Proposed Guidelines for Neurotoxicity Risk Assessment

SUMMARY: The U.S. Environmental Protection Agency (EPA; Agency) is

today issuing proposed guidelines for assessing the risks for

neurotoxicity from exposure to environmental agents. As background

information for this guidance, this notice describes the scientific

basis for concern about exposure to agents that cause neurotoxicity and

outlines the general process for assessing potential risk to humans

because of environmental contaminants.

These proposed Guidelines for Neurotoxicity Risk Assessment

(hereafter ``Guidelines'') are intended to guide Agency evaluation of

agents that are suspected to cause neurotoxicity in line with the

policies and procedures established in the statutes administered by the

EPA. The Guidelines were developed as part of an interoffice guidelines

development program under the auspices of the Risk Assessment Forum,

within EPA's Office of Research and Development. Draft Guidelines were

developed by an Agency work group composed of scientists from

throughout the Agency, and selected drafts were peer reviewed

internally and by experts from universities, environmental groups,

industry, and other governmental agencies. A subsequent draft has

undergone peer review in a workshop held on June 2-3, 1992, and has

received internal review by the Concordance and Oversight Subcommittees

of the Risk Assessment Forum. Most recently, the Committee on the

Environment and Natural Resources of the Office of Science and

Technology Policy reviewed the guidelines at a meeting held on August

15, 1995. The proposed Guidelines are based, in part, on

recommendations derived from these reviews and on those made at various

scientific meetings and workshops on neurotoxicology.

The public is invited to comment, and public comments will be

considered in EPA decisions in formulating the final Guidelines.

Commenters are asked to focus on several special issues, particularly,

(1) the issue of compensation and recovery of function in

neurotoxicological studies and how to account for compensation in

neurotoxicology risk assessment; (2) the use of blood and/or brain

acetylcholinesterase activity as an indication of neurotoxicity for

risk assessment; (3) endpoints indicative of neurotoxicity that may not

be covered by these guidelines, i.e., endocrine disruption or

neuroendocrine-mediated neurotoxicity; and (4) the possibility of no

threshold for some neurotoxic agents.

The EPA Science Advisory Board (SAB) also will review these

proposed Guidelines at a meeting to be announced in a future Federal

Register. Agency staff will prepare summaries of the public and SAB

comments, analyses of major issues presented by commenters, and Agency

responses to those comments. Appropriate comments will be incorporated,

and the revised Guidelines will be submitted to the Risk Assessment

Forum for review. The Agency will consider comments from the public,

the SAB, and the Risk Assessment Forum in its recommendations to the

EPA Administrator.

DATES: The Proposed Guidelines are being made available for a 120-day

public review and comment period. Comments must be in writing and must

be postmarked by February 1, 1996. Please submit one unbound original

with pages consecutively numbered, and three copies. If there are

attachments, include an index numbered consecutively with comments, and

three copies.

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

Fax: 919-541-4849.

ADDRESSES: Comments on the proposed Guidelines may be mailed or

delivered to: Dr. Hugh A. Tilson, Neurotoxicology Division (MD-74B),

National Health and Environmental Effects Research Laboratory, U.S.

Environmental Protection Agency, Research Triangle Park, NC 27711.

Please note that all comments received in response to this notice will

be placed in a public record. Commenters should not send any item of

personal information, such as medical information or home address, if

they do not wish it to be part of the public record.

SUPPLEMENTARY INFORMATION: In its 1983 book, Risk Assessment in the

Federal Government: Managing the Process, the National Academy of

Sciences recommended that Federal regulatory agencies establish

``inference guidelines'' (1) to promote consistency and technical

quality in risk assessment, and (2) to ensure that the risk assessment

process is maintained as a scientific effort separate from risk

management. A task force within EPA accepted that recommendation and

requested that Agency scientists begin to develop such guidelines.

In 1984, EPA scientists began work on risk assessment guidelines

for carcinogenicity, mutagenicity, suspect developmental toxicants,

chemical mixtures, and exposure assessment. Following extensive

scientific and public review, these first five guidelines were issued

on September 24, 1986 (51 FR 33992-34054). Since 1986, additional risk

assessment guidelines have been proposed for male and female

reproductive risk (53 FR 24834-847; 53 FR 24850-869), and two of the

1986 guidelines, suspect developmental toxicants (56 FR 63798-826) and

exposure assessment (57 FR 22888-938), have been revised, reproposed,

and finalized.

The Guidelines proposed today continue the guidelines development

process initiated in 1984. These Guidelines set forth principles and

procedures to guide EPA scientists in the conduct of Agency risk

assessments and to inform Agency decision makers and the public about

these procedures. In particular, the Guidelines emphasize that risk

assessments will be conducted on a case-by-case basis, giving full

consideration to all relevant scientific information. This case-by-case

approach means that Agency experts study scientific information on each

chemical under review and use the most scientifically appropriate

interpretation to assess risk. The Guidelines also stress that this

information will be fully presented in Agency risk assessment

documents, and that Agency scientists will identify the strengths and

weaknesses of each assessment by describing uncertainties, assumptions,

and limitations, as well as the scientific basis and rationale for each

assessment.

The Guidelines are formulated in part to bridge gaps in risk

assessment methodology and data. By identifying these gaps and the

importance of the missing information to the risk assessment process,

EPA wishes to encourage research and analysis that will lead to new

risk assessment methods and data.

Dated: September 25, 1995.

Carol M. Browner,

Administrator.

Proposed Guidelines for Neurotoxicity Risk Assessment Contents

I. Introduction

A. Organization of These Guidelines

B. The Role of Environmental Agents in Neurotoxicity

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C. Neurotoxicity Risk Assessment

D. Assumptions

II. Definitions and Critical Concepts

III. Hazard Characterization

A. Neurotoxicological Studies: End Points and Their

Interpretation

1. Human Studies

a. Clinical Evaluations

b. Case Reports

c. Epidemiologic Studies

(1) Cross-sectional studies

(2) Case-control (retrospective) studies

(3) Cohort (prospective, follow-up) studies

d. Human Laboratory Exposure Studies

2. Animal Studies

a. Structural End Points of Neurotoxicity

b. Neurophysiological End Points of Neurotoxicity

(1) Nerve conduction studies

(2) Sensory, motor, and other evoked potentials

(3) Seizures/convulsions

(4) Electroencephalography (EEG)

c. Neurochemical End Points of Neurotoxicity

d. Behavioral End Points of Neurotoxicity

(1) Functional observational battery

(2) Motor activity

(3) Schedule-controlled operant behavior

(4) Convulsions

(5) Specialized tests for neurotoxicity

(a) Motor function

(b) Sensory function

(c) Cognitive function

e. Developmental Neurotoxicity

3. Other Considerations

a. Pharmacokinetics

b. Comparisons of Molecular Structure

c. Statistical Considerations

d. In Vitro Data in Neurotoxicology

B. Dose-Response Evaluation

C. Characterization of the Health-Related Data Base

IV. Dose-Response Analysis

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

B. Determination of the Reference Dose or Reference

Concentration

V. Exposure Assessment

VI. Risk Characterization

A. Overview

B. Integration of Hazard Characterization, Dose-Response

Analysis, and Exposure Assessment

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

Assessment

D. Descriptors of Neurotoxicity Risk

1. Estimation of the Number of Individuals

2. Presentation of Specific Scenarios

3. Risk Characterization for Highly Exposed Individuals

4. Risk Characterization for Highly Sensitive or Susceptible

Individuals

5. Other Risk Descriptors

E. Communicating Results

F. Summary and Research Needs

VII. References

List of Tables

Table 1. Examples of possible indicators of a neurotoxic effect

Table 2. Neurotoxicants and diseases with specific neuronal targets

Table 3. Examples of neurophysiological measures of neurotoxicity

Table 4. Examples of neurotoxicants with known neurochemical

mechanisms

Table 5. Summary of measures in a representative functional

observational battery, and the type of data produced by each

Table 6. Examples of specialized behavioral tests to measure

neurotoxicity

Table 7. Examples of developmental neurotoxicants

Table 8. Characterization of the Health-Related Database

I. Introduction

These proposed Guidelines describe the principles, concepts, and

procedures that the U.S. Environmental Protection Agency (EPA; Agency)

would follow in evaluating data on potential neurotoxicity associated

with exposure to environmental toxicants. The Agency's authority to

regulate substances that have the potential to interfere with human

health is derived from a number of statutes that are implemented

through multiple offices within the EPA. The procedures outlined here

are intended to help develop a sound scientific basis for neurotoxicity

risk assessment, promote consistency in the Agency's assessment of

toxic effects on the nervous system, and inform others of the

approaches used by the Agency in those assessments.

A. Organization of These Guidelines

This Introduction (section I) summarizes the purpose of these

proposed Guidelines within the overall framework of risk assessment at

the EPA. It also outlines the organization of the guidance and

describes several default assumptions to be used in the risk assessment

process as discussed in the recent National Research Council report

``Science and Judgment in Risk Assessment (NRC, 1994).''

Section II sets forth definitions of particular terms widely used

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

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

discussions concerning reversible and irreversible effects and direct

versus indirect effects.

Risk assessment is the process by which scientific judgments are

made concerning the potential for toxicity to occur in humans. The

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

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

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

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

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

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

the importance of an approach that is less fragmented and more

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

recurring conceptual issues that cut across all stages of risk

assessment. These Guidelines propose a more interactive approach by

organizing the process around components that focus on evaluation of

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

response analysis, the exposure assessment, and the risk

characterization. This is done because, in practice, hazard

identification for neurotoxicity and other noncancer health effects is

usually done in conjunction with an evaluation of dose-response

relationships in the studies used to identify the hazard. Determining a

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

(Kimmel et al., 1990). Thus, the hazard characterization provides an

evaluation of a hazard within the context of the dose, route, duration,

and timing of exposure. This approach combines the information

important in comparing the toxicity of a chemical to potential human

exposure scenarios (Section V). Secondly, it avoids the potential for

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

This organization of the risk assessment process is similar to that

discussed in the Guidelines for Developmental Toxicity Risk Assessment

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

response analysis is discussed under a separate section in these

guidelines.

Hazard characterization involves examining all available

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

timing, and durations of exposure to determine if an agent causes

neurotoxicity in that species and under what conditions. From the

hazard characterization and criteria provided in these Guidelines, the

health-related data base can be characterized as sufficient or

insufficient for use in risk assessment (section III.C). Combining

hazard identification and some aspects of dose-response evaluation into

hazard characterization does not preclude the evaluation and use of

data when quantitative information for setting reference doses (RfDs)

and reference concentrations (RfCs) are not available.

The next step, the dose-response analysis (section IV) is the

quantitative analysis, and includes determining the no-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, i.e., the

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benchmark dose approach (Crump, 1984; U.S. EPA, 1995a), for more

quantitative dose-response evaluation when sufficient data are

available. The benchmark dose approach takes into account the

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

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

data are considered sufficient for risk assessment, and if

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

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

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

using the NOAEL or benchmark dose divided by uncertainty factors to

account for interspecies differences in response, intraspecies

variability and other factors of study design or the data base. A

statement of the potential for human risk and the consequences of

exposure can come only from integrating the hazard characterization and

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

risk characterization.

The section on exposure assessment (section V) identifies human

populations exposed or potentially exposed to an agent, describes their

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

and durations of exposure to the agent. The exposure assessment

provides an estimate of human exposure levels for particular

populations from all potential sources.

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

dose-response analysis, and the exposure assessment for given

populations are combined to estimate some measure of the risk for

neurotoxicity. As part of risk characterization, a summary of the

strengths and weaknesses of each component of the risk assessment is

given along with major assumptions, scientific judgments, and, to the

extent possible, qualitative and quantitative estimates of the

uncertainties. This characterization of the health-related data base is

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

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

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

the exposure basis used for the risk assessment is clearly described,

e.g., highly exposed individuals or highly sensitive or susceptible

individuals. The NOAEL may be compared to the various estimates of

human exposure to calculate the margin(s) of exposure (MOE). The

considerations for judging the acceptability of the MOE are similar to

those for determining the appropriate size of the uncertainty factor

for calculating the RfD or RfC.

The Agency recently issued a policy statement and associated

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

currently being implemented throughout EPA. This policy statement is

designed to ensure that critical information from each stage of a risk

assessment is used in forming conclusions about risk and that this

information is communicated from risk assessors to risk managers

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

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

clarity, transparency, reasonableness, and consistency in risk

assessments across Agency programs. Final neurotoxicity risk assessment

guidelines may reflect additional changes in risk characterization

practices resulting from implementation activities.

Risk assessment is just one component of the regulatory process and

defines the potential adverse health consequences of exposure to a

toxic agent. The other component, risk management, combines risk

assessment with statutory directives regarding socioeconomic,

technical, political, and other considerations, to decide whether to

control future exposure to the suspected toxic agent and, if so, the

nature and level of control. One major objective of these risk

assessment Guidelines is to help the risk assessor determine whether

the experimental animal or human data indicate the potential for a

neurotoxic effect. Such information can then be used subsequently to

categorize evidence to identify and characterize neurotoxic hazards as

described in section III.3.C, Characterization of the Health-Related

Data Base, and Table 8 of these Guidelines. Risk management is not

dealt with directly in these Guidelines because the basis for decision

making goes beyond scientific considerations alone, but the use of

scientific information in this process is discussed. For example, the

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

scientific bases for establishing this value are discussed here.

B. The Role of Environmental Agents in Neurotoxicity

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

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

to chemicals with neurotoxic properties in air, water, foods,

cosmetics, household products, and drugs used therapeutically or

illicitly. Naturally occurring neurotoxins, such as animal and plant

toxins, present additional hazards. During daily life, a person

experiences a multitude of exposures, both voluntary and unintentional,

to neuroactive substances, singly and in combination. Levels of

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

route, and duration of exposure.

A link between human exposure to some chemical substances and

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

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

environment, there is growing scientific and regulatory interest in the

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

sufficient exposure occurs, the effects resulting from such exposures

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

how many chemicals may be neurotoxic in humans (Reiter, 1987). The

EPA's inventory of toxic chemicals is greater than 65,000 and

increasing yearly. An overwhelming majority of the materials in

commercial use have not been tested for their neurotoxic potential

(NRC, 1984). Estimates of the number of chemicals with neurotoxic

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

large percentage of the more than 500 registered active pesticide

ingredients are neurotoxic to varying degrees. Of 588 chemicals listed

by the American Conference of Governmental Industrial Hygienists, 167

affected the nervous system or behavior at some exposure level (Anger,

1984). Anger (1990) estimated that of the approximately 200 chemicals

to which one million or more American workers are exposed, more than

one-third may have adverse effects on the nervous system, if sufficient

exposure occurs. Anger (1984) also recognized neurotoxic effects as one

of the 10 leading workplace disorders. A number of therapeutic

substances, including some anticancer and antiviral agents and abused

drugs, can cause adverse or neurotoxicological side effects at

therapeutic levels (OTA, 1990). Thus, estimating the risks of exposure

to chemicals with neurotoxic potential is of concern with regard to the

overall impact of these exposures on human health.

C. Neurotoxicity Risk Assessment

In addition to its primary role in cognitive functions, the nervous

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

sensitive to perturbation from various sources and has limited ability

to regenerate. There is evidence that even small anatomical,

biochemical, or physiological insults to the nervous system may result

in

[[Page 52035]]

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

consistent guidance on how to evaluate data on neurotoxic substances

and assess to what degree, if any, they have the potential to cause

transient or persistent, direct or indirect effects on human health.

To help address these needs, these Guidelines develop principles

and concepts in several areas. First, these Guidelines outline the

scientific basis for evaluating effects due to exposure to

neurotoxicants and discuss principles and methods for evaluating data

from human and animal studies on behavior, neurochemistry,

neurophysiology, and neuropathology. This guidance document also

discusses adverse effects on neurological development and function in

infants and children following prenatal and perinatal exposure to

chemical agents. Other sections of these Guidelines outline the method

for calculating reference doses or reference concentrations when

neurotoxicity is the critical effect, discuss the availability of

alternative mathematical approaches to dose-response analyses,

characterize the health-related data base for neurotoxicity risk

assessment, and discuss integration of exposure information with the

results of the dose-response assessment to characterize risks of

exposures of concern. These Guidelines do not advocate developing

reference doses specific for neurotoxicity, but rather the use of

neurotoxicity as one possible end point to develop reference doses.

EPA offices have published guidelines for neurotoxicity testing in

animals (U.S. EPA, 1986, 1987, 1988a, 1991a). The testing guidelines

address the development of new data for use in risk assessment. These

proposed neurotoxicity risk assessment Guidelines provide the Agency's

first comprehensive guidance on the use and interpretation of

neurotoxicity data. These proposed Guidelines are part of the Agency's

risk assessment guidelines development process, which was initiated in

1984. As part of its neurotoxicity guidelines development program, the

EPA has sponsored or participated in several conferences on relevant

issues (Tilson, 1990); these and other sources (see references) provide

the scientific basis for these proposed risk assessment Guidelines.

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

separate and distinct from the recently published document on

principles of neurotoxicity risk assessment (U.S. EPA, 1993). The

document on principles was prepared under the auspices of the

Subcommittee on Risk Assessment of the Federal Coordinating Council for

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

specific directives for how neurotoxicity risk assessment should be

performed.

It is expected that, like other EPA risk assessment guidelines

(U.S. EPA, 1991b), this document will encourage research and analysis

leading to new risk assessment methods and data, which in turn would be

used to revise and improve the Guidelines and better guide Agency risk

assessors.

D. Assumptions

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

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

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

relevance of effects to potential human risk. Default assumptions

should not be applied indiscriminantly. First, all available

mechanistic and pharmacokinetic data should be considered. If these

data indicate that an alternative assumption is appropriate or obviate

the need for applying an assumption, such information should be used in

the risk assessment of that agent. The following default assumptions

form the basis of the approaches taken in these Guidelines.

It is assumed that an agent that produces detectable adverse

neurotoxic effects in experimental animal studies will pose a potential

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

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

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

data are frequently predictive of a neurotoxic effect in humans.

It is assumed that behavioral, neurophysiological, neurochemical,

and neuroanatomical manifestations are of concern. In the past, the

tendency has been to consider only neuropathological changes as end

points of concern. Based on the data on agents that are known human

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

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

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

tested, the type of neurotoxic effect may be different or absent. Thus,

a biologically significant increase in any of the manifestations is

considered indicative of an agent's potential for disrupting the

structure or function of the human nervous system.

It is assumed that the types of neurotoxic effects seen in animal

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

Therefore, it may be difficult to determine which will be the most

appropriate species in terms of predicting the specific types of

effects seen in humans. The fact that every species may not react in

the same way is probably due to species-specific differences in

maturation of the nervous system, differences in timing of exposure,

metabolism, or mechanisms of action.

It is assumed that the most appropriate species will be used when

data are available to estimate human risk. In the absence of such data,

the most sensitive species is used, based on the fact that for the

majority of known human neurotoxicants, humans are as sensitive or more

so than the most sensitive animal species tested.

In general, a threshold is assumed for the dose-response curve for

most neurotoxicants. This is based on the known capacity of the nervous

system to compensate for or to repair a certain amount of damage at the

cellular, tissue, or organ level. In addition, because of the

multiplicity of cells in the nervous system, multiple insults at the

molecular or cellular level may be required to produce an effect on the

whole organism.

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

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

scientific knowledge about the effects of concern.

II. Definitions and Critical Concepts

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

use in the identification and evaluation of neurotoxicity. The various

health effects that fall within the broad classification of

neurotoxicity are described and examples are provided.

Adverse effects include alterations from baseline that diminish an

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

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

central and/or peripheral nervous system following exposure to a

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

effects include changes in somatic/autonomic, sensory, motor, and/or

cognitive function. Structural effects are defined as neuroanatomical

changes occurring at any level of nervous system organization;

functional changes are defined as neurochemical, neurophysiological, or

behavioral alterations. Changes in function can also result from

toxicity to other specific organ systems, and these indirect changes

may be considered adverse but not necessarily neurotoxic.

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The risk assessor also should know that there are different levels

of concern based on the magnitude of effect and reversibility of some

neurotoxic effects. Neurotoxic effects may be irreversible, i.e.,

cannot return to the state prior to exposure, resulting in a permanent

change in the organism, or reversible, i.e., can return to the pre-

exposure condition, allowing the organism to return to its state prior

to exposure. Clear or demonstrable irreversible change in either the

structure or function of the nervous system causes greater concern than

do reversible changes. If neurotoxic effects are observed at some time

during the life span of the organism but are slowly reversible, the

concern is also high. There is lesser concern for effects that are

rapidly reversible or transient, i.e., measured in minutes, hours, or

days, and appear to be associated with the pharmacokinetics of the

causal agent and its presence in the body. Reversible changes that

occur in the occupational setting or environment, however, may be of

high concern if, for example, exposure to a short-acting solvent

interferes with operation of heavy equipment in an industrial plant.

The context of the exposure should be considered in evaluating

reversible effects. The risk assessor should note that once damaged,

neurons, particularly in the central nervous system, have a limited

capacity for regeneration. Reversibility of effects resulting from cell

death or from the destruction of cell processes may represent an

activation of repair capacity, decreasing future potential

adaptability. Therefore, even reversible neurotoxic changes should be

of concern. Evidence of progressive effects, i.e., those that continue

to worsen even after the causal agent has been removed; or delayed

effects, i.e., those that occur at a time distant from the last contact

with the causal agent; or residual effects, i.e., those that persist

beyond a recovery period; or latent effects, i.e., those that become

evident only after an environmental challenge or aging, have a high

level of concern. Environmental challenges can include stress,

increased physical or cognitive workload, pharmacological

manipulations, and nutritional deficiency or excess.

Neurotoxic effects can be observed at various levels of

organization of the nervous system, including neurochemical,

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

for example, an agent that causes neurotoxicity might inhibit

macromolecule or transmitter synthesis, alter the flow of ions across

cellular membranes, or prevent release of neurotransmitter from the

nerve terminals. Anatomical changes may include alterations of the cell

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

chemical might change the thresholds for neural activation or reduce

the speed of neurotransmission. Behavioral alterations can include

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

alterations in simple or complex reflexes and motor functions;

alterations in cognitive functions such as learning, memory or

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

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

as delusions and hallucinations. At present, relatively few neurotoxic

syndromes have been thoroughly characterized in terms of the initial

neurochemical change, structural alterations, physiological

consequence, and behavioral effects. Knowledge of exact mechanisms of

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

change is a neurotoxic effect.

Neurotoxic effects can be produced by chemicals that do not require

metabolism prior to interacting with their target sites in the nervous

system, i.e., primary neurotoxic agents, or those that require

metabolism prior to interacting with their target sites in the nervous

system, i.e., secondary neurotoxic agents. Chemically induced

neurotoxic effects can be direct, i.e., due to an agent or its

metabolites acting directly on target sites in the nervous system, or

indirect, i.e., due to agents or metabolites that produce their effects

primarily by interacting with target sites outside the nervous system,

which subsequently affect target sites in the nervous system.

Excitatory amino acids such as domoic acid damage specific neurons

directly by activating excitatory amino acid receptors in the nervous

system, while carbon monoxide decreases oxygen availability, which

indirectly kills neurons. Other examples of indirect effects of

chemicals that could lead to altered structure and/or function of the

nervous system include cadmium-induced spasms in blood vessels

supplying the nervous system, dichloroacetate-induced perturbation of

metabolic pathways, and chemically induced alterations in

skeletomuscular function or structure and effects on the endocrine

system. Professional judgment may be required in making determinations

about direct versus indirect effects.

The interpretation of data as indicative of a potential neurotoxic

effect involves the evaluation of the validity of the data base. This

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

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

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

different measures of intelligence or other abilities, aptitudes, or

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

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

the effects are adverse or toxicologically significant (construct

validity); whether there are correlative measures among behavioral,

physiological, neurochemical, and morphological end points (concurrent

validity); and whether the effects are predictive of what will happen

under various conditions (predictive validity). Addressing these issues

can provide a useful framework for evaluating either human or animal

studies or the weight of evidence for a chemical (Sette, 1987; Sette

and MacPhail, 1992). The next sections indicate the extent to which

chemically induced changes can be interpreted as providing evidence of

neurotoxicity.

III. Hazard Characterization

A. Neurotoxicological Studies: End Points and Their Interpretation

Identification and characterization of neurotoxic hazard can be

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

EPA, 1993). Such data can result from accidental, inappropriate, or

controlled experimental exposures. This section describes many of the

general and some of the specific characteristics of human studies and

reports of neurotoxicity. It then describes some features of animal

studies of neuroanatomical, neurochemical, neurophysiological, and

behavioral effects relevant to risk assessment. The process of

characterizing the sufficiency or insufficiency of neurotoxic effects

for risk assessment is described in section III.C. Additional sources

of information relevant to hazard characterization, such as comparisons

of molecular structure among compounds and in vitro screening methods,

are also discussed.

The hazard characterization should:

a. Identify strengths and limitations of the database:

--Epidemiological studies (case reports, cross-sectional, case-control,

cohort, or human laboratory exposure studies);

--Animal studies including (structural or neuropathological,

neurochemical, neurophysiological, behavioral or neurological, or

developmental end points).

[[Page 52037]]

b. Evaluate the validity of the database:

--Content validity (effects result from exposure);

--Construct validity (effects are adverse or toxicologically

significant);

--Concurrent validity (correlative measures among behavioral,

physiological, neurochemical, or morphological end points);

--Predictive validity (effects are predictive of what will happen under

various conditions).

c. Identify and describe key toxicological studies.

d. Describe the type of effects:

--Structural (neuroanatomical alternations);

--Functional (neurochemical, neurophysiological, behavioral

alterations).

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

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

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

mechanism) the chemical produces adverse effects.

g. Discuss other health end points of concern.

h. Comment on any non-positive data in humans or animals.

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

available for further dose-response analysis.

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

studies demonstrating neurotoxicity as compared to expected human

exposures.

k. Summarize the hazard characterization:

--Confidence in conclusions;

--Alternative conclusions also supported by the data;

--Significant data gaps; and

--Highlight of major assumptions.

1. Human Studies

It is well established that information from the evaluation of

human exposure can identify neurotoxic hazards (Anger and Johnson,

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

neurotoxicity in human populations are the outbreaks of methylmercury

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

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

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

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

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

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

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

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

known neurotoxicants. They may pose occupational risks to manufacturing

and formulation workers, pesticide applicators and farm workers, and

consumers through home application or consumption of residues in foods.

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

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

including clinical evaluations, case reports, and epidemiologic

studies. A more extensive description of issues concerning human

neurotoxicology and risk assessment has been published elsewhere (U.S.

EPA, 1993).

a. Clinical Evaluations. Clinical methods are used extensively in

neurology and neuropsychology to evaluate patients suspected of having

neurotoxicity. An extensive array of examiner-administered and paper-

and-pencil tasks are used to assess sensory, motor, cognitive, and

affective functions and personality states/traits. Neurobehavioral data

are synthesized with information from neurophysiologic studies and

medical history to derive a working diagnosis. Brain imaging techniques

based on magnetic resonance imaging or emission tomography may also be

useful in helping diagnose neurodegenerative disorders following

chemical exposures in humans (Omerod et al., 1994; Callender et al.,

1994). Clinical diagnostic approaches have provided a rich conceptual

framework for understanding the functions (and malfunctions) of the

central and peripheral nervous systems and have formed the basis for

the development of methods for measuring the behavioral expression of

nervous system disorders. Human neurobehavioral toxicology has borrowed

heavily from neurology and neuropsychology for concepts of nervous

system impairment and functional assessment methods. Neurobehavioral

toxicology has adopted the neurologic/neuropsychologic model, using

adverse changes in behavioral function to assist in identifying

chemically or drug-induced changes in nervous system processes.

Neurologic and neuropsychologic methods have long been employed to

identify the adverse health effects of environmental workplace

exposures (Sterman and Schaumburg, 1980). Peripheral neuropathies (with

sensory and motor disturbances), encephalopathies, organic brain

syndromes, extrapyramidal syndromes, demyelination, autonomic changes,

and dementia are well-characterized consequences of acute and chronic

exposure to chemical agents. The range of exposure conditions that

produce clinical signs of neurotoxicity also has been defined by these

clinical methods. It is very important to make external/internal dose

measurements in humans to determine the actual dose(s) that can cause

unwanted effects.

Aspects of the neurologic examination approach limit its usefulness

for neurotoxicologic risk assessment. Information obtained from the

neurologic exam is mostly qualitative and descriptive rather than

quantitative. Estimates of the severity of functional impairment can be

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

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

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

muscle strength are often judged by having the patient push against the

resistance of the examiner's hands. The end points are therefore the

absolute and relative amount of muscle load sensed by the examiner in

his or her arms.

Compared with other methods, the neurologic exam may be less

sensitive in detecting early neurotoxicity in peripheral sensory and

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

quantitative methods in assessing the amplitude of tremor, tremor

frequency is poorly quantified by clinicians. Thus, important aspects

of the clinical neurologic exam may be insufficiently quantified and

lack sufficient sensitivity for detecting early neurobehavioral

toxicity produced by environmental or workplace exposure conditions.

However, a neurologic evaluation of persons with documented

neurobehavioral impairment would be helpful for identifying nonchemical

causes of neurotoxicity, such as diabetes and cardiovascular

insufficiency.

Administration of a neuropsychological battery also requires a

trained technician, and interpretation requires a trained and

experienced neuropsychologist. Depending on the capabilities of the

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

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

considerations may limit the usefulness of neuropsychological

assessment in large field studies of suspected neurotoxicity.

In addition to logistical problems in administration and

interpretation, neuropsychological batteries and neurologic exams share

two disadvantages with respect to neurotoxicity risk assessment. First,

[[Page 52038]]

neurologic exams and neuropsychological test batteries are designed to

confirm and classify functional problems in individuals selected on the

basis of signs and symptoms identified by the patient, family, or other

health professionals. Their usefulness in detecting low base-rate

impairment in workers or the general population is generally thought to

be limited, decreasing the usefulness of clinical assessment approaches

for epidemiologic risk assessment.

Second, neurologic exams and neuropsychologic test batteries were

developed to assess the functional correlates of the most common forms

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

degenerative conditions. The clinical tests were validated against

these neurologic disease states. With a few notable exceptions,

chemicals are not believed to produce impairment similar to that from

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

of degenerative disease. There has been insufficient research to

demonstrate which tests designed to assess functional expression of

neurologic disease are useful in characterizing the modes of central

nervous system impairment produced by chemical agents and drugs.

b. Case reports. The first type of human data available is often

the case report or case series, which can identify cases of a disease

and are reported by clinicians or discerned through active or passive

surveillance, usually in the workplace. However, case reports where

exposure involved a single neurotoxic agent, although informative, are

rare in the literature; for example, farmers are likely to be exposed

to a wide variety of potentially neurotoxic pesticides. Careful case

histories assist in identifying common risk factors, especially when

the association between the exposure and disease is strong, the mode of

action of the agent is biologically plausible, and clusters occur in a

limited period of time.

Case reports are inexpensive compared with epidemiologic studies

and can be obtained more quickly than more complex studies. However,

they provide little information about disease frequency or population

at risk, but their importance has been clearly demonstrated,

particularly in accidental poisoning or acute exposure to high levels

of toxicant. They remain an important source of index cases of new

diseases and for surveillance.

c. Epidemiologic Studies. Epidemiology has been defined as ``the

study of the distributions and determinants of disease and injuries in

human populations'' (Mausner and Kramer, 1985). Knowing the frequency

of illness in groups and the factors that influence the distribution is

the tool of epidemiology that allows the evaluation of causal inference

with the goal of prevention and cure of disease (Friedlander and Hearn,

1980). Epidemiologic studies are a means of evaluating the effects of

neurotoxic substances on human populations, but such studies are

limited because they must be performed shortly after exposure if the

effect is acute. Most often these effects are suspected to be a result

of occupational exposures due to the increased opportunity for exposure

to industrial and other chemicals. Frequently, determining the precise

dose or exposure concentration can be difficult in epidemiological

studies.

(1) Cross-sectional studies. In cross-sectional studies or surveys,

both the disease and suspected risk factors are ascertained at the same

time, and the findings are useful in generating hypotheses. A group of

people are interviewed, examined, and tested at a single point in time

to ascertain a relationship between a disease and a neurotoxic

exposure. This study design does not allow the investigator to

determine whether the disease or the exposure came first, rendering it

less useful in estimating risk. These studies are intermediate in cost

and time required to complete compared with case reports and more

complex analytical studies but should be augmented with additional

data.

(2) Case-control (retrospective) studies. Last (1986) defines a

case-control study as one that ``starts with the identification of

persons with the disease (or other outcome variable) of interest, and a

suitable control population (comparison, reference group) of persons

without the disease.'' He states that the relationship of an

``attribute'' to the disease is measured by comparing the diseased with

the nondiseased with regard to how frequently the attribute is present

in each of the groups. The cases are assembled from a population of

persons with and without exposure, and the comparison group is selected

from the same population; the relative distribution of the potential

risk factor (exposure) in both groups is evaluated by computing an odds

ratio that serves as an estimate of the strength of the association

between the disease and the potential risk factor. The statistical

significance of the ratio is determined by calculating a p-value and is

used to approximate relative risk.

The case-control approach to the study of potential neurotoxicants

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

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

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

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

cases with the disease or a representative sample of them have been

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

representative of the nondiseased population (or that the prevalence of

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

the general population). Failure to satisfy these assumptions may

result in selection bias, but violation of assumptions does not

necessarily invalidate the study results.

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

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

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

These must be controlled either in the design of the study by matching

cases to controls on the basis of the confounding factor or in the

analysis of the data by using statistical techniques such as

stratification or regression. Matching requires time to identify an

adequate number of potential controls to distinguish those with the

proper characteristics, while statistical control of confounding

factors requires a larger study.

The definition of exposure is critical in epidemiologic studies. In

occupational settings, exposure assessment often is based on the job

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

company records allow the development of exposure profiles. Positive

results from a properly controlled retrospective

[[Page 52039]]

study should weigh heavily in the risk assessment process.

(3) Cohort (prospective, followup) studies. In a prospective study

design, a healthy group of people is assembled and followed forward in

time and observed for the development of disease. Such studies are

invaluable for determining the time course for development of disease

(e.g., followup studies performed in various cities on the effects of

lead on child development). This approach allows the direct estimate of

risks attributed to a particular exposure since disease incidence rates

in the cohort can be determined. Prospective study designs also allow

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

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

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

by obtaining information before the disease develops. This approach,

however, can be very time-consuming and costly.

In cohort studies information bias can be introduced when

individuals provide distorted information about their health because

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

health effects of the exposure under study.

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

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

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

involve specially exposed groups and have provided much assistance in

estimating risks due to occupational exposures. Occupational

retrospective cohort studies rely on company records of past and

current employees that include information on the dates of employment,

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

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

duration and degree of exposure. Positive results from a properly

controlled prospective study should weigh heavily in the risk

assessment process.

d. Human Laboratory Exposure Studies. Neurotoxicity assessment has

an advantage not afforded the evaluation of other toxic end points,

such as cancer or reproductive toxicity, in that the effects of some

chemicals are short in duration and reversible. This makes it ethically

possible to perform human laboratory exposure studies and obtain data

relevant to the risk assessment process. Information from experimental

human exposure studies has been used to set occupational exposure

limits, mostly for organic solvents that can be inhaled. Laboratory

exposure studies have contributed to risk assessment and the setting of

exposure limits for several solvents and other chemicals with acute

reversible effects.

Human exposure studies sometime offer advantages over epidemiologic

field studies. Combined with appropriate sampling of biologic fluids

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

examine toxicokinetics, and identify metabolites. Bioavailability,

elimination, dose-related changes in metabolic pathways, individual

variability, time course of effects, interactions between chemicals,

and interactions between chemical and environmental/biobehavioral

processes (stressors, workload/respiratory rate) are factors that are

generally easier to collect under controlled conditions.

Other goals of laboratory studies include the indepth

characterization of effects, the development of new assessment methods,

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

neurobehavioral assessment methods across chemical classes. The

laboratory is the most appropriate setting for the study of

environmental and biobehavioral variables that affect the action of

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

rate of ongoing behavior, conditioning variables, tolerance/

sensitization, sleep deprivation, motivation, and so forth are

sometimes studied.

From a methodologic standpoint, human laboratory studies can be

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

designs. In the former, the neurobehavioral performance of exposed

volunteers is compared with that of nonexposed participants. In the

latter, preexposure performance is compared with neurobehavioral

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

designs have the advantage of requiring fewer participants, eliminating

individual differences as a source of variability, and controlling for

chronic mediating variables, such as caffeine use and educational

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

neurobehavioral tests must be administered more than once. Practice on

many neurobehavioral tests often leads to improved performance that may

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

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

performance on all tests to achieve a relatively stable baseline level.

Participants in laboratory exposure studies may have been recruited

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

from naive populations. Although the use of exposed volunteers has

ethical advantages, can mitigate against novelty effects, and allows

evaluation of tolerance/sensitization, finding an accessible exposed

population in reasonable proximity to the laboratory is difficult.

Naive participants are more easily recruited but may differ

significantly in important characteristics from a representative sample

of exposed persons. Naive volunteers are often younger, healthier, and

better educated than the populations exposed environmentally, in the

workplace, or pharmacotherapeutically.

Compared with workplace and environmental exposures, laboratory

exposure conditions can be controlled more precisely, but exposure

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

chemicals are studied for several hours while the population of

interest may be exposed to multiple chemicals containing impurities for

months or years. Laboratory studies are therefore better at identifying

and characterizing effects with acute onset and the selective effects

of pure agents.

Neurobehavioral test methods may have been selected according to

several strategies. A test battery that examines multiple

neurobehavioral functions may be more useful for screening and the

initial characterization of acute effects. Selected neurobehavioral

tests that measure a more limited number of functions in multiple ways

may be more useful for elucidating mechanisms or validating specific

effects.

Both chemical and behavioral control procedures are valuable for

examining the specificity of the effects. A concordant effect among

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

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

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

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

nonspecific motor function. Likewise, finding concordant effects among

similar chemical or drug classes along with different effects from

dissimilar classes would support the specificity of chemical effect.

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

those of ethanol but not caffeine would support the specificity of

solvent effects on a given measure of neurotoxicity.

2. Animal Studies

This section provides an overview of the major types of end points

that may be evaluated in animal neurotoxicity

[[Page 52040]]

studies, describes the kinds of effects that may be observed and some

of the tests used to detect and quantify these effects, and provides

guidance for interpreting data. Compared with human studies, animal

studies are more often available for specific chemicals, provide more

precise exposure information, and control environmental factors better

(Anger, 1984). For these reasons, risk assessments tend to rely heavily

on animal studies.

Many tests that can measure some aspect of neurotoxicity have been

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

Prevention, Pesticides and Toxic Substances (OPPTS) has published

animal testing guidelines that were developed in cooperation with the

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

end points included serve as a convenient focus for this section, there

are many other end points for which there are no current EPA

guidelines. The goal of this document is to provide a framework for

interpreting data collected with tests frequently used by

neurotoxicologists.

Five categories of end points will be described: Structural or

neuropathological, neurophysiological, neurochemical, behavioral, and

developmental end points. Table 1 lists a number of end points in each

of these categories.

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

I. Structural or Neuropathological End Points

1. Gross changes in morphology, including brain weight

2. Hemorrhage in nerve tissue

3. Breakdown of neurons, glial cells

4. Accumulation, proliferation, or rearrangement of structural

elements

5. Glial fibrillary acidic protein increases (in adults)

II. Neurochemical End Points

1. Alterations in synthesis, release, uptake, degradation of

neurotransmitters

2. Alterations in second messenger associated signal

transduction

3. Alterations in membrane-bound enzymes regulating neuronal

activity

4. Inhibition of neuropathy target enzyme (40%)

III. Neurophysiological End Points

1. Change in velocity, amplitude, or refractory period of nerve

conduction

2. Change in latency or amplitude of sensory-evoked potential

3. Change in electroencephalographic pattern

IV. Behavioral and Neurological End Points

1. Increases or decreases in motor activity

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

3. Changes in motor coordination, weakness, paralysis, abnormal

movement or posture, tremor, ongoing performance

4. Absence or decreased occurrence, magnitude, or latency of

sensorimotor reflex

5. Altered magnitude of neurological measurement, including grip

strength, hindlimb splay

6. Seizures

7. Changes in rate or temporal patterning of schedule-controlled

behavior

8. Changes in learning, memory, intelligence, attention

V. Developmental End Points

1. Chemically induced changes in the time of appearance of

behaviors during development

2. Chemically induced changes in the growth or organization of

structural or neurochemical elements.

a. Structural End Points of Neurotoxicity. Structural end points

are typically defined as neuropathological changes measured through

gross observation or with the aid of a microscope. Gross changes in

morphology can include discrete or widespread lesions in nerve tissue.

Changes in brain size (weight, width, or length) are considered to be

indicative of neurotoxic events. This is true regardless of changes in

body weight, because brain size is generally protected during

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

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

weight and thereby dismiss changes in absolute brain weight. The risk

assessor should be aware that a unit of measurement that is

biologically meaningful should be used for analysis. Brain length

measurements, for example, expressed to 1 or 10 micron units is

biologically meaningless. The same is true for brain width.

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

processes. Various types of neuropathological lesions may be classified

according to the site where they occur (WHO, 1986; Krinke, 1989;

Griffin, 1990). Neurodegenerative lesions in the central or peripheral

nervous system may be classified as a neuronopathy (changes in the

neuronal cell body), axonopathy (changes in the axons), myelinopathy

(changes in the myelin sheaths), or terminal degeneration. For

axonopathies, a more precise location of the changes may also be

described (i.e., proximal, central, or distal axonopathy). In the case

of some developmental exposures, a neurotoxic chemical might delay or

accelerate the differentiation or proliferation of cells or cell types.

Alteration in the axonal termination site might also occur with

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

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

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

neuropathic conditions resembling neurodegenerative disorders in humans

such as Parkinson's disease (WHO, 1986). Table 2 lists examples of such

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

neuropathology produced, and the disease or condition that each

typifies.

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

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

Corresponding

Site of action Neuropathology Neurotoxicant neurodegenerative disease

or condition

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

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

acid 3-Acetylpyridine. Huntington's disease,

Cerebellar ataxia.

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

1,2,3,6-

tetrahydropyridine

(dopaminergic).

Schwann cell Myelin......... Myelinopathy................ Hexachlorophene......... Congenital

hypomyelinogenesis.

Central-peripheral distal Distal axonopathy........... Acrylamide Carbon Peripheral neuropathy.

axon. disulfide n-Hexane.

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

myeloopticoneuropathy.

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

iminodipropionitrile.

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

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

neuronopathy, axonopathy, myelinopathy, terminal degeneration) are

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

note that pathological changes in many cases require time for the

perturbation to become observable, especially with evaluation at the

light

[[Page 52041]]

microscopic level. Neuropathological studies should control for

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

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

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

nervous system tissues derives from its functional specialization and

the lack of regenerative capacity in the central nervous system.

In general, chemical effects can lead to two types of structural

alteration at the cellular level: the breakdown of cells, in whole or

in part, or the accumulation, proliferation, or rearrangement of

structural elements (e.g., intermediate filaments, microtubules) or

organelles (e.g., mitochondria). Some changes may be associated with

regenerative processes that reflect adaptive changes associated with

exposure to a toxicant.

Chemically induced injury to the central nervous system may be

associated with astrocytic hypertrophy. Such changes may be seen using

immunocytochemical techniques visualized by light microscopy or

quantified more precisely by radioimmunoassay (RIA) procedures. Assays

of glial fibrillary acidic protein (GFAP), the major intermediate

filament protein of astrocytes, have been proposed as a biomarker of

this response (O'Callaghan, 1988). The interpretation of a 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 in the Neurotoxicity

Test Battery testing guidelines (U.S. EPA, 1991a).

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

those necessary to produce damage seen by standard microscopic or

histopathological techniques. Because increases in GFAP reflect an

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

considered to be evidence that a neurotoxic effect has occurred.

Decreases in GFAP are not clearly interpretable as indicative of

neurotoxicity. The absence of a change in GFAP following exposure does

not necessarily mean that the chemical is devoid of neurotoxic

potential. Known neurotoxicants such as cholinesterase-inhibiting

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

of GFAP. Interpretation of GFAP changes prior to weaning is confounded

by the possibility that chemically induced increases in GFAP may be

masked by changes in the concentration of this protein associated with

maturation of the central nervous system, and these data may be

difficult to interpret.

b. Neurophysiological End Points of Neurotoxicity.

Neurophysiological studies are those that measure the electrical

activity of the nervous system. The term ``neurophysiology'' is often

used synonymously with ``electrophysiology'' (Dyer, 1987).

Neurophysiological techniques provide information on the integrity of

defined portions of the nervous system. Several neurophysiological

procedures are available for application to neurotoxicological studies.

Examples of neurophysiological measures of neurotoxicity are listed in

Table 3. They range in scale from procedures that employ

microelectrodes to study the function of single nerve cells or

restricted portions of them, to procedures that employ macroelectrodes

to perform simultaneous recordings of the summed activity of many

cells. Microelectrode procedures typically are used to study mechanisms

of action and are frequently performed in vitro. Macroelectrode

procedures are generally used in studies to detect or characterize the

potential neurotoxic effects of agents of interest because of potential

environmental exposure. The present discussion concentrates on

macroelectrode neurophysiological procedures because it is more likely

that they will be the focus of decisions regarding critical effects in

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

animals also have been used in humans to determine chemically induced

alterations in neurophysiological function.

Table 3.--Examples of Neurophysiological Measures of Neurotoxicity

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

System/function Procedure Representative agents

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

Retina................. Electroretinography Developmental lead.

(ERG).

Visual pathway......... Flash evoked potential Carbon disulfide.

(FEP).

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

potential (PEP)

pattern size and

contrast).

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

evoked potential Antibiotics, Toluene,

(BAER) (clicks). styrene.

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

Antibiotics, Toluene,

styrene.

Somatosensory pathway.. Somatosensory evoked Acrylamide, n-Hexane.

potential (SEP)

(shocks).

Somatosensory function. SEP (tactile).......... Acrylamide n-Hexane.

Spinocerebellar pathway SEP recorded from Acrylamide n-Hexane.

cerebellum.

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

compound action

potential (PNAP).

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

components.

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

components.

Neuromuscular.......... Electromyography (EMG), Dithiobiuret.

H-reflex, M-response.

General central nervous Electroencephalography Anesthetics.

system/level of (EEG).

arousal.

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

(1) Nerve conduction studies. Nerve conduction studies, generally

performed on peripheral nerves, can be useful in investigations of

possible peripheral neuropathy. Most peripheral nerves contain mixtures

of individual sensory and motor nerve fibers, which may or may not be

differentially sensitive to neurotoxicants. It is possible to

distinguish sensory from motor effects in peripheral nerve studies by

measuring activity in purely sensory

[[Page 52042]]

nerves such as the sural nerve or by measuring the muscle response

evoked by nerve stimulation to measure motor effects. While a number of

end points can be recorded, the most critical variables are (1) nerve

conduction velocity, (2) response amplitude, and (3) refractory period.

Nerve conduction measurements are influenced by a number of

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

conduction study will either measure the temperature of the limb under

study and mathematically adjust the results according to well-

established temperature factors or control limb temperature within

narrow limits. Studies that measure peripheral nerve function without

regard for temperature are not adequate for risk assessment.

In well-controlled studies, statistically significant decreases in

nerve conduction velocity are indicative of a neurotoxic effect. While

a decrease in nerve conduction velocity is indicative of demyelination,

it frequently occurs later in the course of axonal degradation because

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

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

conduction velocity does not rule out peripheral axonal degeneration if

other signs of peripheral nerve dysfunction are present.

Decreases in response amplitude reflect a loss of active nerve

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

course of peripheral neuropathy. Hence, changes in response amplitude

may be more sensitive measurements of axonal degeneration than

conduction velocity. Measurements of response amplitude, however, can

be more variable and require careful application of experimental

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

measurements of velocity to detect changes. The refractory period

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

again and provides a measure reflecting the functional status of nerve

membrane ion channels. Chemically induced changes in refractory periods

in a well-controlled study indicate a neurotoxic effect.

In summary, alterations in peripheral nerve response amplitude and

refractory period in studies that are well controlled for temperature

are indicative of a neurotoxic effect. Alterations in peripheral nerve

function are frequently associated with clinical signs such as

numbness, tingling, or burning sensations or with motor impairments

such as weakness. Examples of compounds that alter peripheral nerve

function in humans or experimental animals include acrylamide, carbon

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

(2) Sensory, motor, and other evoked potentials. Evoked potential

studies are electrophysiological procedures that measure the response

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

electrical pulse. Evoked potentials reflect the function of the system

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

involving motor nerves and innervated muscles; or other neural pathways

in the central or peripheral nervous system (Rebert, 1983; Dyer, 1985;

Mattsson and Albee, 1988; Mattsson et al., 1992; Boyes, 1992, 1993).

Evoked potential studies should be interpreted with respect to the

known or presumed neural generators of the responses, and their likely

relationships with behavioral outcomes, when such information is

available. Such correlative information strengthens the confidence in

electrophysiological outcomes. In the absence of such supportive

information, the extent to which evoked potential studies provide

convincing evidence of neurotoxicity is a matter of professional

judgment on a case-by-case basis. Judgments should consider the nature,

magnitude, and duration of such effects, along with other factors

discussed elsewhere in this document.

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

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

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

voltage peaks are identified and measured. Alternative measurement

schemes may involve substitution of spectral phase or template shifts

for peak latency and spectral power, spectral amplitude, root-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 must be interpreted in light of the behavioral status

of the subject and would generally be considered evidence of a

neurotoxic effect.

(3) Seizures/convulsions. Neurophysiological recordings of brain

electrical activity that demonstrate seizure-like activity are

indicative of a neurotoxic effect. Occasionally, behaviors resembling

convulsions might follow actions outside the nervous system, such as

direct effects on muscle. When convulsion-like behaviors are observed,

as described in the behavioral section, neurophysiological recordings

can determine if these behaviors

[[Page 52043]]

originate from seizure activity in the brain.

In addition to producing seizures directly, neurotoxicants also may

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

seizures produced through other means. Such changes can occur after

acute exposure or after repeated exposure to dose levels below the

acute threshold and are considered to be neurotoxic effects. Examples

of agents that produce convulsions include lindane, DDT (dichloro-

diphenyl-trichloroethane), pyrethroids, and trimethyltin.

(4) Electroencephalography (EEG). EEG analysis is used widely in

clinical settings for the diagnosis of neurological disorders and less

often for the detection of subtle toxicant-induced dysfunction (WHO,

1986; Eccles, 1988). The basis for using EEG in either setting is the

relationship between specific patterns of EEG waveforms and specific

behavioral states. Because states of alertness and stages of sleep are

associated with distinct patterns of electrical activity in the brain,

it is generally thought that arousal level can be evaluated by

monitoring the EEG.

Dissociation of EEG activity and behavior can, however, occur after

exposure to certain chemicals. Normal patterns of transition between

sleep stages or between sleeping and waking states are known to remain

disturbed for prolonged periods of time after exposure to some

chemicals. Changes in the pattern of the EEG can be elicited by stimuli

producing arousal (e.g., lights, sounds) and anesthetic drugs. In

studies with toxicants, changes in EEG pattern can sometimes precede

alterations in other objective signs of neurotoxicity (Dyer, 1987).

EEG studies must be done under highly controlled conditions, and

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

seizure activity detected in the EEG pattern is evidence of a

neurotoxic effect.

c. Neurochemical End Points of Neurotoxicity. Many different

neurochemical end points have been measured in neurotoxicological

studies, and some have proven useful in advancing the understanding of

mechanisms of action of neurotoxic chemicals (Bondy, 1986; Mailman,

1987; Morell and Mailman, 1987; Costa, 1988). Normal functioning of the

nervous system depends on the synthesis and release of specific

neurotransmitters and activation of their receptors at specific

presynaptic and postsynaptic sites. Chemicals can interfere with the

ionic balance of a neuron, act as a cytotoxicant after transport into a

nerve terminal, block reuptake of neurotransmitters and their

precursors, act as a metabolic poison, overstimulate receptors, block

transmitter release, and inhibit transmitter synthetic or catabolic

enzymes. Table 4 lists several chemicals that produce neurotoxic

effects at the neurochemical level (Bondy, 1986; Mailman, 1987; Morell

and Mailman, 1987; Costa, 1988).

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

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

Site of action Examples

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

1. Neurotoxicants Acting on Ionic

Balance:

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

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

channel.

C. Increase permeability to Batrachotoxin.

sodium.

D. Increase intracellular Chlordecone.

calcium.

2. Cytotoxicants--Depend on uptake MPTP.

into nerve terminal.

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

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

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

6. Blocks transmitter release Botulinum toxin.

(Acetylcholine [ACh]).

7. Inhibition of transmitter Pesticides of the organophosphate

degradation (ACh). and carbamate classes.

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

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

As stated previously, any neurochemical change is potentially

neurotoxic, but each determination requires professional judgment.

Persistent or irreversible chemically induced neurochemical changes are

indicative of neurotoxicity. Because the ultimate functional

significance of some biochemical changes is not known at this time,

neurochemical studies should be interpreted with reference to the

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

example, many neuroactive agents can increase or decrease

neurotransmitter levels, but such changes are not necessarily

indicative of a neurotoxic effect. If, however, these neurochemical

changes may be expected to have neurophysiological, neuropathological,

or neurobehavioral correlates, then the neurochemical changes could be

classified as neurotoxic effects.

Some neurotoxicants, such as the organophosphate and carbamate

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

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

the neurotransmitter acetylcholine. Inhibition of the enzyme prolongs

the action of the acetylcholine at the neuron's synaptic receptors and

is responsible for the autonomic stimulation and death that these

agents cause.

Within EPA and elsewhere, questions have arisen as to whether

inhibition of cholinesterase activity constitutes an adverse effect for

defining hazard potential and evaluating risk. There is agreement among

scientists that statistically significant inhibition of cholinesterase

activity in multiple organs and tissues accompanied by clinical effects

constitutes a hazard. However, there is scientific uncertainty and

related controversy about the risk assessment implications of data

describing inhibition of cholinesterase enzyme activity in the absence

of observable clinical effects. While there is agreement that such

inhibition is a biomarker of exposure, there is continued disagreement

over whether cholinesterase inhibition, especially in blood,

constitutes an adverse effect.

At this point, it can be stated that there is general agreement

among scientists that objective clinical measures of dysfunction/

impairment can be overt manifestations of inhibition of cholinesterase

in the nervous system. On the basis of clinical manifestations, e.g.,

muscle weakness, tremor, blurred vision, one should be able to evaluate

dose-response and dose-effect relationships and define the presence and

absence of given effects. A relationship between the effect and

cholinesterase inhibition should be

[[Page 52044]]

confirmed by biochemical measures of reduced cholinesterase activity.

In addition, a reduction in brain cholinesterase activity may or

may not be accompanied by clinical manifestations. Most experts in the

field acknowledge that when significant reductions in brain

cholinesterase activity alone occur, reduced cholinesterase levels

either are themselves toxic or would lead to a neurotoxic effect if

exposure were to persist over time or increase in magnitude. Therefore,

statistically significant decreases in brain cholinesterase could be

considered to be a biologically significant effect.

A reduction in RBC and/or plasma cholinesterase activity also may

or may not be accompanied by clinical manifestations. At this time,

there is general agreement that the observation of inhibition of RBC

and/or plasma cholinesterase contributes to the overall hazard

identification of cholinesterase inhibiting agents by serving as

biomarkers. As such, these enzyme parameters can provide information

that will help scientists evaluate whether reported clinical effects

are associated with cholinesterase inhibition. There remains, however,

a lack of consensus as to whether RBC and/or plasma cholinesterase

represent biologically significant events. Discussions on this topic

are continuing within the Agency.

A subset of organophosphate agents also produces organophosphate-

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

Prolonged inhibition (i.e., aging) of neurotoxic esterase (or

neuropathy target enzyme) has been associated with agents that produce

OPIDN (Johnson, 1990), a clear neurotoxic effect.

d. Behavioral End Points of Neurotoxicity. EPA's testing guidelines

developed for the Toxic Substances Control Act and the Federal

Insecticide, Fungicide and Rodenticide Act describe the use of

functional observational batteries (FOB), motor activity, and schedule-

controlled behavior for assessing neurotoxic potential (U.S. EPA,

1991a). There are many other measures of behavior, including

specialized tests of motor and sensory function and of learning and

memory (Tilson, 1987; Anger, 1984). Examples of behavioral end points

that have been used to detect neurotoxicity are included in Table 1.

The risk assessor should know that the literature is clear that a

number of other behaviors besides those listed in Tables 1 and 5 could

be affected by chemical exposure. For example, alterations in food and

water intake, reproduction, sleep, temperature regulation, and

circadian rhythmicity are controlled by specific regions of the brain

and chemical-induced alterations in these behaviors could be indicative

of neurotoxicity. It is reasonable to assume that a NOAEL or LOAEL

could be based on one or more of these end points.

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

Produced by Each

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

Home cage and open field Manipulative Physiologic

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

Posture (D) Ease of removal (R) Body temperature (I).

Convulsions, tremors (D) Handling reactivity (R) Body weight (I).

Palpebral closure (R) Palpebral closure (R). .................................

Lacrimation (R) Approach response (R).

Piloerection (Q) Click response (R).

Salivation (R) Touch response (R).

Vocalizations (Q) Tail pinch response (R).

Rearing (C) Righting reflex (R).

Urination (C) Landing foot splay (I).

Defecation (C) Forelimb grip strength (I).

Gait (D, R) Hindlimb grip strength (I).

Arousal (R) Pupil response (Q).

Mobility (R).

Stereotypy (D).

Bizarre behavior (D)

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

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

Behavior is an indication of the overall well-being of the

organism. Changes in behavior can arise from a direct effect of a

toxicant on the nervous system or indirectly from its effects on other

physiological systems. Understanding the interrelationship between

systemic toxicity and behavioral changes is extremely important (e.g.,

the relationship between liver damage and motor activity). The presence

of systemic toxicity may complicate, but does not necessarily preclude,

interpretation of behavioral changes as evidence of neurotoxicity. In

addition, a number of behaviors (e.g., schedule-controlled behavior)

may require a motivational component for successful completion of the

task. In such cases, experimental paradigms designed to assess the

motivation of an animal during behavior might be necessary to interpret

the meaning of some chemical-induced changes in behavior.

The following sections describe in general behavioral tests and

their uses and offer guidance on interpreting data.

(1) Functional observational battery. A functional observational

battery is designed to detect and quantify major overt behavioral,

physiological, and neurological signs (Gad, 1982; O'Donoghue, 1989;

Moser, 1989). A number of batteries have been developed, each

consisting of tests generally intended to evaluate various aspects of

sensorimotor function (Tilson and Moser, 1992). Many FOB tests are

essentially clinical neurological examinations that rate the presence

or absence, and in many cases the severity, of specific neurological

signs. Some FOBs in animals are similar to clinical neurological

examinations used with human patients. Most FOBs have several

components or tests. A typical FOB is summarized in Table 5 and

evaluates several functional domains, including neuromuscular (i.e.,

weakness, incoordination, gait, and tremor), sensory (i.e., audition,

vision, and somatosensory), and autonomic (i.e., pupil response and

salivation) function. FOB data may be in the form of interval, ordinal,

or continuous measurements.

The relevance of statistically significant test results from an FOB

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

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

of the effects and their

[[Page 52045]]

incidence in relation to control animals. If only a few unrelated

measures in the FOB are affected, or the effects are unrelated to dose,

the results are not considered evidence of a neurotoxic effect. If

several neurological signs are affected but only at the high dose and

in conjunction with other overt signs of toxicity, including systemic

toxicity, large decreases in body weight, decreases in body

temperature, or debilitation, there is no conclusive evidence of a

direct neurotoxic effect. In cases where several related measures in a

battery of tests are affected and the effects appear to be dose

dependent, the data are considered to be evidence of a neurotoxic

effect, especially in the absence of systemic toxicity. Recently, it

was proposed that data from FOB studies be grouped into several

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

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

visual, somatosensory), and autonomic functions (Tilson and Moser,

1992). This statistical technique is useful when separating changes

that occur on the basis of chance or in conjunction with systemic

toxicity from those treatment-related changes indicative of neurotoxic

effects. In the case of the developing organism, chemicals may alter

the maturation or appearance of sensorimotor reflexes. Significant

alterations in or delay of such reflexes is evidence of a neurotoxic

effect.

Examples of chemicals that affect neuromuscular function are 3-

acetylpyridine, acrylamide, and triethyltin. Organophosphate and

carbamate insecticides produce autonomic dysfunction, while

organochlorine and pyrethroid insecticides increase sensorimotor

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

convulsions (Spencer and Schaumberg, 1980).

(2) Motor activity. Motor activity represents a broad class of

behaviors involving coordinated participation of sensory, motor, and

integrative processes. Assessment of motor activity is noninvasive and

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

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

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

agents, including nonneurotoxic agents. Motor activity measurements

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

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

Motor activity is usually quantified as the frequency of movements

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

will depend on the recording mechanism and size and configuration of

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

expressed as absolute activity counts or as a percentage of control

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

to achieve a normal distribution of the data. The frequency of motor

activity within a session usually decreases and is reported as the

average number of counts occurring in each successive block of time.

The EPA's Office of Prevention, Pesticides and Toxic Substances

guidelines (U.S. EPA, 1991a), for example, call for test sessions of

sufficient duration to allow motor activity to approach steady-state

levels during the last 20 percent of the session for control animals. A

sum of the counts in each epoch will add up to the total number of

counts per session.

In the adult, neurotoxic agents generally decrease motor activity

(MacPhail et al., 1989). Examples include many pesticides (e.g.,

carbamates, chlorinated hydrocarbons, organophosphates, and

pyrethroids), heavy metals (lead, tin, and mercury), and other agents

(3-acetylpyridine, acrylamide, and 2,4-dithiobiuret). Some

neurotoxicants (e.g., toluene, xylene, triadimefon) produce transient

increases in activity by presumably stimulating neurotransmitter

release, while others (e.g., trimethyltin) produce persistent increases

in motor activity by destroying specific regions of the brain (e.g.,

hippocampus).

Following developmental exposures, neurotoxic effects are often

observed as a change in the developmental profile or maturation of

motor activity patterns. Frequently, developmental exposure to

neurotoxic agents will produce an increase in motor activity that

persists into adulthood or that results in changes in other behaviors.

This type of effect is evidence of a neurotoxic effect. Like other

organ systems, the nervous system may be differentially sensitive to

toxicants in groups such as the young. For example, toxicants

introduced to the developing nervous system may kill stem cells and

thus cause profound effects on adult structure and function. Moreover,

toxicants may have greater access to the developing nervous system

before the blood-brain barrier is completely formed or before metabolic

detoxifying systems are functional.

Motor activity measurements are typically used with other tests

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

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

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

related fashion are of less concern than changes that are dose

dependent, related to structural or other functional changes in the

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

(3) Schedule-controlled operant behavior. Schedule-controlled

operant behavior (SCOB) involves the maintenance of behavior (e.g.,

performance of a lever-press or key-peck response) by reinforcement.

Different rates and patterns of responding are controlled by the

relationship between response and subsequent reinforcement. SCOB

provides a measure of performance of a learned behavior (e.g., lever

press or key peck) and involves training and motivational variables

that must be considered in evaluating the data. Agents may interact

with sensory processing, motor output, motivational variables (i.e.,

related to reinforcement), training history, and baseline

characteristics (Rice, 1988; Cory-Slechta, 1989). Rates and patterns of

SCOB display remarkable species and experimental generality.

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

neurotoxicants, including methylmercury, many pesticides, carbon

disulfide, organic and inorganic lead, and triethyl and trimethyltin

(MacPhail, 1985; Tilson, 1987; Rice, 1988). The primary SCOB end points

for evaluation are response rate and the temporal pattern of

responding. These end points may vary as a function of the contingency

between responding and reinforcement presentation (i.e., schedule of

reinforcement). While most chemicals decrease the efficiency of

responding at some dose, some agents may increase response efficiency

on schedules requiring high response rates due to a stimulant effect or

an increase in central nervous system excitability. Agent-induced

changes in responding between reinforcements (i.e., the temporal

pattern of responding) may occur independently of changes in the

overall rate of responding. Chemicals may also affect the reaction time

to respond following presentation of a stimulus. Agent-induced changes

in response rate or temporal patterning associated with other overt

signs of toxicity (e.g., body weight loss, systemic toxicity, or

occurring in a non-dose-related fashion) are of less concern than

changes that are dose dependent, related to structural or other

functional changes in the nervous system, or occur in the absence of

life-threatening toxicity.

(4) Convulsions. Observable convulsions in animals are indicative

of an adverse effect. These events can

[[Page 52046]]

reflect central nervous system activity comparable to that of epilepsy

in humans and could be defined as neurotoxicity. Occasionally, other

toxic actions of compounds, such as direct effects on muscle, might

mimic some convulsion-like behaviors. In some cases, convulsions or

convulsion-like behaviors may be observed in animals that are otherwise

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

convulsions might reflect an indirect effect of systemic toxicity and

are less clearly indicative of neurotoxicity. As discussed in the

section on neurophysiological measures, electrical recordings of brain

activity could be used to determine specificity of effects on the

nervous system.

(5) Specialized tests for neurotoxicity. Several procedures have

been developed to measure agent-induced changes in specific

neurobehavioral functions such as motor, sensory, or cognitive function

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

behavioral tests, the neurobehavioral functions they were designed to

assess, and agents known to affect the response. Many of these tests in

animals have been designed to assess neural functions in humans using

similar testing procedures.

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

Neurotoxicity

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

Function Procedure Representative agents

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

Neuromuscular:

Weakness........... Grip strength; swimming n-Hexane, methl n-

endurance; suspension butylketone,

rod; discriminative carbaryl.

motor function.

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

measurements; righting ethanol.

reflex.

Tremor............. Rating scale, spectral Chlordecone, Type I

analysis. pyrethroids, DDT.

Myoclonia spasms... Rating scale, spectral DDT, Type II

analysis. pyrethroids.

Sensory:

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

conditioning Reflex

modification.

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

conditioning.

Somatosensory...... Discrimination Acrylamide.

conditioning.

Pain sensitivity... Discrimination Parathion.

conditioning

(titration);

functional

observational battery.

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

conditioning. methylbromide.

Learning/Memory:

Habituation........ Startle reflex......... Diisopropyl-

fluorophosphate (DFP)

Pre/neonatal

methylmercury.

Classical Nictitating membrane... Aluminum.

conditioning. Conditioned flavor Carbaryl.

aversion. Trimethyltin, IDPN.

Passive avoidance...... Neonatal trimethyltin.

Olfactory conditioning.

Operant One-way avoidance...... Chlordecone.

conditioning. Two-way avoidance...... Pre/neonatal lead.

Y-maze avoidance....... Hypervitaminois A.

Biel water maze........ Styrene.

Morris water maze...... DFP.

Radial arm maze........ Trimethyltin.

Delayed matching to DFP.

sample. Carbaryl.

Repeated acquisition... Lead.

Visual discrimination..

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

A statistically significant chemically induced change in any

measure in Table 6 is presumptive evidence of adverse effect. Judgments

of neurotoxicity may involve not only the analysis of changes seen but

the structure and class of the chemical and other available

neurochemical, neurophysiological, and neuropathological evidence. In

general, behavioral changes seen across broader dose ranges indicate

more specific actions on the systems underlying those changes, i.e.,

the nervous system. Changes that are not dose dependent or that are

confounded with body weight changes and/or other systemic toxicity may

be more difficult to interpret as neurotoxic effects.

(a) Motor function: Neurotoxicants commonly affect motor function.

These effects can be categorized generally into (1) weakness or

decreased strength, (2) tremor, (3) incoordination, and (4) spasms,

myoclonia, or abnormal motor movements (Tilson, 1987; Cory-Slechta,

1989). Specialized tests used to assess weakness include measures of

grip strength, swimming endurance, suspension from a hanging rod, and

discriminative motor function. Rotarod and gait assessments are used to

measure incoordination, while rating scales and spectral analysis

techniques can be used to quantify tremor and other abnormal movements.

(b) Sensory function: Gross perturbations of sensory function can

be observed in simple neurological assessments such as the FOB.

However, these tests may not be sufficiently sensitive to detect subtle

sensory changes. Psychophysical procedures that study the relationship

between a physical dimension (e.g., intensity, frequency) of a stimulus

and behavior may be necessary to quantify agent-induced alterations in

sensory function. Examples of psychophysical procedures include

discriminated conditioning and startle reflex modification.

(c) Cognitive function: Alterations in learning and memory in

experimental animals must be inferred from changes in behavior

following exposure when compared with that either seen prior to

exposure or with a nonexposed control group. Learning is defined as a

relatively lasting change in behavior due to experience, and memory is

defined as the persistence of a learned behavior over time. Table 6

lists several examples of learning and memory tests and representative

neurotoxicants known to affect these tests. Measurement of changes in

learning and memory must be separated from other changes in behavior

that do not involve cognitive or associative processes (i.e., motor

function, sensory capabilities, motivational factors). In addition, any

apparent toxicant-induced change in learning or memory should ideally

be

[[Page 52047]]

demonstrated over a range of stimulus and response conditions and

testing conditions. In developmental exposures, it should be shown that

the animals have matured enough to perform the specified task.

Developmental neurotoxicants can accelerate or delay the ability to

learn a response or interfere with cognitive function at the time of

testing. Older animals frequently perform poorly on some types of

tests, and it must be demonstrated that control animals in this

population are capable of performing the procedure. Neurotoxicants

might accelerate age-related dysfunction or alter motivational

variables that are important for learning to occur. Further, it is not

necessarily the case that a decrease in responding on a learning task

is adverse while an increase in performance on a learning task is not.

It is well known that lesions in certain regions of the brain can

facilitate the acquisition of certain types of behaviors by removing

preexisting response tendencies (e.g., inhibitory responses due to

stress) that moderate the rate of learning under normal circumstances.

Examples of learning and memory procedures include simple habituation,

classical conditioning, and operant (or instrumental) conditioning,

including tests for spatial learning and memory.

e. Developmental Neurotoxicity. Although the previous discussion of

various neurotoxicity end points and tests applies to studies in which

developmental exposures are used, there are particular issues of

importance in the evaluation of developmental neurotoxicity studies.

Exposure to chemicals during development can result in a spectrum of

effects, including death, structural abnormalities, altered growth, and

functional deficits (U.S. EPA, 1991b). Children are often

differentially sensitive to chemical exposure. A number of agents have

been shown to cause developmental neurotoxicity when exposure occurred

during the period between conception and sexual maturity (e.g., Riley

and Vorhees, 1986; Vorhees, 1987). Table 7 lists several examples of

agents known to produce developmental neurotoxicity in experimental

animals. Animal models of developmental neurotoxicity have been shown

to be sensitive to several environmental agents known to produce

developmental neurotoxicity in humans, including lead, ethanol, 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 to

be adverse effects.

Table 7.--Examples of Developmental Neurotoxicants

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

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

Alcohols........................... Methanol, ethanol.

Antimitotics....................... X-radiation, azacytidine.

Insecticides....................... DDT, kepone.

Metals............................. Lead, methylmercury, cadmium.

Polyhalogenated hydrocarbons....... PCBs, PBBs.

Solvents........................... Carbon disulfide, toluene.

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

Testing for developmental neurotoxicity has not been required

routinely by regulatory agencies in the United States, but is required

by the EPA when other information indicates the potential for

developmental neurotoxicity (U.S. EPA, 1986, 1988a, 1988b, 1989, 1991a,

1991b). Useful data for decision making may be derived from well-

conducted adult neurotoxicity studies, standard developmental toxicity

studies, and multigeneration studies, although the dose levels used in

the latter may be lower than that in studies with shorter term

exposure.

Important design issues to be evaluated for developmental

neurotoxicity studies are similar to those for standard developmental

toxicity studies (e.g., a dose-response approach with the highest dose

producing minimal overt maternal or perinatal toxicity, number of

litters large enough for adequate statistical power, randomization of

animals to dose groups and test groups, litter generally considered as

the statistical unit). In addition, the use of a replicate study design

provides added confidence in the interpretation of data. A

pharmacological/physiological challenge may also be valuable in

evaluating neurologic function and ``unmasking'' effects not otherwise

detectable. For example, a challenge with a psychomotor stimulant such

as d-amphetamine may unmask latent developmental neurotoxicity (Hughes

and Sparber, 1978; Adams and Buelke-Sam, 1981; Buelke-Sam et al.,

1985).

Direct extrapolation of developmental neurotoxicity to humans is

limited in the same way as for other end points of toxicity, i.e., by

the lack of knowledge about underlying toxicological mechanisms and

their significance (U.S. EPA, 1991b). However, comparisons of human and

animal data for several agents known to cause developmental

neurotoxicity in humans showed many similarities in effects (Kimmel et

al., 1990). Comparisons at the level of functional category (sensory,

motivational, cognitive, and motor function and social behavior) showed

close agreement across species for the agents evaluated, even though

the specific end points used to assess these functions varied

considerably across species (Stanton and Spear, 1990). Thus, it can be

assumed that developmental neurotoxicity effects in animal studies

indicate the potential for altered neurobehavioral development in

humans, although the specific types of developmental effects seen in

experimental animal studies will not necessarily be the same as those

that may be produced in humans. Therefore, when data suggesting adverse

effects in developmental neurotoxicity studies are encountered for

particular agents, they should be considered in the risk assessment

process.

Functional tests with a moderate degree of background variability

(e.g., a coefficient of variability of 20 percent or less) may be more

sensitive to the effects of an agent on behavioral end points than are

tests with low variability that may be impossible to disrupt without

using life-threatening doses. A battery of functional tests, in

contrast to a single test, is usually needed to evaluate the full

complement of nervous system functions in an animal. Likewise, a series

of tests conducted in animals in several age groups may provide more

information about maturational changes and their persistence than tests

conducted at a single age.

It is a well-established principle that there are critical

developmental periods for the disruption of functional competence,

which include both the prenatal and postnatal periods to the time of

sexual maturation, and the effect of a toxicant is likely to vary

depending on the time and degree of exposure (Rodier, 1978, 1990). It

is also important to consider the data from studies in which postnatal

exposure is included, as there may be an interaction of the agent with

maternal behavior, milk composition, pup suckling behavior, as well as

possible direct exposure of pups via dosed food or water (Kimmel et

al., 1992).

Agents that produce developmental neurotoxicity at a dose that is

not toxic to the maternal animal are of special concern. However,

adverse developmental effects are often produced at doses that cause

maternal toxicity (e.g., 20

percent reduction in weight gain during gestation and lactation),

interpretation of developmental effects may be confounded. Current

information is inadequate to assume that developmental effects at doses

causing minimal maternal toxicity result only from maternal toxicity;

rather, it may be that the mother and developing organism are equally

sensitive to that dose level. Moreover, whether developmental effects

are secondary to maternal toxicity or not, the maternal effects may be

reversible while the effects on the offspring may be permanent. These

are important considerations for agents to which humans may be exposed

at minimally toxic levels either voluntarily or involuntarily, because

several agents are known to produce adverse developmental effects at

minimally toxic doses in adult humans (e.g., alcohol) (Coles et al.,

1991).

Although interpretation of developmental neurotoxicity data may be

limited, it is clear that functional effects must be evaluated in light

of other toxicity data, including other forms of developmental toxicity

(e.g., structural abnormalities, perinatal death, and growth

retardation). For example, alterations in motor performance may be due

to a skeletal malformation rather than nervous system change. Changes

in learning tasks that require a visual cue might be influenced by

structural abnormalities in the eye. The level of confidence that an

agent produces an adverse effect may be as important as the type of

change seen, and confidence may be increased by such factors as

reproducibility of the effect either in another study of the same

function or by convergence of data from tests that purport to measure

similar functions. A dose-response relationship is an extremely

important measure of a chemical's effect; in the case of developmental

neurotoxicity both monotonic and biphasic dose-response curves are

likely, depending on the function being tested. The EPA Guidelines for

Developmental Toxicity Risk Assessment (U.S. EPA, 1991b) may be

consulted for more information on interpreting developmental toxicity

studies. The endpoints frequently used to assess developmental

neurotoxicity in exposed children was recently reviewed by Winneke

(1995).

3. Other Considerations

a. Pharmacokinetics. Extrapolation of test results between species

can be aided considerably by data on the pharmacokinetics of a

particular agent in the species tested and, if possible, in humans.

Information on a toxicant's half-life, metabolism, absorption,

excretion, and distribution to the peripheral and central nervous

system may be useful in predicting risk. Of particular importance for

the pharmacokinetics of neurotoxicants is the blood-brain barrier,

which ordinarily excludes ionic and nonlipid soluble chemicals from the

central nervous system. The brain contains circumventricular organs

whose purpose seems to be to sense the chemical composition of the

peripheral circulation and activate mechanisms to bring the composition

of the blood back to equilibrium if disturbed. These areas are

technically inside the brain, but they lie outside of the blood-brain-

barrier. Therefore, chemicals from the periphery can pass directly into

the brain at these sites. The majority of these structures are located

within or near the hypothalamus, an area that is crucial for

maintenance of neuroendocrine function. Pharmacokinetic data may be

helpful in defining the dose-response curve, developing a more accurate

basis for comparing species sensitivity (including that of humans),

determining dosimetry at target sites, and comparing pharmacokinetic

profiles for various dosing regimens or routes of administration. The

correlation of pharmacokinetic parameters and neurotoxicity data may be

useful in determining the contribution of specific pharmacokinetic

processes to the effects observed.

b. Comparisons of Molecular Structure. Comparisons of the chemical

or physical properties of an agent with those of known neurotoxicants

may provide some indication of the potential for neurotoxicity. Such

information may be helpful for evaluating potential toxicity when only

minimal data are available. The structure-activity relationships (SAR)

of some chemical classes have been studied, including hexacarbons,

organophosphates, carbamates, and pyrethroids. Therefore, class

relationships or SAR may help predict neurotoxicity or interpret data

from neurotoxicological studies. Under certain circumstances (e.g., in

the case of new chemicals), this procedure is one of the primary

methods used to evaluate the potential for toxicity when little or no

empirical toxicity data are available. It should be recognized,

however, that effects of chemicals in the same class can vary widely.

Moser (1994), for example, reported that the behavioral effects of

prototypic cholinesterase-inhibiting pesticides differed qualitatively

in a battery of behavioral tests.

c. Statistical Considerations. Properly designed studies on the

neurotoxic effects of compounds will include appropriate statistical

tests of significance. In general, the likelihood of obtaining a

significant effect will depend jointly on the magnitude of the effect

and the variability obtained in control and treated groups. A number of

texts are available on standard statistical tests (e.g., Siegel, 1956;

Winer, 1971; Sokal and Rohlf, 1969; Salsburg, 1986; Gad and Weil,

1988).

Neurotoxicity data present some unique features that must be

considered in selecting statistical tests for analysis. Data may

involve several different measurement scales, including categorical

(affected or not), rank (more or less affected), and interval and ratio

scales of measurement (affected by some percentage). For example,

convulsions are usually recorded as being present or absent

(categorical), whereas neuropathological changes are frequently

described in terms of the degree of damage (rank). Many tests of

neurotoxicity involve interval or ratio measurements (e.g., frequency

of photocell interruptions or amplitude of an evoked potential), which

are the most powerful and sensitive scales of measurement. In addition,

measurements are frequently made repeatedly in control and treated

subjects, especially in the case of behavioral and neurophysiological

end points. For example, OPPTS guidelines for FOB assessment call for

evaluations before exposure and at several times during exposure in a

subchronic study (U.S. EPA, 1991a).

Descriptive data (categorical) and rank order data can be analyzed

using standard nonparametric techniques (Siegel, 1956). In some cases,

if it is determined that the data fit the linear model, the categorical

modeling procedure can be used for weighted least-squares estimation of

parameters for a wide range of general linear models, including

repeated-measures analyses. The weighted least-squares approach to

categorical and rank data allows computation of statistics for testing

the significance of sources of variation as reflected by the model. In

the case of studies assessing effects in the same animals at several

time points, univariate analyses can be carried out at each time point

when the overall dose effect or the dose-by-time interaction is

significant.

[[Page 52049]]

Continuous data (e.g., magnitude, rate, amplitude), if found to be

normally distributed, can be analyzed with general linear models using

a grouping factor of dose and, if necessary, repeated measures across

time (Winer, 1971). Univariate analyses of dose, comparing dose groups

to the control group at each time point, are performed when there is a

significant overall dose effect or a dose-by-time interaction. Post hoc

comparisons between control and treatment groups can be made following

tests for overall significance. In the case of multiple end points

within a series of evaluations, some type of correction for multiple

observations is warranted (Winer, 1971).

d. In Vitro Data in Neurotoxicology. Methods and procedures that

fall under the general heading of short-term tests include an array of

in vitro tests that have been proposed as alternatives to whole-animal

tests (Goldberg and Frazier, 1989). In vitro approaches use animal or

human cells, tissues, or organs and maintain them in a nutritive

medium. Various types of in vitro techniques produce data for

evaluating potential and known neurotoxic substances, including primary

cell cultures, cell lines, and cloned cells. While such procedures are

important in studying the mechanism of action of toxic agents, their

use in hazard identification in human health risk assessment has not

been explored to any great extent.

Data from in vitro procedures are generally based on simplified

approaches that require less time to yield information than do many in

vivo techniques. However, in vitro methods generally do not take into

account the distribution of the toxicant in the body, the route of

administration, or the metabolism of the substance. It also is

difficult to extrapolate in vitro data to animal or human neurotoxicity

end points, which include behavioral changes, motor disorders, sensory

and perceptual disorders, lack of coordination, and learning deficits.

In addition, data from in vitro tests cannot duplicate the complex

neuronal circuitry characteristic of the intact animal.

Many in vitro systems are now being evaluated for their ability to

predict the neurotoxicity of various agents seen in intact animals.

This validation process requires considerations in study design,

including defined end points of toxicity and an understanding of how a

test agent would be handled in vitro as compared to the intact

organism. Demonstrated neurotoxicity in vitro in the absence of in vivo

data is suggestive but inadequate evidence of a neurotoxic effect. In

vivo data supported by in vitro data enhance the reliability of the in

vivo results.

B. Dose-Response Evaluation

Dose-response evaluation is a critical part of hazard

characterization and involves the description of the dose response

relationship in the available data. Human studies covering a range of

exposures are rarely available and therefore animal data are typically

used for estimating exposure levels likely to produce adverse effects

in humans. Evidence for a dose-response relationship is an important

criterion in establishing a neurotoxic effect, although this analysis

may be limited when based on standard studies using three dose groups

or fewer. The evaluation of dose-response relationships includes

identifying effective dose levels as well as doses associated with no

increase in incidence of adverse effects when compared with controls.

Much of the focus is on identifying the critical effect(s) observed at

the lowest-observed-adverse-effect-level and the no-observed-adverse-

effect-level associated with that effect. The NOAEL is defined as the

highest dose at which there is no statistically or biologically

significant increase in the frequency of an adverse neurotoxic effect

when compared with the appropriate control group in a data base

characterized as having sufficient evidence for use in a risk

assessment (see section C). Although a threshold is assumed for

neurotoxic effects, the existence of a NOAEL in an animal study does

not prove or disprove the existence or level of a biological threshold.

Alternatively, mathematical modeling of the dose-response relationship

may be performed to determine a quantitative estimate of responses in

the experimental range. This approach can be used to determine a BMD,

which may be used in place of the NOAEL (Crump, 1994) (see Dose-

Response Analysis, Section IV).

In addition to identifying the NOAEL/LOAEL or BMD, the dose-

response evaluation defines the range of doses that are neurotoxic for

a given agent, species, route of exposure, and duration of exposure. In

addition to these considerations, pharmacokinetic factors and other

aspects that might influence comparisons with human exposure scenarios

should be taken into account. For example, dose-response curves may

exhibit not only monotonic but also U-shaped or inverted U-shaped

functions (Davis and Svendsgaard, 1990). Such curves are hypothesized

to reflect multiple mechanisms of action, the presence of homeostatic

mechanisms, and/or activation of compensatory or protective mechanisms.

In addition to considering the shape of the dose-response curve, it

should also be recognized that neurotoxic effects vary in terms of

nature and severity across dose or exposure level. At high levels of

exposure, frank lesions accompanied by severe functional impairment may

be observed. Such effects are widely accepted as adverse. At

progressively lower levels of exposure, however, the lesions may become

less severe and the impairments less obvious. At levels of exposure

near the NOAEL and LOAEL, the effects will often be mild, possibly

reversible, and inconsistently found. In addition, the end points

showing responses may be at levels of organization below the whole

organism (e.g., neurochemical or electrophysiological end points). The

adversity of such effects can be contentious (e.g., cholinesterase

inhibition), yet it is such effects that are likely to be the focus of

risk assessment decisions. To the extent possible, this document

provides guidance on determining the adversity of neurotoxic effects.

However, the identification of a critical adverse effect often requires

considerable professional judgment and should consider factors such as

the biological plausibility of the effect, the evidence of a dose-

effect continuum, and the likelihood for progression of the effect with

continued exposure.

C. Characterization of the Health-Related Data Base

This section describes a scheme for characterizing the sufficiency

of evidence for neurotoxic effects. This scheme defines two broad

categories: sufficient and insufficient (Table 8). Categorization is

aimed at providing certain criteria for the Agency to use to define the

minimum evidence necessary to define hazards and to conduct dose-

response analyses. It does not address the issues related to

characterization of risk, which requires analysis of potential human

exposures and their relation to potential hazards to estimate the risks

of those hazards from anticipated or estimated exposures.

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

Sufficient Evidence

The sufficient evidence category includes data that collectively

provide enough information to judge whether or not a human

neurotoxic hazard could exist. This category may include both human

and experimental animal evidence.

Sufficient Human Evidence

This category includes agents for which there is sufficient

evidence from

[[Page 52050]]

epidemiologic studies, e.g., case control and cohort studies, to judge

that some neurotoxic effect is associated with exposure. A case

series in conjunction with other supporting evidence may also be

judged ``sufficient evidence.'' Epidemiologic and clinical case

studies should discuss whether the observed effects can be

considered biologically plausible in relation to chemical exposure.

(Historically, much often has been made of the notion of causality

in epidemiologic studies. Causality is a more stringent criterion

than association and has become a topic of scientific and

philosophical debate. See Susser [1986], for example, for a

discussion of inference in epidemiology.)

Sufficient Experimental Animal Evidence/Limited or No Human Data

This category includes agents for which there is sufficient

evidence from experimental animal studies and/or limited human data

to judge whether a potential neurotoxic hazard may exist. Generally,

agents that have been tested according to current test guidelines

would be included in this category. The minimum evidence necessary

to judge that a potential hazard exists would be data demonstrating

an effect in a single appropriate, well-executed study in a single

experimental animal species, whereas the minimum evidence needed to

judge that a potential hazard does not exist would include data from

appropriate, well-executed laboratory animal studies that evaluated

a variety of the potential manifestations of neuroxtoxicity and

showed no effects at doses that were at least minimally toxic.

Information on pharmacokinetics, mechanisms, or known properties of

the chemical class may also strengthen the evidence.

Insufficient Evidence

This category includes agents for which there is less than the

minimum evidence sufficient for identifying whether or not a

neurotoxic hazard exists, such as agents for which there are no data

on neurotoxicity or agents with data bases from studies in animals

or humans that are limited by study design or conduct (e.g.,

inadequate conduct or report of clinical signs). Many general

toxicity studies, for example, are considered insufficient in terms

of the conduct of clinical neurobehavioral observations or the

number of samples taken for histopathology of the nervous system.

Thus, a battery of negative toxicity studies with these shortcomings

would be regarded as providing insufficient evidence of the lack of

a neurotoxic effect of the test material. Further, most screening

studies based on simple observations involving autonomic and motor

function provide insufficient evaluation of many sensory or

cognitive functions. Data, which by itself would likely fall in this

category, would also include information on structure-activity

relationships or data from in vitro tests. While such information

would be insufficient by itself to proceed further in the assessment

it could be used to support the need for additional testing.

Data from all potentially relevant studies, whether indicative

of potential hazard or not, should be included in this

characterization. The primary sources of data are human studies and

case reports, experimental animal studies, other supporting data,

and in vitro and/or structure-activity relationship data. Because a

complex interrelationship exists among study design, statistical

analysis, and biological significance of the data, a great deal of

scientific judgment, based on experience with neurotoxicity data and

with the principles of study design and statistical analysis, is

required to adequately evaluate the data base on neurotoxicity. In

many cases, interaction with scientists in specific disciplines

either within or outside the field of neurotoxicology (e.g.,

epidemiology, statistics) may be appropriate.

The adverse nature of different neurotoxicity end points may be

a complex judgment. In general, most neuropathological and many

neurobehavioral changes are regarded as adverse. However, there are

adverse behavioral effects that may not reflect a direct action on

the nervous system. Neurochemical and electrophysiological changes

may be regarded as adverse as a function of their known or presumed

relation to neuropathological and/or neurobehavioral consequences.

In the absence of supportive information, a professional judgment

must be made regarding the adversity of such outcomes, considering

factors such as the nature, magnitude, and duration of the effects

reported. Thus, correlated measures of neurotoxicity strengthen the

evidence for a hazard. Correlations between functional and

morphological effects, such as the correlation between leg weakness

and paralysis and peripheral nerve damage from exposure to tri-

ortho-cresyl phosphate, are the most common and striking example of

this form of validity. Correlations support a coherent and logical

link between behavioral effects and biochemical mechanisms.

Replication of a finding also strengthens the evidence for a hazard.

Some neurotoxicants cause similar effects across most species. Many

chemicals shown to produce neurotoxicity in laboratory animals have

similar effects in humans. Some neurologic effects may be considered

adverse even if they are small in magnitude, reversible, or the

result of indirect mechanisms.

Because of the inherent difficulty in ``proving any negative,''

it is more difficult to document a finding of no apparent adverse

effect than a finding of an adverse effect. Neurotoxic effects (and

most kinds of toxicity) can be observed at many different levels, so

that only a single end point needs to be found to demonstrate a

hazard, but many end points need to be examined to demonstrate no

effect. For example, to judge that a hazard for neurotoxicity could

exist for a given agent, the minimum evidence sufficient would be

data on a single adverse end point from a well-conducted study. In

contrast, to judge that an agent is unlikely to pose a hazard for

neurotoxicity, the minimum evidence would include data from a host

of end points that revealed no neurotoxic effects. This may include

human data from appropriate studies that could support a conclusion

of no evidence of a neurotoxic effect. With respect to clinical

signs and symptoms, human exposures can reveal far more about the

absence of effects than animal studies, which are confined to the

signs examined.

In some cases, it may be that no individual study is judged

sufficient to establish a hazard, but the total available data may

support such a conclusion. Pharmacokinetic data and structure-

activity considerations, data from other toxicity studies, as well

as other factors may affect the strength of the evidence in these

situations. For example, given that gamma diketones are known to

cause motor system neurotoxicity, a marginal data set on a candidate

gamma diketone, e.g., \1/10\ animals affected, might be more likely

to be judged sufficient than equivalent data from a member of a

chemical class about which nothing is known.

A judgment that the toxicology data base is sufficient to

indicate a potential neurotoxic hazard is not the end of analysis.

The circumstances of expression of hazard are essential to

describing human hazard potential. Thus, reporting should contain

the details of the circumstances under which effects have been

observed, e.g., ``long-term oral exposures of adult rodents to

compound X at levels of roughly 1 mg/kg have been associated with

ataxia and peripheral nerve damage.''

IV. Dose-Response Analysis

This section describes several approaches (including the LOAEL/

NOAEL and BMD) for determining the reference dose or reference

concentration. The NOAEL or BMD/uncertainty factor approach results in

a RfD or RfC, which is an estimate (with uncertainty spanning perhaps

an order of magnitude) of a daily exposure to the human population

(including sensitive subgroups) that is likely to be without an

appreciable risk of deleterious effects during a lifetime.

The dose-response analysis characterization should:

a. Describe how the RfD/RfC was calculated;

b. Discuss the confidence in the estimates;

c. Describe the assumptions or uncertainty factors used; and

d. Discuss the route and level of exposure observed, as compared to

expected human exposures. (Specifically, are the available data from

the same route of exposure as the expected human exposures? How many

orders of magnitude do you need to extrapolate from the observed data

to environmental exposures?)

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

As indicated earlier, the LOAEL and NOAEL are determined for

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

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

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

For example, the NOAEL is derived from a single end point from a single

study (the critical study) and

[[Page 52051]]

ignores both the slope of the dose-response function and baseline

variability in the end point of concern. Because the baseline

variability is not taken into account, the NOAEL from a study using

small group sizes may be higher than the NOAEL from a similar study in

the same species that uses larger group sizes. The NOAEL is also

directly dependent on the dose spacing used in the study. Finally, and

perhaps most importantly, use of the NOAEL does not allow estimates of

risk or extrapolation of risk to lower dose levels.

Because of these and other limitations in the NOAEL approach,

mathematical curve-fitting techniques (Crump, 1984; Gaylor and Slikker,

1990; Glowa, 1991; U.S. EPA, 1995a) are beginning to be used with, or

as an alternative to, the NOAEL in calculating the RfD or RfC. The

Agency is in the process of implementing these newer techniques and

strongly encourages the calculation of BMDs for neurotoxicity and other

health effect end points. These techniques typically apply a

mathematical function that describes the dose-response relationship and

then interpolate to a level of exposure associated with a small

increase in effect over that occurring in the control group or under

baseline conditions. The BMD has been defined as a lower confidence

limit on the effective dose associated with some defined level of

effect, e.g., a 5 percent or 10 percent increase in response (i.e., a

BMD05 or BMD10 for a particular effect). Because the model is

only used to interpolate within the dose range of the study, no

assumptions about the existence (or nonexistence) of a threshold are

needed. Thus, any model that fits the data well is likely to provide a

reasonable estimate of the BMD.

Many neurotoxic end points provide continuous measures of response,

such as response speed, nerve conduction velocity, IQ score, degree of

enzyme inhibition, or the accuracy of task performance. Although it is

possible to impose a dichotomy on a continuous effects distribution and

to classify some level of response as ``affected'' and the remainder as

``unaffected,'' it may be very difficult and inappropriate to establish

such clear distinctions, because such a dichotomy would misrepresent

the true nature of the neurotoxic response. Alternatively, quantitative

models designed to analyze continuous effect variables may be

preferable. Other techniques that allow this approach, with

transformation of the information into estimates of the incidence or

frequency of affected individuals in a population, have been proposed

(Crump, 1984; Gaylor and Slikker, 1990). Categorical regression

analysis has been proposed since it can evaluate different types of

data and derive estimates for short-term exposures (Rees and Hattis,

1994). Decisions about the most appropriate approach require

professional judgment, taking into account the biological nature of the

continuous effect variable and its distribution in the population under

study.

Although dose-response functions in neurotoxicology are generally

linear or monotonic, curvilinear functions, especially U-shaped or

inverted U-shaped curves, have been reported as noted earlier (Section

III B). Dose-response analyses should consider the uncertainty that U-

shaped dose-response functions might contribute to the estimate of the

NOAEL/LOAEL or BMD. Typically, estimates of the NOAEL/LOAEL are taken

from the lowest part of the dose-response curve associated with

impaired function or adverse effect.

B. Determination of the Reference Dose or Reference Concentration

Since the availability of dose-response data in humans is limited,

extrapolation of data from animals to humans usually involves the

application of uncertainty factors to the NOAEL/LOAEL or BMD. The NOAEL

or BMD/uncertainty factor approach results in a RfD or RfC, which is an

estimate (with uncertainty spanning perhaps an order of magnitude) of a

daily exposure to the human population (including sensitive subgroups)

that is likely to be without an appreciable risk of deleterious effects

during a lifetime. The oral RfD and inhalation RfC are applicable to

chronic exposure situations and are based on an evaluation of all the

noncancer health effects, including neurotoxicity data. RfDs and RfCs

in the Integrated Risk Information System (IRIS-2) data base for

several agents are based on neurotoxicity end points and include a few

cases in which the RfD or RfC is calculated using the BMD approach

(e.g., methylmercury, carbon disulfide). The size of the final

uncertainty factor used will vary from agent to agent and will require

the exercise of scientific judgment, taking into account interspecies

differences, the shape of the dose-response curve, and the

neurotoxicity end points observed. Default uncertainty factors are

typically multiples of 10 and are used to compensate for human

variability in sensitivity, the need to extrapolate from animals to

humans, and the need to extrapolate from less than lifetime (e.g.,

subchronic) to lifetime exposures. An additional factor of up to 10 may

be included when only a LOAEL (and not a NOAEL) is available from a

study, or depending on the completeness of the data base, a modifying

factor of up to 10 may be applied, depending on the confidence one has

in the data base. Barnes and Dourson (1988) provide a more complete

description of the calculation, use, and significance of RfDs in

setting exposure limits to toxic agents by the oral route. Jarabek et

al. (1990) provide a more complete description of the calculation, use,

and significance of RfCs in setting exposure limits to toxic agents in

air. Neurotoxicity can result from acute, shorter term exposures, and

it may be appropriate in some cases, e.g., for air pollutants or water

contaminants, to set shorter term exposure limits for neurotoxicity as

well as for other noncancer health effects.

V. Exposure Assessment

Exposure assessment describes the magnitude, duration, frequency,

and routes of exposure to the agent of interest. This information may

come from hypothetical values, models, or actual experimental values,

including ambient environmental sampling results. Guidelines for

exposure assessment have been published separately (U.S. EPA, 1992) and

will, therefore, be discussed only briefly here.

The exposure assessment should include an exposure characterization

that:

a. Provides a statement of the purpose, scope, level of detail, and

approach used in the exposure assessment;

b. Presents the estimates of exposure and dose by pathway and route

for individuals, population segments, and populations in a manner

appropriate for the intended risk characterization;

c. Provides an evaluation of the overall level of confidence in the

estimate of exposure and dose and the conclusions drawn; and

d. Communicates the results of the exposure assessment to the risk

assessor, who can then use the exposure characterization, along with

the characterization of the other risk assessment elements, to develop

a risk characterization.

A number of considerations are relevant to exposure assessment for

neurotoxicants. An appropriate evaluation of exposure should consider

the potential for exposure via ingestion, inhalation, and dermal

penetration from relevant sources of exposures, including multiple

avenues of intake from the same source. On-going Agency activities that

support neurotoxicity exposure

[[Page 52052]]

assessment include characterizing cumulative risk and revising the

Guidelines for the Health Risk Assessment of Chemical Mixtures.

In addition, neurotoxic effects may result from short-term (acute),

high-concentration exposures as well as from longer term (subchronic),

lower level exposures. Neurotoxic effects may occur after a period of

time following initial exposure or be obfuscated by repair mechanisms

or apparent tolerance. The type and severity of effect may depend

significantly on the pattern of exposure rather than on the average

dose over a long period of time. For this reason, exposure assessments

for neurotoxicants may be much more complicated than those for long-

latency effects such as carcinogenicity. It is rare for sufficient data

to be available to construct such patterns of exposure or dose, and

professional judgment may be necessary to evaluate exposure to

neurotoxic agents.

VI. Risk Characterization

A. Overview

Risk characterization, the culmination of the risk assessment

process, consists of an integrative analysis and a risk

characterization summary. The integrative analysis (a) involves

integration of the toxicity information from the hazard

characterization and dose-response analysis with the human exposure

estimates, (b) provides an evaluation of the overall quality of the

assessment and the degree of confidence in the estimates of risk and

conclusions drawn, and (c) describes risk in terms of the nature and

extent of harm. The risk characterization summary communicates the

results of the risk assessment to the risk manager.

This summary should include but is not limited to a discussion of

the following elements:

a. Quality of and confidence in the available data;

b. Uncertainty analysis;

c. Justification of defaults or assumptions;

d. Related research recommendations;

e. Contentious issues and extent of scientific consensus;

f. Effect of reasonable alternative assumptions on conclusions and

estimates;

g. Highlight reasonable plausible ranges;

h. Reasonable alternative models; and

i. Perspective through analogy.

The risk manager can then use the risk assessment, along with other

risk management elements, to make public health decisions.

An effective risk characterization must fully, openly, and clearly

characterize risks and disclose the scientific analyses, uncertainties,

assumptions, and science policies that underlie decisions throughout

the risk assessment and risk management processes. The risk

characterization must feature values such as transparency in the

decision-making process; clarity in communicating with each other and

the public regarding environmental risk and the uncertainties

associated with assessments of environmental risk; and consistency

across program offices in core assumptions and science policies, which

are well grounded in science and reasonable.

The following sections describe these four aspects of the risk

characterization in more detail.

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

Exposure Assessment

In developing the hazard characterization, dose-response analysis

and exposure portions of the risk assessment, the assessor must take

into account many judgments concerning human relevance of the toxicity

data, including the appropriateness of the various animal models for

which data are available and the route, timing, and duration of

exposure relative to expected human exposure. These judgments should be

summarized at each stage of the risk assessment process (e.g., the

biological relevance of anatomical variations may be established in the

hazard characterization process, or the influence of species

differences in metabolic patterns in the dose-response analysis). In

integrating the information from the assessment, the risk assessor must

determine if some of these judgments have implications for other

portions of the assessment and whether the various components of the

assessment are compatible.

The risk characterization should not only examine the judgments but

also explain the constraints of available data and the state of

knowledge about the phenomena studied in making them, including (1) the

qualitative conclusions about the likelihood that the chemical may pose

a specific hazard to human health, the nature of the observed effects,

under what conditions (route, dose levels, time, and duration) of

exposure these effects occur, and whether the health-related data are

sufficient to use in a risk assessment; (2) a discussion of the dose-

response characteristics of the critical effects(s), data such as the

shapes and slopes of the dose-response curves for the various end

points, the rationale behind the determination of the NOAEL and LOAEL

and calculation of the benchmark dose, and the assumptions underlying

the estimation of the RfD or RfC; and (3) the estimates of the

magnitude of human exposure; the route, duration, and pattern of the

exposure; relevant pharmacokinetics; and the number and characteristics

of the population(s) exposed.

If data to be used in a risk characterization are from a route of

exposure other than the expected human exposure, then pharmacokinetic

data should be used, if available, to make extrapolations across routes

of exposure. If such data are not available, the Agency makes certain

assumptions concerning the amount of absorption likely or the

applicability of the data from one route to another (U.S. EPA, 1992).

The level of confidence in the hazard characterization should be

stated to the extent possible, including the appropriate category

regarding sufficiency of the health-related data. A comprehensive risk

assessment ideally includes information on a variety of end points that

provide insight into the full spectrum of potential neurotoxicological

responses. A profile that integrates both human and test species data

and incorporates a broad range of potential adverse neurotoxic effects

provides more confidence in a risk assessment for a given agent.

The ability to describe the nature of the potential human exposure

is important to predict when certain outcomes can be anticipated and

the likelihood of permanence or reversibility of the effect. An

important part of this effort is a description of the nature of the

exposed population and the potential for sensitive, highly susceptible,

or highly exposed populations. For example, the consequences of

exposure to the developing individual versus the adult can differ

markedly and can influence whether the effects are transient or

permanent. Other considerations relative to human exposures might

include the likelihood of exposures to other agents, concurrent

disease, and nutritional status.

The presentation of the integrated results of the assessment should

draw from and highlight key points of the individual characterizations

of component analyses performed under these Guidelines. The overall

risk characterization represents the integration of these component

characterizations. If relevant risk assessments on the agent or an

analogous agent have been done by EPA or other Federal agencies, these

should

[[Page 52053]]

be described and the similarities and differences discussed.

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

The risk characterization should summarize the kinds of data

brought together in the analysis and the reasoning on which the

assessment is based. The description should convey the major strengths

and weaknesses of the assessment that arise from availability of data

and the current limits of our understanding of the mechanisms of

toxicity.

Health risk is a function of the hazard characterization, dose-

response analysis, and exposure assessment. Confidence in the results

of a risk assessment is, thus, a function of confidence in the results

of the analysis of these elements. Each of these elements should have

its own characterization as a part of the assessment. Within each

characterization, the important uncertainties of the analysis and

interpretation of data should be explained, and the risk manager should

be given a clear picture of consensus or lack of consensus that exists

about significant aspects of the assessment. Whenever more than one

view is supported by the data and choosing between them is difficult,

all views should be presented. If one has been selected over the

others, the rationale should be given; if not, then all should be

presented as plausible alternative results.

D. Descriptors of Neurotoxicity Risk

There are a number of ways to describe risks. Several ways that are

relevant to describing risks for neurotoxicity are as follows:

1. Estimation of the Number of Individuals

The RfD or RfC is taken to be a chronic exposure level at or below

which no significant risk occurs. Therefore, presentation of the

population in terms of those at or below the RfD or RfC (``not at

risk'') and above the RfD or RfC (``may be at risk'') may be useful

information for risk managers. This method is particularly useful to a

risk manager considering possible actions to ameliorate risk for a

population. If the number of persons in the at-risk category can be

estimated, then the number of persons removed from the at-risk category

after a contemplated action is taken can be used as an indication of

the efficacy of the action.

2. Presentation of Specific Scenarios

Presenting specific scenarios in the form of ``what if?'' questions

is particularly useful to give perspective to the risk manager,

especially where criteria, tolerance limits, or media quality limits

are being set. The question being asked in these cases is, at this

proposed limit, what would be the resulting risk for neurotoxicity

above the RfD or RfC?

3. Risk Characterization for Highly Exposed Individuals

This measure is one example of the just-discussed descriptor. This

measure describes the magnitude of concern at the upper end of the

exposure distribution. This allows risk managers to evaluate whether

certain individuals are at disproportionately high or unacceptably high

risk.

The objective of looking at the upper end of the exposure

distribution is to derive a realistic estimate of a relatively highly

exposed individual or individuals. This measure could be addressed by

identifying a specified upper percentile of exposure in the population

and/or by estimating the exposure of the highest exposed individual(s).

Whenever possible, it is important to express the number of individuals

who comprise the selected highly exposed group and discuss the

potential for exposure at still higher levels.

If population data are absent, it will often be possible to

describe a scenario representing high-end exposures using upper

percentile or judgment-based values for exposure variables. In these

instances caution should be used not to compound a substantial number

of high-end values for variables if a ``reasonable'' exposure estimate

is to be achieved.

4. Risk Characterization for Highly Sensitive or Susceptible

Individuals

This measure identifies populations sensitive or susceptible to the

effect of concern. Sensitive or susceptible individuals are those

within the exposed population at increased risk of expressing the toxic

effect. All stages of nervous system maturation might be considered

highly sensitive or susceptible, but certain subpopulations can

sometimes be identified because of critical periods for exposure, for

example, pregnant or lactating women, infants, children.

In general, not enough is understood about the mechanisms of

toxicity to identify sensitive subgroups for all agents, although

factors such as nutrition, personal habits (e.g., smoking, alcohol

consumption, illicit drug abuse), or preexisting disease (e.g.,

diabetes, sexually transmitted diseases) may predispose some

individuals to be more sensitive to the neurotoxic effects of various

agents.

5. Other Risk Descriptors

In risk characterization, dose-response information and the human

exposure estimates may be combined either by comparing the RfD or RfC

and the human exposure estimate or by calculating the margin of

exposure (MOE). The MOE is the ratio of the NOAEL from the most

appropriate or sensitive species to the estimated human exposure level.

If a NOAEL is not available, a LOAEL may be used in calculating the

MOE. Alternatively, a benchmark dose may be compared with the estimated

human exposure level to obtain the MOE. Considerations for the

evaluation of the MOE are similar to those for the uncertainty factor

applied to the LOAEL/NOAEL or the benchmark dose. The MOE is presented

along with a discussion of the adequacy of the data base, including the

nature and quality of the hazard and exposure data, the number of

species affected, and the dose-response information.

The RfD or R

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