

but are slowly reversible, the concern is also high. There is lesser

concern for effects that are rapidly reversible or ``transient,'' i.e.,

measured in minutes, hours, or days, and that appear to be associated

with the pharmacokinetics of the causal agent and its presence in the

body. Reversible changes that occur in the occupational setting or

environment, however, may be of high concern if, for example, exposure

to a short-acting solvent interferes with operation of heavy equipment

in an industrial plant. The context of the exposure should be

considered in evaluating reversible effects. Setting of exposure limits

is not always associated with the determination of a reference dose,

which is based on chronic dosing. Data from acute or subacute dosing

can be used for health advisories or in studies involving developmental

exposures.

It should also be noted that the nervous system is known for its

reserve capacity (Tilson and Mitchell, 1983). That is, repeated insult

to the nervous system could lead to an adaptation. There are, however,

limits to this capacity, and when these limits are exceeded, further

exposure could lead to frank manifestations of neurotoxicity at the

structural or functional level. The risk assessor should be aware that

once damaged, neurons, particularly in the central nervous system, have

a limited capacity for regeneration. Reversibility of effects resulting

from cell death or from the destruction of cell processes may represent

an activation of repair capacity, decreasing future potential

adaptability. Therefore, even reversible neurotoxic changes should be

of concern. Evidence of progressive effects (those that continue to

worsen even after the causal agent has been removed), delayed-onset

effects (those that occur at a time distant from the last contact with

the causal agent), residual effects (those that persist beyond a

recovery period), or latent effects (those that become evident only

after an environmental challenge or aging) have a high level of

concern.

Environmental challenges can include stress, increased physical or

cognitive workload, pharmacological manipulations, and nutritional

deficiency or excess. Evidence for reversibility may depend on the

region of the nervous system affected, the chemical involved, and

organismic factors such as the age of the exposed population. Some

regions of the nervous system, such as peripheral nerves, have a high

capacity for regeneration, while regions in the brain such as the

hippocampus are known for their ability to compensate or adapt to

neurotoxic insult. For example, compensation is likely to be seen with

solvents (e.g., n-hexane) that produce peripheral neuropathy because of

the repair capacity of the peripheral nerve. In addition, tolerance to

some cholinergic effects of cholinesterase-inhibiting compounds may be

due to compensatory down-regulation of muscarinic receptors. Younger

individuals may have more capacity to adapt than older individuals,

suggesting that the aged may be at greater risk to neurotoxic exposure.

Neurotoxic effects can be observed at various levels of

organization of the nervous system, including neurochemical,

anatomical, physiological, or behavioral. At the neurochemical level,

for example, an agent that causes neurotoxicity might inhibit

macromolecule or transmitter synthesis, alter the flow of ions across

cellular membranes, or prevent release of neurotransmitter from the

nerve terminals. Anatomical changes may include alterations of the cell

body, the axon, or the myelin sheath. At the physiological level, a

chemical might change the thresholds for neural activation or reduce

the speed of neurotransmission. Behavioral alterations can include

significant changes in sensations of sight, hearing, or touch;

alterations in simple or complex reflexes and motor functions;

alterations in cognitive functions such as learning, memory, or

attention; and changes in mood, such as fear or rage, disorientation as

to person, time, or place, or distortions of thinking and feeling, such

as delusions and hallucinations. At present, relatively few neurotoxic

syndromes have been thoroughly characterized in terms of the initial

neurochemical change, structural alterations, physiological

consequence, and behavioral effects. Knowledge of exact mechanisms of

action is not, however, necessary to conclude that a chemically induced

change is a neurotoxic effect.

Neurotoxic effects can be produced by chemicals that do not require

metabolism prior to interacting with their sites in the nervous system

(primary neurotoxic agents) or those that require metabolism prior to

interacting with their sites (secondary neurotoxic agents). Chemically

induced neurotoxic effects can be direct (due to an agent or its

metabolites acting directly on sites in the nervous system) or indirect

(due to agents or metabolites that produce their effects primarily by

interacting with sites outside the nervous system). For example,

excitatory amino acids such as domoic acid damage specific neurons

directly by activating excitatory amino acid receptors in the nervous

system, whereas carbon monoxide decreases oxygen availability, which

can indirectly kill neurons. Other examples of indirect effects include

cadmium-induced spasms in blood vessels supplying the nervous system,

dichloroacetate-induced perturbation of metabolic pathways, and

chemically induced alterations in skeletomuscular function or structure

and effects on the endocrine system. Professional judgment may be

required in making determinations about direct versus indirect effects.

The interpretation of data as indicative of a potential neurotoxic

effect involves the evaluation of the validity of the database. This

approach and these terms have been adapted from the literature on human

psychological testing (Sette, 1987; Sette and MacPhail, 1992), where

they have long been used to evaluate the level of confidence in

different measures of intelligence or other abilities, aptitudes, or

feelings. There are four principal questions that should be addressed:

whether the effects result from exposure (content validity); whether

the effects are adverse or toxicologically significant (construct

validity); whether there are correlative measures among behavioral,

physiological, neurochemical, and

[[Page 26931]]

morphological endpoints (concurrent validity); and whether the effects

are predictive of what will happen under various conditions (predictive

validity). Addressing these issues can provide a useful framework for

evaluating either human or animal studies or the weight of evidence for

a chemical (Sette, 1987; Sette and MacPhail, 1992). The next sections

indicate the extent to which chemically induced changes can be

interpreted as providing evidence of neurotoxicity.

3. Hazard Characterization

3.1. Neurotoxicological Studies: Endpoints and Their Interpretation

The qualitative characterization of neurotoxic hazard can be based

on either human or animal data (Anger, 1984; Reiter, 1987; U.S. EPA,

1994). Such data can result from accidental, inappropriate, or

controlled experimental exposures. This section describes many of the

general and some of the specific characteristics of human studies and

reports of neurotoxicity. It then describes some features of animal

studies of neuroanatomical, neurochemical, neurophysiological, and

behavioral effects relevant to risk assessment. The process of

characterizing the sufficiency or insufficiency of neurotoxic effects

for risk assessment is described in section 3.3. Additional sources of

information relevant to hazard characterization, such as comparisons of

molecular structure among compounds and in vitro screening methods, are

also discussed.

The hazard characterization should:

a. Identify strengths and limitations of the database:

Epidemiological studies (case reports, cross-sectional,

case-control, cohort, or human laboratory exposure studies);

Animal studies (including structural or neuropathological,

neurochemical, neurophysiological, behavioral or neurological, or

developmental endpoints).

b. Evaluate the validity of the database:

Content validity (effects result from exposure);

Construct validity (effects are adverse or toxicologically

significant);

Concurrent validity (correlative measures among

behavioral, physiological, neurochemical, or morphological endpoints);

Predictive validity (effects are predictive of what will

happen under various conditions).

c. Identify and describe key toxicological studies.

d. Describe the type of effects:

Structural (neuroanatomical alternations);

Functional (neurochemical, neurophysiological, behavioral

alterations).

e. Describe the nature of the effects (irreversible, reversible,

transient, progressive, delayed, residual, or latent).

f. Describe how much is known about how (through what biological

mechanism) the chemical produces adverse effects.

g. Discuss other health endpoints of concern.

h. Comment on any nonpositive data in humans or animals.

I. Discuss the dose-response data (epidemiological or animal)

available for further dose-response analysis.

j. Discuss the route, level, timing, and duration of exposure in

studies demonstrating neurotoxicity as compared to expected human

exposures.

k. Summarize the hazard characterization:

Confidence in conclusions;

Alternative conclusions also supported by the data;

Significant data gaps; and

Highlights of major assumptions.

3.1.1. Human Studies

It is well established that information from the evaluation of

human exposure can identify neurotoxic hazards (Anger and Johnson,

1985; Anger, 1990). Prominent among historical episodes of

neurotoxicity in human populations are the outbreaks of methylmercury

poisoning in Japan and Iraq and the neurotoxicity seen in miners of

metals, including mercury, manganese, and lead (Carson et al., 1987;

Silbergeld and Percival, 1987; OTA, 1990). In the past decade, lead

poisoning in children has been a prominent issue of concern (Silbergeld

and Percival, 1987). Neurotoxicity in humans has been studied and

reviewed for many pesticides (Hayes, 1982; NRDC, 1989; Ecobichon and

Joy, 1982; Ecobichon et al., 1990). Organochlorines, organophosphates,

carbamates, pyrethroids, certain fungicides, and some fumigants are all

known neurotoxicants. They may pose occupational risks to manufacturing

and formulation workers, pesticide applicators and farm workers, and

consumers through home application or consumption of residues in foods.

Families of workers may also be exposed by transport into the home from

workers' clothing. Data on humans can come from a number of sources,

including clinical evaluations, case reports, epidemiologic studies,

and human laboratory exposure studies. A more extensive description of

issues concerning human neurotoxicology and risk assessment has been

published elsewhere (U.S. EPA, 1993). A review of the types of tests

used to assess cognitive and neurological function in children, in

addition to a discussion of methodological issues in the design of

prospective, longitudinal studies of developmental neurotoxicity in

humans, has recently been published (Jacobson and Jacobson, 1996).

Stanton and Spear (1990) reviewed assessment measures used in

developmental neurotoxicology for their comparability in humans and

laboratory animals and their ability to detect comparable adverse

effects across species. At the level of the various functional

assessments for sensory, motivational, cognitive and motor function,

and social behavior, there was good agreement across species among the

neurotoxic agents reviewed.

3.1.1.1. Clinical Evaluations

Clinical methods are used extensively in neurology and

neuropsychology to evaluate patients suspected of having neurotoxicity.

An array of examiner-administered and paper-and-pencil tasks are used

to assess sensory, motor, cognitive, and affective functions and

personality states/traits. Neurobehavioral data are synthesized with

information from neurophysiological studies and medical history to

derive a working diagnosis. Brain functional imaging techniques based

on magnetic resonance imaging or emission tomography may also be useful

in helping diagnose neurodegenerative disorders following chemical

exposures in humans (Omerand et al., 1994; Callender et al., 1994).

Clinical diagnostic approaches have provided a rich conceptual

framework for understanding the functions (and malfunctions) of the

central and peripheral nervous systems and have formed the basis for

the development of methods for measuring the behavioral expression of

nervous system disorders. Human neurobehavioral toxicology has borrowed

heavily from neurology and neuropsychology for concepts of nervous

system impairment and functional assessment methods. Neurobehavioral

toxicology has adopted the neurologic/neuropsychologic model, using

adverse changes in behavioral function to assist in identifying

chemical-or drug-induced changes in nervous system processes.

Neurological and neuropsychological methods have long been employed

to identify the adverse health effects of environmental workplace

exposures (Sterman and Schaumburg, 1980).

[[Page 26932]]

Peripheral neuropathies (with sensory and motor disturbances),

encephalopathies, organic brain syndromes, extrapyramidal syndromes,

demyelination, autonomic changes, and dementia are well-characterized

consequences of acute and chronic exposure to chemical agents. The

range of exposure conditions that produce clinical signs of

neurotoxicity also has been defined by these clinical methods. It is

very important to make external/internal dose measurements in humans to

determine the actual dose(s) that can cause unwanted effects.

Aspects of the neurological examination approach limit its

usefulness for neurotoxicological risk assessment. Information obtained

from the neurological exam is mostly qualitative and descriptive rather

than quantitative. Estimates of the severity of functional impairment

can be reliably placed into only three or four categories (for example,

mild, moderate, severe). Much of the assessment depends on the

subjective judgment of the examiner. For example, the magnitude and

symmetry of muscle strength are often judged by having the patient push

against the resistance of the examiner's hands. The endpoints are

therefore the absolute and relative amount of muscle load sensed by the

examiner in his or her arms.

Compared with other methods, the neurological exam may be less

sensitive in detecting early neurotoxicity in peripheral sensory and

motor nerves. While clinicians' judgments are equal in sensitivity to

quantitative methods in assessing the amplitude of tremor, tremor

frequency is poorly quantified by clinicians. Thus, important aspects

of the clinical neurologic exam may be insufficiently quantified and

lack sufficient sensitivity for detecting early neurobehavioral

toxicity produced by environmental or workplace exposure conditions.

However, a neurological evaluation of persons with documented

neurobehavioral impairment would be helpful for identifying nonchemical

causes of neurotoxicity, such as diabetes and cardiovascular

insufficiency.

Administration of a neuropsychological battery also requires a

trained technician, and interpretation requires a trained and

experienced neuropsychologist. Depending on the capabilities of the

patient, 2 to 4 hours may be needed to administer a full battery; 1

hour may be needed for the shorter screening versions. These practical

considerations may limit the usefulness of neuropsychological

assessment in large field studies of suspected neurotoxicity.

In addition to logistical problems in administration and

interpretation, neuropsychological batteries and neurological exams

share two disadvantages with respect to neurotoxicity risk assessment.

First, neurological exams and neuropsychological test batteries are

designed to confirm and classify functional problems in individuals

selected on the basis of signs and symptoms identified by the patient,

family, or other health professionals. Their usefulness in detecting

low base-rate impairment in workers or the general population is

generally thought to be limited, decreasing the usefulness of clinical

assessment approaches for epidemiologic risk assessment.

Second, neurological exams and neuropsychological test batteries

were developed to assess the functional correlates of the most common

forms of nervous system dysfunction: brain trauma, focal lesions, and

degenerative conditions. The clinical tests were validated against

these neurological disease states. With a few notable exceptions,

chemicals are not believed to produce impairment similar to that from

trauma or lesions; neurotoxic effects are more similar to the effects

of degenerative disease. There has been insufficient research to

demonstrate which tests designed to assess functional expression of

neurologic disease are useful in characterizing the modes of central

nervous system impairment produced by chemical agents and drugs.

It should be noted that alternative approaches are available that

avoid many of the limitations of clinical and neurological and

traditional neuropsychological methods. Computerized behavioral

assessment systems designed for field testing of populations exposed to

chemicals in the community or workplace have been developed during the

past decade. The most widely used system is the Neurobehavioral

Evaluation System (NES) developed by Baker et al. (1985). Advantages of

computerized tests include (1) standardized administration to eliminate

intertester variability and minimize subject-experimenter interaction;

(2) automated data collection and scoring, which is faster, easier, and

less error-prone than traditional methods; and (3) test administration

requires minimal training and experience. NES tests have proven

sensitive to a variety of solvents, metals, and pesticides (Otto,

1992). Computerized systems available for human neurotoxicity testing

are critically reviewed in Anger et al. (1996).

3.1.1.2. Case Reports

The first type of human data available is often the case report or

case series, which can identify cases of a disease and are reported by

clinicians or discerned through active or passive surveillance, usually

in the workplace. However, case reports involving a single neurotoxic

agent, although informative, are rare in the literature; for example,

farmers are likely to be exposed to a wide variety of potentially

neurotoxic pesticides. Careful case histories assist in identifying

common risk factors, especially when the association between the

exposure and disease is strong, the mode of action of the agent is

biologically plausible, and clusters occur in a limited period of time.

Case reports can be obtained more quickly than more complex

studies. Case reports of acute high-level exposure to a toxicant can be

useful for identifying signs and symptoms that may also apply to lower

exposure. Case reports can also be useful when corroborating

epidemiological data are available.

3.1.1.3. Epidemiologic Studies

Epidemiology has been defined as ``the study of the distributions

and determinants of disease and injuries in human populations''

(Mausner and Kramer, 1985). Knowing the frequency of illness in groups

and the factors that influence the distribution is the tool of

epidemiology that allows the evaluation of causal inference with the

goal of prevention and cure of disease (Friedlander and Hearn, 1980).

Epidemiologic studies are a useful means of evaluating the effects of

neurotoxic substances on human populations, particularly if effects of

exposure are cumulative or exposures are repeated. Such studies are

less useful in cases of acute exposure, where the effects are short-

term. Frequently, determining the precise dose or exposure

concentration in epidemiological studies can be difficult.

3.1.1.3.1. Cross-Sectional Studies.

In cross-sectional studies or surveys, both the disease and

suspected risk factors are ascertained at the same time, and the

findings are useful in generating hypotheses. A group of people are

interviewed, examined, and tested at a single point in time to

ascertain a relationship between a disease and a neurotoxic exposure.

This study design does not allow the investigator to determine whether

the disease or the exposure came first, rendering it less useful in

estimating risk. These studies are intermediate in cost and time

[[Page 26933]]

required to complete compared with case reports and more complex

analytical studies, but should be augmented with additional data.

3.1.1.3.2. Case-Control (Retrospective) Studies.

Last (1986) defines a case-control study as one that ``starts with

the identification of persons with the disease (or other outcome

variable) of interest, and a suitable control population (comparison,

reference group) of persons without the disease.'' He states that the

relationship of an ``attribute'' to the disease is measured by

comparing the diseased with the nondiseased with regard to how

frequently the attribute is present in each of the groups. The cases

are assembled from a population of persons with and without exposure,

and the comparison group is selected from the same population; the

relative distribution of the potential risk factor (exposure) in both

groups is evaluated by computing an odds ratio that serves as an

estimate of the strength of the association between the disease and the

potential risk factor. The statistical significance of the ratio is

determined by calculating a p-value and is used to approximate relative

risk.

The case-control approach to the study of potential neurotoxicants

in the environment provides a great deal of useful information for the

risk assessor. In his textbook, Valciukas (1991) notes that the case-

control approach is the strategy of choice when no other environmental

or biological indicator of neurotoxic exposure is available. He further

states: ``Considering the fact that for the vast majority of neurotoxic

chemical compounds, no objective biological indicators of exposure are

available (or if they are, their half-life is too short to be of any

practical value), the case-control paradigm is a widely accepted

strategy for the assessment of toxic causation.'' The case-control

study design, however, can be very susceptible to bias. The potential

sources of bias are numerous and can be specific to a particular study.

Many of these biases also can be present in cross-sectional studies.

For example, recall bias or faulty recall of information by study

subjects in a questionnaire-based study can distort the results.

Analysis of the case-comparison study design assumes that the selected

cases are representative persons with the disease--either all cases

with the disease or a representative sample of them have been

ascertained. It further assumes that the control or comparison group is

representative of the nonexposed population (or that the prevalence of

the characteristic under study is the same in the control group as in

the general population). Failure to satisfy these assumptions may

result in selection bias that may invalidate study results.

An additional source of bias in case-control studies is the

presence of confounding variables, i.e., factors known to be associated

with the exposure and causally related to the disease under study.

These should be controlled, either in the design of the study by

matching cases to controls on the basis of the confounding factor, or

in the analysis of the data by using statistical techniques such as

stratification or regression. Matching requires time to identify an

adequate number of potential controls to distinguish those with the

proper characteristics, while statistical control of confounding

factors requires a larger study.

The definition of exposure is critical in epidemiologic studies. In

occupational settings, exposure assessment often is based on the job

assignment of the study subjects, but can be more precise if detailed

company records allow the development of exposure profiles. Positive

results from a properly controlled retrospective study should weigh

heavily in the risk assessment process.

3.1.1.3.3. Cohort (Prospective, Follow-Up) Studies.

In a prospective study design, a healthy group of people is

assembled and followed forward in time and observed for the development

of dysfunction. Such studies are invaluable for determining the time

course for development of dysfunction (e.g., follow-up studies

performed in various cities on the effects of lead on child

development). This approach allows the direct estimate of risks

attributed to a particular exposure, since toxic incidence rates in the

cohort can be determined. Prospective study designs also allow the

study of chronic effects of exposure. One major strength of the cohort

design is that it allows the calculation of rates to determine the

excess risk associated with an exposure. Also, biases are reduced by

obtaining information before the disease develops. This approach,

however, can be very time-consuming and costly.

In cohort studies information bias can be introduced when

individuals provide distorted information about their health because

they know their exposure status and may have been told of the expected

health effects of the exposure under study. More credence should be

given to those studies in which both observer and subject bias are

carefully controlled (e.g., double-blind studies).

A special type of cohort study is the retrospective cohort study,

in which the investigator goes back in time to select the study groups

and traces them over time, often to the present. The studies usually

involve specially exposed groups and have provided much assistance in

estimating risks due to occupational exposures. Occupational

retrospective cohort studies rely on company records of past and

current employees that include information on the dates of employment,

age at employment, date of departure, and whether diseased (or dead in

the case of mortality studies). Workers can then be classified by

duration and degree of exposure. Positive or negative results from a

properly controlled prospective study should weigh heavily in the risk

assessment process.

3.1.1.4. Human Laboratory Exposure Studies

Neurotoxicity assessment has an advantage not afforded to the

evaluation of other toxic endpoints, such as cancer or reproductive

toxicity, in that the effects of some chemicals are short in duration

and reversible. This makes it ethically possible to perform human

laboratory exposure studies and obtain data relevant to the risk

assessment process. Information from experimental human exposure

studies has been used to set occupational exposure limits, mostly for

organic solvents that can be inhaled. Laboratory exposure studies have

contributed to risk assessment and the setting of exposure limits for

several solvents and other chemicals with acute reversible effects.

Human exposure studies sometimes offer advantages over

epidemiologic field studies. Combined with appropriate sampling of

biological fluids (urine or blood), it is possible to calculate body

concentrations, examine toxicokinetics, and identify metabolites.

Bioavailability, elimination, dose-related changes in metabolic

pathways, individual variability, time course of effects, interactions

between chemicals, and interactions between chemical and environmental/

biobehavioral processes (stressors, workload/respiratory rate) are

factors that are generally easier to collect under controlled

conditions.

Other goals of laboratory studies include the in-depth

characterization of effects, the development of new assessment methods,

and the examination of the sensitivity, specificity, and reliability of

neurobehavioral assessment methods across chemical classes. The

laboratory is the most appropriate setting for the

[[Page 26934]]

study of environmental and biobehavioral variables that affect the

action of chemical agents. The effects of ambient temperature, task

difficulty, rate of ongoing behavior, conditioning variables,

tolerance/sensitization, sleep deprivation, motivation, and so forth

are sometimes studied.

From a methodological standpoint, human laboratory studies can be

divided into two categories: between-subjects and within-subjects

designs. In the former, the neurobehavioral performance of exposed

volunteers is compared with that of nonexposed participants. In the

latter, preexposure performance is compared with neurobehavioral

function under the influence of the chemical or drug. Within-subjects

designs have the advantage of requiring fewer participants, eliminating

individual differences as a source of variability, and controlling for

chronic mediating variables, such as caffeine use and educational

achievement. A disadvantage of the within-subjects design is that

neurobehavioral tests must be administered more than once. Practice on

many neurobehavioral tests often leads to improved performance that may

confound the effect of the chemical/drug. There should be a sufficient

number of test sessions in the pre-exposure phase to allow performance

on all tests to achieve a relatively stable baseline level.

Participants in laboratory exposure studies may have been recruited

from populations of persons already exposed to the chemical/drug or

from chemical-naive populations. Although the use of exposed volunteers

has ethical advantages, can mitigate against novelty effects, and

allows evaluation of tolerance/sensitization, finding an accessible

exposed population in reasonable proximity to the laboratory can be

difficult. Chemical-naive participants are more easily recruited but

may differ significantly in important characteristics from a

representative sample of exposed persons. Chemical-naive volunteers are

often younger, healthier, and better educated than the populations

exposed environmentally, in the workplace, or pharmacotherapeutically.

Compared with workplace and environmental exposures, laboratory

exposure conditions can be controlled more precisely, but exposure

periods are much shorter. Generally only one or two relatively pure

chemicals are studied for several hours, whereas the population of

interest may be exposed to multiple chemicals containing impurities for

months or years. Laboratory studies are therefore better at identifying

and characterizing effects with acute onset and the selective effects

of pure agents. In all cases, the potential for participant bias should

be as carefully controlled for as possible. Even the consent form can

lead to participant bias, as toxic effects have been reported in some

individuals who were warned of such effects in an informed consent

form. In addition, double-blind studies have been shown to provide some

control for observer bias that may occur in single-blind studies. More

credence should be given to those studies in which both observer and

subject bias are carefully controlled (Benignus, 1993).

A test battery that examines multiple neurobehavioral functions may

be more useful for screening and the initial characterization of acute

effects. Selected neurobehavioral tests that measure a limited number

of functions in multiple ways may be more useful for elucidating

mechanisms or validating specific effects.

Both chemical and behavioral control procedures are valuable for

examining the specificity of the effects. A concordant effect among

different measures of the same neurobehavioral function (e.g., reaction

time) and a lack of effect on some other measures of psychomotor

function (e.g., untimed manual dexterity) would increase the confidence

in a selective effect on motor speed and not on attention or another

nonspecific motor function. Likewise, finding concordant effects among

similar chemical or drug classes along with different effects from

dissimilar classes would support the specificity of chemical effect.

For example, finding that the effects of a solvent were similar to

those of ethanol but not caffeine would support the specificity of

solvent effects on a given measure of neurotoxicity.

3.1.2. Animal Studies

This section provides an overview of the major types of endpoints

that may be evaluated in animal neurotoxicity studies, describes the

kinds of effects that may be observed and some of the tests used to

detect and quantify these effects, and provides guidance for

interpreting data. Compared with human studies, animal studies are more

often available for specific chemicals, provide more precise exposure

information, and control environmental factors better (Anger, 1984).

For these reasons, risk assessments tend to rely heavily on animal

studies.

Many tests that can measure some aspect of neurotoxicity have been

used in the field of neurobiology in the past 50 years. The Office of

Prevention, Pesticides and Toxic Substances (OPPTS) has published

animal testing guidelines that were developed in cooperation with the

Office of Research and Development (U.S. EPA, 1991a). While the test

endpoints included in the 1991 document serve as a convenient focus for

this section, there are many other endpoints for which there are no

current EPA guidelines. The goal of the current document is to provide

a framework for interpreting data collected in tests frequently used by

neurotoxicologists.

Five categories of endpoints will be described: structural or

neuropathological, neurophysiological, neurochemical, behavioral, and

developmental. Table 1 lists a number of endpoints in each of these

categories. It is imperative for the risk assessor to understand that

the interpretation of the indicators listed in Table 1 as neurotoxic

effects is dependent on the dose at which such changes occur and the

possibility that damage to other organ systems may contribute to or

cause such changes indirectly.

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

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

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

Structural or neuropathological endpoints:

Gross changes in morphology, including brain weight.

Histologic changes in neurons or glia (neuronopathy, axonopathy,

myelinopathy).

Neurochemical endpoints:

Alterations in synthesis, release, uptake, degradation of

neurotransmitters.

Alterations in second-messenger-associated signal transduction.

Alterations in membrane-bound enzymes regulating neuronal activity.

Inhibition and aging of neuropathy enzyme.

Increases in glial fibrillary acidic protein in adults.

Neurophysiological endpoints:

Change in velocity, amplitude, or refractory period of nerve

conduction.

[[Page 26935]]

Change in latency or amplitude of sensory-evoked potential.

Change in electroencephalographic pattern.

Behavioral and neurological endpoints:

Increases or decreases in motor activity.

Changes in touch, sight, sound, taste, or smell sensations.

Changes in motor coordination, weakness, paralysis, abnormal

movement or posture, tremor, ongoing performance.

Absence or decreased occurrence, magnitude, or latency of

sensorimotor reflex.

Altered magnitude of neurological measurement, including grip

strength, hindlimb splay.

Seizures.

Changes in rate or temporal patterning of schedule-controlled

behavior.

Changes in learning, memory, and attention.

Developmental endpoints:

Chemically induced changes in the time of appearance of behaviors

during development.

Chemically induced changes in the growth or organization of

structural or neurochemical elements.

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

3.1.2.1. Structural Endpoints of Neurotoxicity

Structural endpoints are typically defined as neuropathological

changes evident by gross observation or light microscopy, although most

neurotoxic changes will be detectable only at the light microscopic

level. Gross changes in morphology can include discrete or widespread

lesions in nerve tissue. A change in brain weight is considered to be a

biologically significant effect. This is true regardless of changes in

body weight, because brain weight is generally protected during

undernutrition or weight loss, unlike many other organs or tissues. It

is inappropriate to express brain weight changes as a ratio of body

weight and thereby dismiss changes in absolute brain weight. Changes in

brain weight are a more reliable indicator of alteration in brain

structure than are measurements of length or width in fresh brain,

because there is little historical data in the toxicology literature.

Neurons are composed of a neuronal body, axon, and dendritic

processes. Various types of neuropathological lesions may be classified

according to the site where they occur (Spencer and Schaumburg, 1980;

WHO, 1986; Krinke, 1989; Griffin, 1990). Neurotoxicant-induced lesions

in the central or peripheral nervous system may be classified as a

neuronopathy (changes in the neuronal cell body), axonopathy (changes

in the axons), myelinopathy (changes in the myelin sheaths), or nerve

terminal degeneration. Nerve terminal degeneration represents a very

subtle change that may not be detected by routine histopathology, but

requires detection by special procedures such as silver staining or

neurotransmitter-specific immunohistochemistry. For axonopathies, a

more precise location of the changes may also be described (i.e.,

proximal, central, or distal axonopathy). In the case of some

developmental exposures, a neurotoxic chemical might delay or

accelerate the differentiation or proliferation of cells or cell types.

Alteration in the axonal termination site might also occur with

exposure. In an aged population, exposure to some neurotoxicants might

accelerate the normal loss of neurons associated with aging (Reuhl,

1991). In rare cases, neurotoxic agents have been reported to produce

neuropathic conditions resembling neurodegenerative disorders, such as

Parkinson's disease, in humans (WHO, 1986). Table 2 lists examples of

such neurotoxic chemicals, their putative site of action, the type of

neuropathology produced, and the disorder or condition that each

typifies. Inclusion of any chemical in any of the following tables is

for illustrative purposes, i.e., it has been reported that the chemical

will produce a neurotoxic effect at some dose; any individual chemical

listed may also adversely affect other organs at lower doses. It is

important that the severity of each structural union be graded

objectively and the grading criteria reported.

Table 2.--Neurotoxicants and Disorders With Specific Neurological Targets

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

Corresponding

Site of action Neurotoxic change Neurotoxic chemical neurodegenerative

disorder

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

Neuron cell body.................... Neuronopathy........... Methylmercury.......... Minamata disease.

Quinolinic acid........ Huntington's disease.

3-Acetylpyridine....... Cerebellar ataxia.

Nerve terminal...................... Terminal destruction... 1-Methyl-4-phenyl 1,2,. Parkinson's disease.

3,6-tetrahydro-........

pyridine (MPTP)

(dopaminergic).

Schwann cell myelin................. Myelinopathy........... Hexachlorophene........ Congenital

hypomyelinogenesis.

Centra-peripheral distal axon....... Distal axonopathy...... Acrylamide, carbon Peripheral neuropathy.

disulfide, n-hexane.

Central axons....................... Central axonopathy..... Clioquinol............. Subacute

myeloopticoneuro-pathy.

Proximal axon....................... Proximal axonopathy.... B,B'- Motor neuron disease.

Iminodipropionitrile.

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

Alterations in the structure of the nervous system (i.e.,

neuronopathy, axonopathy, myelinopathy, terminal degeneration) are

regarded as evidence of a neurotoxic effect. The risk assessor should

note that pathological changes in many cases require time for the

perturbation to become observable, especially with evaluation at the

light microscopic level. Neuropathological studies should control for

potential differences in the area(s) and section(s) of the nervous

system sampled; in the age, sex, and body weight of the subject; and in

fixation artifacts (WHO, 1986). Concern for the structural integrity of

nervous system tissues derives from

[[Page 26936]]

their functional specialization and lack of regenerative capacity.

Within general class of nervous system structural alteration, there

are various histological changes that can result after exposure to

neurotoxicants. For example, specific changes in nerve cell bodies

include chromatolysis, vacuolization, and cell death. Axons can undergo

swelling, degeneration, and atrophy, while myelin sheath changes

include folding, edematous splitting, and demyelination. Although

terminal degeneration does occur, it is not readily detectable by light

microscopy. Many of these changes are a result of complex effects at

specific subcellular organelles, such as the axonal swelling that

occurs as a result of neurofilament accumulation in acrylamide

toxicity. Other changes may be associated with regenerative or adaptive

processes that occur after neurotoxicant exposure.

3.1.2.2. Neurophysiological Endpoints of Neurotoxicity

Neurophysiological studies measure the electrical activity of the

nervous system. The term ``neurophysiology'' is often used synonymously

with ``electrophysiology'' (Dyer, 1987). Neurophysiological techniques

provide information on the integrity of defined portions of the nervous

system. Several neurophysiological procedures are available for

application to neurotoxicological studies. Examples are listed in Table

3. They range in scale from procedures that employ microelectrodes to

study the function of single nerve cells or restricted portions of

them, to procedures that employ macroelectrodes to perform simultaneous

recordings of the summed activity of many cells. Microelectrode

procedures typically are used to study mechanisms of action and are

frequently performed in vitro. Macroelectrode procedures are generally

used in studies to detect or characterize the potential neurotoxic

effects of agents of interest because of potential environmental

exposure. The present discussion concentrates on macroelectrode

neurophysiological procedures because it is more likely that they will

be the focus of decisions regarding critical effects in risk

assessment. All of the procedures described below for use in animals

also have been used in humans to determine chemically induced

alterations in neurophysiological function.

Table 3.--Examples of Neurophysiological Measures of Neurotoxicity

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

Representative

System/function Procedure agents

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

Retina.......................... Electroretinograph Developmental

y (ERG). lead.

Visual pathway.................. Flash-evoked Carbon disulfide.

potential (FEP).

Visual function................. Pattern-evoked Carbon disulfide.

potential (PEP)

(pattern size and

contrast).

Auditory pathway................ Brain stem Aminoglycoside,

auditory evoked antibiotics,

potential (BAER) toluene, styrene.

(clicks).

Auditory function............... BAER (tones)...... Aminoglycoside,

antibiotics,

toluene, styrene.

Somatosensory pathway........... Somatosensory Acrylamide, n-

provoked. hexane.

Somatosensory function.......... Sensory-evoked Acrylamide, n-

potential (SEP) hexane.

(tactile).

Spinocerebellar pathway......... SEP recorded from Acrylamide, n-

cerebellum. hexane.

Mixed nerve..................... Peripheral nerve Triethyltin.

compound action

potential (PNAP).

Motor axons..................... PNAP isolate motor Triethyltin.

components.

Sensory axons................... PNAP isolate Triethyltin.

sensory

components.

Neuromuscular................... Electromyography Dithiobiuret.

(EMG).

General central nervous system/ Electroencephalogr Toluene.

level of arousal. aphy (EEG).

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

3.1.2.2.1. Nerve Conduction Studies. Nerve conduction studies,

generally performed on peripheral nerves, can be useful in

investigations of possible peripheral neuropathy. Most peripheral

nerves contain mixtures of individual sensory and motor nerve fibers,

which may or may not be differentially sensitive to neurotoxicants. It

is possible to distinguish sensory from motor effects in peripheral

nerve studies by measuring activity in sensory nerves or by measuring

the muscle response evoked by nerve stimulation to measure motor

effects. While a number of endpoints can be recorded, the most critical

variables are nerve conduction velocity, response amplitude, and

refractory period. It is important to recognize that damage to nerve

fibers may not be reflected in changes in these endpoints if the damage

is not sufficiently extensive. Thus, the interpretation of data from

such studies may be enhanced if evaluations such as nerve pathology

and/or other structural measures are also included.

Nerve conduction measurements are influenced by a number of

factors, the most important of which is temperature. An adequate nerve

conduction study will either measure the temperature of the limb under

study and mathematically adjust the results according to well-

established temperature factors or will control limb temperature within

narrow limits. Studies that measure peripheral nerve function without

regard for temperature are not adequate for risk assessment.

In well-controlled studies, statistically significant decreases in

nerve conduction velocity are indicative of a neurotoxic effect. While

a decrease in nerve conduction velocity is indicative of demyelination,

it frequently occurs later in the course of axonal degradation because

normal conduction velocity may be maintained for some time in the face

of axonal degeneration. For this reason, a measurement of normal nerve

conduction velocity does not rule out peripheral axonal degeneration if

other signs of peripheral nerve dysfunction are present.

Decreases in response amplitude reflect a loss of active nerve

fibers and may occur prior to decreases in conduction velocity in the

course of peripheral neuropathy. Hence, changes in response amplitude

may be more sensitive measurements of axonal degeneration than is

conduction velocity. Measurements of response amplitude, however, can

be more variable and require careful application of experimental

techniques, a larger sample size, and greater statistical power than

measurements of velocity to detect changes. The refractory period

refers to the time required after stimulation before a nerve can fire

again and reflects the functional status of nerve membrane ion

channels. Chemically induced changes in

[[Page 26937]]

refractory periods in a well-controlled study indicate a neurotoxic

effect.

In summary, alterations in peripheral nerve response amplitude and

refractory period in studies that are well controlled for temperature

are indicative of a neurotoxic effect. Alterations in peripheral nerve

function are frequently associated with clinical signs such as

numbness, tingling, or burning sensations or with motor impairments

such as weakness. Examples of compounds that alter peripheral nerve

function in humans or experimental animals include acrylamide, carbon

disulfide, n-hexane, lead, and some organophosphates.

3.1.2.2.2. Sensory, Motor, and Other Evoked Potentials. Evoked

potential studies are electrophysiological procedures that measure the

response elicited from a defined stimulus such as a tone, a light, or a

brief electrical pulse. Evoked potentials reflect the function of the

system under study, including visual, auditory, or somatosensory;

motor, involving motor nerves and innervated muscles; or other neural

pathways in the central or peripheral nervous system (Rebert, 1983;

Dyer, 1985; Mattsson and Albee, 1988; Mattsson et al., 1992; Boyes,

1992, 1993). Evoked potential studies should be interpreted with

respect to the known or presumed neural generators of the responses,

and their likely relationships with behavioral outcomes, when such

information is available. Such correlative information strengthens the

confidence in electrophysiological outcomes. In the absence of such

supportive information, the extent to which evoked potential studies

provide convincing evidence of neurotoxicity is a matter of

professional judgment on a case-by-case basis. Judgments should

consider the nature, magnitude, and duration of such effects, along

with other factors discussed elsewhere in this document.

Data are in the form of a voltage record collected over time and

can be quantified in several ways. Commonly, the latency (time from

stimulus onset) and amplitude (voltage) of the positive and negative

voltage peaks are identified and measured. Alternative measurement

schemes may involve substitution of spectral phase or template shifts

for peak latency and spectral power, spectral amplitude, root-mean-

square, or integrated area under the curve for peak amplitude. Latency

measurements are dependent on both the velocity of nerve conduction and

the time of synaptic transmission. Both of these factors depend on

temperature, as discussed in regard to nerve conduction, and similar

caveats apply for sensory evoked potential studies. In studies that are

well controlled for temperature, increases in latencies or related

measures can reflect deficits in nerve conduction, including

demyelination or delayed synaptic transmission, and are indicators of a

neurotoxic effect.

Decreases in peak latencies, like increases in nerve conduction

velocity, are unusual, but the neural systems under study in sensory

evoked potentials are complex, and situations that might cause a peak

measurement to occur earlier are conceivable. Two such situations are a

reduced threshold for spatial or temporal summation of afferent neural

transmission and a selective loss of cells responding late in the peak,

thus making the measured peak occur earlier. Decreases in peak latency

should not be dismissed outright as experimental or statistical error,

but should be examined carefully and perhaps replicated to assess

possible neurotoxicity. A decrease in latency is not conclusive

evidence of a neurotoxic effect.

Changes in peak amplitudes or equivalent measures reflect changes

in the magnitude of the neural population responsive to stimulation.

Both increases and decreases in amplitude are possible following

exposure to chemicals. Whether excitatory or inhibitory neural activity

is translated into a positive or negative deflection in the sensory

evoked potential is dependent on the physical orientation of the

electrode with respect to the tissue generating the response, which is

frequently unknown. Comparisons should be based on the absolute change

in amplitude. Therefore, either increases or decreases in amplitude may

be indicative of a neurotoxic effect.

Within any given sensory system, the neural circuits that generate

various evoked potential peaks differ as a function of peak latency. In

general, early latency peaks reflect the transmission of afferent

sensory information. Changes in either the latency or amplitude of

these peaks are considered convincing evidence of a neurotoxic effect

that is likely to be reflected in deficits in sensory perception. The

later-latency peaks, in general, reflect not only the sensory input but

also the more nonspecific factors such as the behavioral state of the

subject, including such factors as arousal level, habituation, or

sensitization (Dyer, 1987). Thus, changes in later-latency evoked

potential peaks should be interpreted in light of the behavioral status

of the subject and would generally be considered evidence of a

neurotoxic effect.

3.1.2.2.3. Seizures/Convulsions. Some neurotoxicants (e.g.,

lindane, pyrethroids, trimethyltin, dichlorodiphenyltrichloroethane

[DDT]) produce observable convulsions. When convulsionlike behaviors

are observed, as described in the behavioral section on convulsions,

neurophysiological recordings can provide additional information to

help interpret the results. Recordings of brain electrical activity

that demonstrate seizurelike activity are indicative of a neurotoxic

effect.

In addition to producing seizures directly, chemicals may also

alter the frequency, severity, duration, or threshold for eliciting

seizures through other means by a phenomenon known as ``kindling.''

Such alterations can occur after acute exposure or after repeated

exposure to dose levels below the acute threshold. In experiments

demonstrating changes in sensitivity following repeated exposures to

the test compound, information regarding possible changes in the

pharmacokinetic distribution of the compound is required before the

seizure susceptibility changes can be interpreted as evidence of

neurotoxicity. Increases in susceptibility to seizures are considered

adverse.

3.1.2.2.4. Electroencephalography (EEG). EEG analysis is used

widely in clinical settings for the diagnosis of neurological

disorders, and less often for the detection of subtle toxicant-induced

dysfunction (WHO, 1986; Eccles, 1988). The basis for using EEG in

either setting is the relationship between specific patterns of EEG

waveforms and specific behavioral states. Because states of alertness

and stages of sleep are associated with distinct patterns of electrical

activity in the brain, it is generally thought that arousal level can

be evaluated by monitoring the EEG. Dissociation of EEG activity and

behavior can, however, occur after exposure to certain chemicals.

Normal patterns of transition between sleep stages or between sleeping

and waking states are known to remain disturbed for prolonged periods

of time after exposure to some chemicals. Changes in the pattern of the

EEG can be elicited by anesthetic drugs and stimuli producing arousal

(e.g., lights, sounds). In studies with toxicants, changes in EEG

pattern can sometimes precede alterations in other objective signs of

neurotoxicity (Dyer, 1987).

EEG studies should be done under highly controlled conditions, and

the data should be considered on a case-by-case basis. Chemically

induced seizure activity detected in the EEG pattern is evidence of a

neurotoxic effect.

[[Page 26938]]

3.1.2.3. Neurochemical Endpoints of Neurotoxicity

Many different neurochemical endpoints have been measured in

neurotoxicological studies, and some have proven useful in advancing

the understanding of mechanisms of action of neurotoxic chemicals

(Bondy, 1986; Mailman, 1987; Morell and Mailman, 1987; Costa, 1988;

Silbergeld, 1993). Normal functioning of the nervous system depends on

the synthesis and release of specific neurotransmitters and activation

of their receptors at specific presynaptic and postsynaptic sites.

Chemicals can interfere with the ionic balance of a neuron, act as a

cytotoxicant after transport into a nerve terminal, block reuptake of

neurotransmitters and their precursors, act as a metabolic poison,

overstimulate receptors, block transmitter release, and inhibit

transmitter synthetic or catabolic enzymes. Table 4 lists several

chemicals that produce neurotoxic effects at the neurochemical level

(Bondy, 1986; Mailman, 1987; Morell and Mailman, 1987; Costa, 1988).

Table 4.--Examples of Neurotoxicants With Known Neurochemical Mechanisms

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

Site of action Examples

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

Neurotoxicants acting on ionic balance:

Inhibit sodium entry................. Tetrodotoxin.

Block closing of sodium channel...... p,p'-DDT, pyrethroids.

Increase permeability to sodium...... Batrachotoxin.

Increase intracellular calcium....... Chlorodecone.

Synaptic neurotoxicants................ MPTP.

Uptake blockers........................ Hemicholinium.

Metabolic poisons...................... Cyanide.

Hyperactivation of receptors........... Domoic acid.

Blocks transmitter release............. Botulinum toxin.

Inhibition of transmitter degradation.. Pesticides of the

organophosphate and carbamate

classes.

Blocks axonal transport................ Acrylamide.

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

As stated previously, any neurochemical change is potentially

neurotoxic. Persistent or irreversible chemically induced neurochemical

changes are indicative of neurotoxicity. Because the ultimate

functional significance of some biochemical changes is not known at

this time, neurochemical studies should be interpreted with reference

to the presumed neurotoxic consequence(s) of the neurochemical changes.

For example, many neuroactive agents can increase or decrease

neurotransmitter levels, but such changes are not indicative of a

neurotoxic effect. If, however, these neurochemical changes may be

expected to have neurophysiological, neuropathological, or

neurobehavioral correlates, then the neurochemical changes could be

classified as neurotoxic effects.

Some neurotoxicants, such as the organophosphate and carbamate

pesticides, are known to inhibit the activity of a specific enzyme,

acetylcholinesterase (for a review see Costa, 1988), which hydrolyzes

the neurotransmitter acetylcholine. Inhibition of the enzyme in either

the central or peripheral nervous system prolongs the action of the

acetylcholine at the neuron's synaptic receptors and is thought to be

responsible for the range of effects these chemicals produce, although

it is possible that these compounds have other modes of action

(Eldefrawi et al., 1992; Greenfield et al., 1984; Small, 1990).

There is agreement that objective clinical measures of cholinergic

overstimulation (e.g., salivation, sweating, muscle weakness, tremor,

blurred vision) can be used to evaluate dose-response and dose-effect

relationships and define the presence and absence of effects. A given

depression in peripheral and central cholinesterase activity may or may

not be accompanied by clinical manifestations. A depression in RBC and/

or plasma cholinesterase activity may or may not be accompanied by

clinical manifestations. It should be noted, however, that reduction in

cholinesterase activity, even if the anticholinesterase exposure is not

severe enough to precipitate clinical signs or symptoms, may impair the

organism's ability to adapt to additional exposures to

anticholinesterase compounds. Inhibition of RBC and/or plasma

cholinesterase activity is a biomarker of exposure, as well as a

reflection of cholinesterase inhibition in other peripheral tissues

(e.g., neuromuscular junction, peripheral nerve, or ganglia) (Maxwell

et al., 1987; Nagymajtenyi et al., 1988; Padilla et al., 1994), thereby

contributing to the overall hazard identification of cholinesterase-

inhibiting compounds.

The risk assessor should also be aware that tolerance to the

cholinergic overstimulation may be observed following repeated exposure

to cholinesterase-inhibiting chemicals. It has been reported, however,

that although tolerance can develop to some effects of cholinesterase

inhibition, the cellular mechanisms responsible for the development of

tolerance may also lead to the development of other effects, i.e.,

cognitive dysfunction, not present at the time of initial exposure

(Bushnell et al., 1991). These adaptive biochemical changes in the

tolerant animal may render it supersensitive to subsequent exposure to

cholinergically active compounds (Pope et al., 1992).

In general, the risk assessor should understand that assessment of

cholinesterase-inhibiting chemicals should be done on a case-by-case

basis using a weight-of evidence approach in which all of the available

data (e.g., brain, blood, and other tissue cholinesterase activity, as

well as the presence or absence of clinical signs) is considered in the

evaluation. Generally, the toxic effects of anticholinesterase

compounds are viewed as reversible, but there is human and experimental

animal evidence indicating that there may be residual, if not

permanent, effects of exposure to these compounds (Steenland et al.,

1994; Tandon et al., 1994; Stephens et al., 1995).

A subset of organophosphate agents also produces organophosphate-

induced delayed neuropathy (OPIDN) after acute or repeated exposure.

Inhibition and aging of neurotoxic esterase (or neuropathy enzymes) are

associated with agents that produce OPIDN (Johnson, 1990; Richardson,

1995). The conclusion that a chemical may produce OPIDN should be based

on at least two of three factors: (1) Evidence of a clinical syndrome,

(2) pathological lesions, and (3) neurotoxic esterase (NTE) inhibition.

NTE inhibition is necessary, but not sufficient, evidence

[[Page 26939]]

of the potential to produce OPIDN when there is at least 55%-70%

inhibition after acute exposure (Ehrich et al., 1995) and at least 45%

inhibition following repeated exposure.

Chemically induced injury to the central nervous system may be

accompanied by hypertrophy of astrocytes. In some cases, these

astrocytic changes can be seen light microscopically with

immunohistochemical stains for glial fibrillary acidic protein (GFAP),

the major intermediate filament protein in astrocytes. In addition,

GFAP can be quantified by an immunoassay, which has been proposed as a

marker of astrocyte reactivity (O'Callaghan, 1988). Immunohistochemical

stains have the advantage of better localization of GFAP increases,

whereas immunoassay evaluations are superior at detecting and

quantifying changes in GFAP levels and establishing dose-response

relationships. The ability to detect and quantify changes in GFAP by

immunoassay is improved by dissecting and analyzing multiple brain

regions. The interpretation of a chemical-induced change in GFAP is

facilitated by corroborative data from the neuropathology or

neuroanatomy evaluation. A number of chemicals known to injure the

central nervous system, including trimethyltin, methylmercury, cadmium,

3-acetylpyridine, and methylphenyltetrahydropyridine (MPTP), have been

shown to increase levels of GFAP. Measures of GFAP are now included as

an optional test in the Neurotoxicity Screening Battery (U.S. EPA,

1991a).

Increases in GFAP above control levels may be seen at dosages below

those necessary to produce damage seen by standard microscopic or

histopathological techniques. Because increases in GFAP reflect an

astrocyte response in adults, treatment-related increases in GFAP are

considered to be evidence that a neurotoxic effect has occurred. There

is less agreement as to how to interpret decreases in GFAP relative to

an appropriate control group. The absence of a change in GFAP following

exposure does not mean that the chemical is devoid of neurotoxic

potential. Known neurotoxicants such as cholinesterase-inhibiting

pesticides, for example, would not be expected to increase brain levels

of GFAP. Interpretation of GFAP changes prior to weaning may be

confounded by the possibility that chemically induced increases in GFAP

could be masked by changes in the concentration of this protein

associated with maturation of the central nervous system, and these

data may be difficult to interpret.

3.1.2.4. Behavioral Endpoints of Neurotoxicity

Behavior reflects the integration of the various functional

components of the nervous system. Changes in behavior can arise from a

direct effect of a toxicant on the nervous system, or indirectly from

its effects on other physiological systems. Understanding the

interrelationship between systemic toxicity and behavioral changes

(e.g., the relationship between liver damage and motor activity) is

extremely important. The presence of systemic toxicity may complicate,

but does not preclude, interpretation of behavioral changes as evidence

of neurotoxicity. In addition, a number of behaviors (e.g., schedule-

controlled behavior) may require a motivational component for

successful completion of the task. In such cases, experimental

paradigms designed to assess the motivation of an animal during

behavior might be necessary to interpret the meaning of some chemical-

induced changes in behavior.

EPA's testing guidelines developed for the Toxic Substances Control

Act and the Federal Insecticide, Fungicide and Rodenticide Act describe

the use of functional observational batteries (FOB), motor activity,

and schedule-controlled behavior for assessing neurotoxic potential

(U.S. EPA, 1991a). Examples of measures obtained in a typical FOB are

presented in Table 5. There are many other measures of behavior,

including specialized tests of motor and sensory function and of

learning and memory (Tilson, 1987; Anger, 1984).

Table 5.--Examples of Measures in a Representative Functional

Observational Battery

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

Home cage and open field Manipulative Physiological

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

Arousal......................... Approach response. Body temperature.

Autonomic signs................. Click response.... Body weight.

Convulsions, tremors............ Foot splay........

Gait............................ Grip strength..... ..................

Mobility........................ Righting reflex... ..................

Posture......................... Tail pinch ..................

response.

Rearing.

Stereotypy.

Touch response. .................. ..................

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

Table 6.--Examples of Specialized Behavioral Tests To Measure

Neurotoxicity

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

Representative

Function Procedure agents

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

Motor Function

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

Weakness........................ Grip strength, n-Hexane, methyl.

swimming n-Butylketone,

endurance, carbaryl.

suspension rod,

discriminative

motor function.

Incoordination.................. Rotorod, gait 3-Acetylpyridine,

assessments, ethanol.

righting reflex.

Tremor.......................... Rating scale, Chlordecone, Type

spectral analysis. I.

pyrethroids, DDT.

Myoclonic spasms................ Rating scale...... DDT, Type II

pyrethroids.

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

Sensory Function

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

Auditory........................ Discrimination Toluene,

conditioning. trimethyltin.

Reflex ..................

modification.

Visual.......................... Discrimination Methylmercury.

conditioning.

Somatosensory................... Discrimination Acrylamide.

conditioning.

Pain sensitivity................ Discrimination Parathion.

conditioning.

Olfactory....................... Discrimination 3-Methylindole,

conditioning. methylbromide.

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

Cognitive Function

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

Habituation..................... Startle reflex.... Diisopropylfluorop

hosphate.

Pre/neonatal

methylmercury.

[[Page 26940]]

Classical conditioning.......... Nictitating Aluminum.

membrane.

Conditioned flavor Carbaryl.

aversion.......... Trimethyltin,

IDPN.

Passive avoidance. Neonatal

trimethyltin.

Olfactory ..................

conditioning.

Instrumental conditioning....... One-way avoidance. Chlordecone.

Two-way avoidance. Pre/neonatal lead.

Y-maze avoidance.. Hypervitaminosis

A.

Biel water maze... Styrene.

Morris water maze. DFP.

Radial arm maze... Trimethyltin.

Delayed matching DFP.

to sample.

Repeated Carbaryl.

acquisition.

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

At the present time, there is no clear consensus concerning the use

of specific behavioral tests to assess chemical-induced sensory, motor,

or cognitive dysfunction in animal models. The risk assessor should

also know that the literature is clear that a number of other behaviors

besides those listed in Tables 1, 5, and 6 could be affected by

chemical exposure. For example, alterations in food and water intake,

reproduction, sleep, temperature regulation, and circadian rhythmicity

are controlled by specific regions of the brain, and chemical-induced

alterations in these behaviors could be indicative of neurotoxicity. It

is reasonable to assume that an NOAEL or LOAEL could be based on one or

more of these endpoints.

The following sections describe, in general, behavioral tests and

their uses and offer guidance on interpreting data.

3.1.2.4.1. Functional Observational Battery (FOB). An FOB is

designed to detect and quantify major overt behavioral, physiological,

and neurological signs (Gad, 1982; O'Donoghue, 1989; Moser, 1989). A

number of batteries have been developed, each consisting of tests

generally intended to evaluate various aspects of sensorimotor function

(Tilson and Moser, 1992). Many FOB tests are essentially clinical

neurological examinations that rate the presence or absence, and in

many cases the severity, of specific neurological signs. Some FOBs in

animals are similar to clinical neurological examinations used with

human patients. Most FOBs have several components or tests. A typical

FOB is summarized in Table 5 and evaluates several functional domains,

including neuromuscular (i.e., weakness, incoordination, gait, and

tremor), sensory (i.e., audition, vision, and somatosensory), and

autonomic (i.e., pupil response and salivation) function.

The relevance of statistically significant test results from an FOB

is judged according to the number of signs affected, the dose(s) at

which effects are observed, and the nature, severity, and persistence

of the effects and their incidence in relation to control animals. In

general, if only a few unrelated measures in the FOB are affected, or

the effects are unrelated to dose, the results may not be considered

evidence of a neurotoxic effect. If several neurological signs are

affected, but only at the high dose and in conjunction with other overt

signs of toxicity, including systemic toxicity, large decreases in body

weight, decreases in body temperature, or debilitation, there is less

persuasive evidence of a direct neurotoxic effect. In cases where

several related measures in a battery of tests are affected and the

effects appear to be dose dependent, the data are considered to be

evidence of a neurotoxic effect, especially in the absence of systemic

toxicity. The risk assessor should be aware of the potential for a

number of false positive statistical findings in these studies because

of the large number of endpoints customarily included in the FOB.

FOB data can be grouped into one or more of several neurobiological

domains, including neuromuscular (i.e., weakness, incoordination,

abnormal movements, gait), sensory (i.e., auditory, visual,

somatosensory), and autonomic functions (Tilson and Moser, 1992). This

statistical technique may be useful when separating changes that occur

on the basis of chance or in conjunction with systemic toxicity from

those treatment-related changes indicative of neurotoxic effects. In

the case of the developing organism, chemicals may alter the maturation

or appearance of sensorimotor reflexes. Significant alterations in or

delay of such reflexes is evidence of a neurotoxic effect.

Examples of chemicals that affect neuromuscular function are 3-

acetylpyridine, acrylamide, and triethyltin. Organophosphate and

carbamate insecticides produce autonomic dysfunction, while

organochlorine and pyrethroid insecticides increase sensorimotor

sensitivity, produce tremors and, in some cases, cause seizures and

convulsions (Spencer and Schaumburg, 1980).

3.1.2.4.2. Motor Activity. Motor activity represents a broad class

of behaviors involving coordinated participation of sensory, motor, and

integrative processes. Assessment of motor activity is noninvasive and

has been used to evaluate the effects of acute and repeated exposure to

neurotoxicants (MacPhail et al., 1989). An organism's level of activity

can, however, be affected by many different types of environmental

agents, including non-neurotoxic agents. Motor activity measurements

also have been used in humans to evaluate disease states, including

disorders of the nervous system (Goldstein and Stein, 1985).

Motor activity is usually quantified as the frequency of movements

over a period of time. The total counts generated during a test period

will depend on the recording mechanism and the size and configuration

of the testing apparatus. Effects of agents on motor activity can be

expressed as absolute activity counts or as a percentage of control

values. In some cases, a transformation (e.g., square root) may be used

to achieve a normal distribution of the data. In these cases, the

transformed data and not raw data should be used for risk assessment

purposes. The frequency of motor activity within a session usually

decreases and is reported as the average number of counts occurring in

each successive block of time. The EPA's

[[Page 26941]]

Office of Prevention, Pesticides and Toxic Substances guidelines (U.S.

EPA, 1991a), for example, call for test sessions of sufficient duration

to allow motor activity to approach steady-state levels during the last

20 percent of the session for control animals. A sum of the counts in

each epoch will add up to the total number of counts per session.

Motor activity can be altered by a number of experimental factors,

including neurotoxic chemicals. Decreases in activity could occur

following high doses of non-neurotoxic agents (Kotsonis and Klaassen,

1977; Landauer et al., 1984). Examples of neurotoxic agents that

decrease motor activity include many pesticides (e.g., carbamates,

chlorinated hydrocarbons, organophosphates, and pyrethroids), heavy

metals (lead, tin, and mercury), and other agents (3-acetylpyridine,

acrylamide, and 2,4-dithiobiuret). Some neurotoxicants (e.g., toluene,

xylene, triadimefon) produce transient increases in activity by

presumably stimulating neurotransmitter release, while others (e.g.,

trimethyltin) produce persistent increases in motor activity by

destroying specific regions of the brain (e.g., hippocampus).

Following developmental exposures, neurotoxic effects are often

observed as a change in the ontogenetic profile or maturation of motor

activity patterns. Frequently, developmental exposure to neurotoxic

agents will produce an increase in motor activity that persists into

adulthood or that results in changes in other behaviors. This is

evidence of a neurotoxic effect. Like other organ systems, the nervous

system may be differentially sensitive to toxicants in groups such as

the young. For example, toxicants introduced to the developing nervous

system may kill stem cells and thus cause profound effects on adult

structure and function. Moreover, toxicants may have greater access to

the developing nervous system before the blood-brain barrier is

completely formed or before metabolic detoxifying systems are

functional.

Motor activity measurements are typically used with other tests

(e.g., FOB) to help detect neurotoxic effects. Agent-induced changes in

motor activity associated with other overt signs of toxicity (e.g.,

loss of body weight, systemic toxicity) or occurring in non-dose-

related fashion are of less concern than changes that are dose

dependent, are related to structural or other functional changes in the

nervous system, or occur in the absence of life-threatening toxicity.

13.1.2.4.3. Schedule-Controlled Operant Behavior. Schedule-

controlled operant behavior (SCOB) involves the maintenance of behavior

(e.g., performance of a lever-press or key-peck response) by

reinforcement. Different rates and patterns of responding are

controlled by the relationship between response and subsequent

reinforcement. SCOB provides a measure of performance of a learned

behavior (e.g., lever press or key peck) and involves training and

motivational variables that should be considered in evaluating the

data. Agents may interact with sensory processing, motor output,

motivational variables (i.e., related to reinforcement), training

history, and baseline characteristics (Rice, 1988; Cory-Slechta, 1989).

Qualitatively, rates and patterns of SCOB display cross-species

generality, but the quantitative measures of rate and pattern of

performance can vary within and between species.

In laboratory animals, SCOB has been used to study a wide range of

neurotoxicants, including methylmercury, many pesticides, organic and

inorganic lead, triethyltin, and trimethyltin (MacPhail, 1985; Tilson,

1987; Rice, 1988). The primary SCOB endpoints for evaluation are

response rate and the temporal pattern of responding. These endpoints

may vary as a function of the contingency between responding and

reinforcement presentation (i.e., schedule of reinforcement). Schedules

of reinforcement that have been used in toxicology studies include

fixed ratio and fixed interval schedules. Fixed ratio schedules

engender high rates of responding and a characteristic pause after

delivery of each reinforcement. Fixed interval schedules engender a

relatively low rate of responding during the initial portion of the

interval and progressively higher rates near the end of the interval.

For some schedules of reinforcement, the temporal pattern of responding

may play a more important role in defining the performance

characteristics than the rate of responding. For other schedules, the

reverse may be true. For example, the temporal pattern of responding

may be more important than rate of responding for defining performance

on a fixed interval schedule. For a fixed ratio schedule, more

importance might be placed on the rate of responding than on the post-

reinforcement pause.

The overall qualitative patterns are important properties of the

behavior. Substantial qualitative changes in operant performance, such

as elimination of characteristic response patterns, can be evidence of

an adverse effect. Most chemicals, however, can disrupt operant

behavior at some dose, and such adverse effects may be due either to

neurotoxic or non-neurotoxic mechanisms. Unlike large qualitative

changes in operant performance, small quantitative changes are not

adverse. Some changes may actually represent an improvement, e.g., an

increase in the index of curvature with a decrease in fixed interval

rate of responding. Assessing the toxicological importance of these

effects requires considerable professional judgment and evaluation of

converging evidence from other types of toxicological endpoints. While

most chemicals decrease the efficiency of responding at some dose, some

agents may increase response efficiency on schedules requiring high

response rates because of a stimulant effect or an increase in central

nervous system excitability. Agent-induced changes in responding

between reinforcements (i.e., the temporal pattern of responding) may

occur independently of changes in the overall rate of responding.

Chemicals may also affect the reaction time to respond following

presentation of a stimulus. Agent-induced changes in response rate or

temporal patterning associated with other overt signs of toxicity

(e.g., body weight loss, systemic toxicity, or occurring in a non-dose-

related fashion) are of less concern than changes that are dose

dependent, related to structural or other functional changes in the

nervous system, or occur in the absence of life-threatening toxicity.

3.1.2.4.4. Convulsions. Observable convulsions in animals are

indicative of an adverse effect. These events can reflect central

nervous system activity comparable to that of epilepsy in humans and

could be defined as neurotoxicity. Occasionally, other toxic actions

of compounds, such as direct effects on muscle, might mimic some

convulsionlike behaviors. In some cases, convulsions or

convulsionlike behaviors may be observed in animals that are

otherwise severely compromised, moribund, or near death. In such

cases, convulsions might reflect an indirect effect of systemic

toxicity and are less clearly indicative of neurotoxicity. As

discussed in the section on neurophysiological measures, electrical

recordings of brain activity could be used to determine specificity

of effects on the nervous system.

3.1.2.4.5. Specialized Tests for Neurotoxicity. Several procedures

have been developed to measure agent-induced changes in specific

neurobehavioral functions such as motor, sensory, or cognitive function

(Tilson, 1987; Cory-Slechta, 1989). Table 6 lists several behavioral

tests, the neurobehavioral functions they were designed to assess, and

agents known to affect the response. Many of these tests in animals

have been designed to assess neural functions in humans using similar

testing procedures.

A statistically or biologically significant chemically induced

change

[[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:

Describe how the RfD/RfC was calculated;

Discuss the confidence in the estimates;

Describe the assumptions or uncertainty factors used; and

Discuss the route and level of exposure observed, as

compared to expected human exposures.

4.1. LOAEL/NOAEL and BMD Determination

As indicated earlier, the LOAEL and NOAEL are determined for

endpoints that are seen at the lowest dose level (so-called critical

effect). Several limitations in the use of the NOAEL have been

identified and described (e.g., Barnes and Dourson, 1988; Crump, 1984).

For example, the NOAEL is derived from a single endpoint from a single

study (the critical study) and

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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

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