26926 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

ENVIRONMENTAL PROTECTION

AGENCY

[FRL–6011–3]

RIN 2080–AA08

Guidelines for Neurotoxicity Risk

Assessment

AGENCY: Environmental Protection

Agency.

ACTION: Notice of availability of final

Guidelines for Neurotoxicity Risk

Assessment.

SUMMARY: The U.S. Environmental

Protection Agency (EPA) is today

publishing in final form a document

entitled Guidelines for Neurotoxicity

Risk Assessment (hereafter

‘‘Guidelines’’). These Guidelines were

developed as part of an interoffice

guidelines development program by a

Technical Panel of the Risk Assessment

Forum. The Panel was composed of

scientists from throughout the Agency,

and selected drafts were peer-reviewed

internally and by experts from

universities, environmental groups,

industry, and other governmental

agencies. The Guidelines are based, in

part, on recommendations derived from

various scientific meetings and

workshops on neurotoxicology, from

public comments, and from

recommendations of the Science

Advisory Board. An earlier draft

underwent external peer review in a

workshop held on June 2–3, 1992, and

received internal review by the Risk

Assessment Forum. The Risk

Assessment Subcommittee of the

Committee on the Environment and

Natural Resources of Office of Science

and Technology Policy reviewed the

proposed Guidelines during a meeting

held on August 15, 1995. The

Guidelines were revised and proposed

for public comment on October 4, 1995

(60 FR 52032–52056). The proposed

Guidelines were reviewed by the

Science Advisory Board on July 18,

1996. EPA appreciates the efforts of all

participants in the process, and has

tried to address their recommendations

in these Guidelines.

This notice describes the scientific

basis for concern about exposure to

agents that cause neurotoxicity, outlines

the general process for assessing

potential risk to humans because of

environmental contaminants, and

addresses Science Advisory Board and

public comments on the 1995 Proposed

Guidelines for Neurotoxicity Risk

Assessment (60 FR:52032–52056). These

Guidelines are intended to guide

Agency evaluation of agents that are

suspected to cause neurotoxicity, in line

with the policies and procedures

established in the statutes administered

by the Agency.

DATES: The Guidelines will be effective

on April 30, 1998.

ADDRESSES: The Guidelines will be

made available in several ways:

(1) The electronic version will be

accessible from EPA’s National Center

for Environmental Assessment home

page on the Internet at http://

www.epa.gov/ncea.

(2) 31⁄2′′ high-density computer

diskettes in WordPerfect format will be

available from ORD Publications,

Technology Transfer and Support

Division, National Risk Management

Research Laboratory, Cincinnati, OH;

Tel: 513–569–7562; Fax: 513–569–7566.

Please provide the EPA No.: EPA/630/

R–95/001Fa when ordering.

(3) This notice contains the full

document. Copies of the Guidelines will

be available for inspection at EPA

headquarters and regional libraries,

through the U.S. Government

Depository Library program, and for

purchase from the National Technical

Information Service (NTIS), Springfield,

VA; telephone: 703–487–4650, fax: 703–

321–8547. Please provide the NTIS PB

No. (PB98–117831) when ordering.

FOR FURTHER INFORMATION CONTACT: Dr.

Hugh A. Tilson, Neurotoxicology

Division, National Health and

Environmental Effects Research

Laboratory, U.S. Environmental

Protection Agency, Research Triangle

Park, NC 27711, Tel: 919–541–2671;

Fax: 919–541–4849; E-mail:

tilson.hugh@epamail.epa.gov.

SUPPLEMENTARY INFORMATION: In its 1983

book Risk Assessment in the Federal

Government: Managing the Process, the

National Academy of Sciences

recommended that Federal regulatory

agencies establish ‘‘inference

guidelines’’ to promote consistency and

technical quality in risk assessment, and

to ensure that the risk assessment

process is maintained as a scientific

effort separate from risk management. A

task force within EPA accepted that

recommendation and requested that

Agency scientists begin to develop such

guidelines. In 1984, EPA scientists

began work on risk assessment

guidelines for carcinogenicity,

mutagenicity, suspect developmental

toxicants, chemical mixtures, and

exposure assessment. Following

extensive scientific and public review,

these first five guidelines were issued

on September 24, 1986 (51 FR 33992–

34054). Since 1986, additional risk

assessment guidelines have been

proposed, revised, reproposed, and

finalized. These guidelines continue the

process initiated in 1984. As with other

EPA guidelines (e.g., developmental

toxicity, 56 FR 63798–63826; exposure

assessment, 57 FR 22888–22938; and

carcinogenicity, 61 FR 17960–18011),

EPA will revisit these guidelines as

experience and scientific consensus

evolve.

These Guidelines set forth principles

and procedures to guide EPA scientists

in the conduct of Agency risk

assessments and to inform Agency

decision makers and the public about

these procedures. Policies in this

document are intended as internal

guidance for EPA. Risk assessors and

risk managers at EPA are the primary

audience, although these Guidelines

may be useful to others outside the

Agency. In particular, the Guidelines

emphasize that risk assessments will be

conducted on a case-by-case basis,

giving full consideration to all relevant

scientific information. This approach

means that Agency experts study

scientific information on each chemical

under review and use the most

scientifically appropriate interpretation

to assess risk. The Guidelines also stress

that this information will be fully

presented in Agency risk assessment

documents, and that Agency scientists

will identify the strengths and

weaknesses of each assessment by

describing uncertainties, assumptions,

and limitations, as well as the scientific

basis and rationale for each assessment.

The Guidelines are formulated in part to

bridge gaps in risk assessment

methodology and data. By identifying

these gaps and the importance of the

missing information to the risk

assessment process, EPA wishes to

encourage research and analysis that

will lead to new risk assessment

methods and data.

Dated: April 30, 1998.

Carol M. Browner,

Administrator.

Contents

Part A: Guidelines for Neurotoxicity Risk

Assessment

List of Tables

1. Introduction

1.1. Organization of These Guidelines

1.2. The Role of Environmental Agents in

Neurotoxicity

1.3. Neurotoxicity Risk Assessment

1.4. Assumptions

2. Definitions and Critical Concepts

3. Hazard Characterization

3.1. Neurotoxicological Studies: Endpoints

and Their Interpretation

3.1.1. Human Studies

3.1.1.1. Clinical Evaluations

3.1.1.2. Case Reports

3.1.1.3. Epidemiologic Studies

3.1.1.4. Human Laboratory Exposure

Studies
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26927

3.1.2. Animal Studies

3.1.2.1. Structural Endpoints of

Neurotoxicity

3.1.2.2. Neurophysiological Endpoints of

Neurotoxicity

3.1.2.3. Neurochemical Endpoints of

Neurotoxicity

3.1.2.4. Behavioral Endpoints of

Neurotoxicity

3.1.3. Other Considerations

3.1.3.1. Pharmacokinetics

3.1.3.2. Comparisons of Molecular

Structure

3.1.3.3. Statistical Considerations

3.1.3.4. In Vitro Data in Neurotoxicology

3.1.3.5. Neuroendocrine Effects

3.2. Dose-Response Evaluation

3.3. Characterization of the Health-Related

Database

4. Quantitative Dose-Response Analysis

4.1. LOAEL/NOAEL and BMD

Determination

4.2. Determination of the Reference Dose or

Reference Concentration

5. Exposure Assessment

6. Risk Characterization

6.1. Overview

6.2. Integration of Hazard Characterization,

Dose-Response Analysis, and Exposure

Assessment

6.3. Quality of the Database and Degree of

Confidence in the Assessment

6.4. Descriptors of Neurotoxicity Risk

6.4.1. Estimation of the Number of

Individuals

6.4.2. Presentation of Specific Scenarios

6.4.3. Risk Characterization for Highly

Exposed Individuals

6.4.4. Risk Characterization for Highly

Sensitive or Susceptible Individuals

6.5.5. Other Risk Descriptors

6.5. Communicating Results

6.6. Summary and Research Needs

References

Part B: Response to Science Advisory Board

and Public Comments

1. Introduction

2. Response to Science Advisory Board

Comments

3. Response to Public Comments

List of Tables

Table 1. Examples of possible indicators of a

neurotoxic effect

Table 2. Neurotoxicants and disorders with

specific neurological targets

Table 3. Examples of neurophysiological

measures of neurotoxicity

Table 4. Examples of neurotoxicants with

known neurochemical mechanisms

Table 5. Examples of measures in a

representative functional observational

battery

Table 6. Examples of specialized behavioral

tests to measure neurotoxicity

Table 7. Examples of compounds or

treatments producing developmental

neurotoxicity

Table 8. Characterization of the healthrelated database

Part A: Guidelines for Neurotoxicity

Risk Assessment

1. Introduction

These Guidelines describe the

principles, concepts, and procedures

that the U.S. Environmental Protection

Agency (EPA) will follow in evaluating

data on potential neurotoxicity

associated with exposure to

environmental toxicants. The Agency’s

authority to regulate substances that

have the potential to interfere with

human health is derived from a number

of statutes that are implemented through

multiple offices within EPA. The

procedures outlined here are intended

to help develop a sound scientific basis

for neurotoxicity risk assessment,

promote consistency in the Agency’s

assessment of toxic effects on the

nervous system, and inform others of

the approaches used by the Agency in

those assessments. This document is not

a regulation and is not intended for EPA

regulations. The Guidelines set forth

current scientific thinking and

approaches for conducting and

evaluating neurotoxic risk assessments.

They are not intended, nor can they be

relied upon, to create any rights

enforceable by any party in litigation

with the United States.

1.1. Organization of These Guidelines

This introduction (section 1)

summarizes the purpose of these

Guidelines within the overall

framework of risk assessment at EPA. It

also outlines the organization of the

guidance and describes several default

assumptions to be used in the risk

assessment process, as discussed in the

recent National Research Council report

‘‘Science and Judgment in Risk

Assessment’’ (NRC, 1994).

Section 2 sets forth definitions of

particular terms widely used in the field

of neurotoxicology. These include

‘‘neurotoxicity’’ and ‘‘behavioral

alterations.’’ Also included in this

section are discussions concerning

reversible and irreversible effects and

direct versus indirect effects.

Risk assessment is the process by

which scientific judgments are made

concerning the potential for toxicity in

humans. The National Research Council

(NRC, 1983) has defined risk assessment

as including some or all of the following

components (paradigm): hazard

identification, dose-response

assessment, exposure assessment, and

risk characterization. In its 1994 report

‘‘Science and Judgment in Risk

Assessment’’ the NRC extended its view

of the paradigm to include

characterization of each component

(NRC, 1994). In addition, it noted the

importance of an approach that is less

fragmented and more holistic, less

linear and more interactive, and that

deals with recurring conceptual issues

that cut across all stages of risk

assessment. These Guidelines describe a

more interactive approach by organizing

the process around the qualitative

evaluation of the toxicity data (hazard

characterization), the quantitative doseresponse analysis, the exposure

assessment, and the risk

characterization. In these Guidelines,

hazard characterization includes

deciding whether a chemical has an

effect by means of qualitative

consideration of dose-response

relationships, route, and duration of

exposure. Determining a hazard often

depends on whether a dose-response

relationship is present (Kimmel et al.,

1990). This approach combines the

information important in comparing the

toxicity of a chemical with potential

human exposure scenarios (section 3).

In addition, it avoids the potential for

labeling chemicals as ‘‘neurotoxicants’’

on a purely qualitative basis. This

organization of the risk assessment

process is similar to that discussed in

the Guidelines for Developmental

Toxicity Risk Assessment (56 FR

63798), the main difference being that

the quantitative dose-response analysis

is discussed under a separate section in

these Guidelines.

Hazard characterization involves

examining all available experimental

animal and human data and the

associated doses, routes, timing, and

durations of exposure to determine

qualitatively if an agent causes

neurotoxicity in that species and under

what conditions. From the hazard

characterization and criteria provided in

these Guidelines, the health-related

database can be characterized as

sufficient or insufficient for use in risk

assessment (section 3.3). Combining

hazard identification and some aspects

of dose-response evaluation into hazard

characterization does not preclude the

evaluation and use of data for other

purposes when quantitative information

for setting reference doses (RfDs) and

reference concentrations (RfCs) is not

available.

The next step in the dose-response

analysis (section 4) is the quantitative

analysis, which includes determining

the no-observed-adverse-effect-level

(NOAEL) and/or the lowest-observedadverse-effect-level (LOAEL) for each

study and type of effect. Because of the

limitations associated with the use of

the NOAEL, the Agency is beginning to

use an additional approach, the

benchmark dose approach (BMD)

(Crump, 1984; U.S. EPA, 1995a), for
26928 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

more quantitative dose-response

evaluation when sufficient data are

available. The benchmark dose

approach takes into account the

variability in the data and the slope of

the dose-response curve, and provides a

more consistent basis for calculation of

the RfD or RfC. If data are considered

sufficient for risk assessment, and if

neurotoxicity is the effect occurring at

the lowest dose level (i.e., the critical

effect), an oral or dermal RfD or an

inhalation RfC, based on neurotoxic

effects, is then derived. This RfD or RfC

is derived using the NOAEL or

benchmark dose divided by uncertainty

factors to account for interspecies

differences in response, intraspecies

variability, and other factors of study

design or the database. A statement of

the potential for human risk and the

consequences of exposure can come

only from integrating the hazard

characterization and dose-response

analysis with the human exposure

estimates in the final risk

characterization.

The section on exposure assessment

(section 5) identifies human populations

exposed or potentially exposed to an

agent, describes their composition and

size, and presents the types,

magnitudes, frequencies, and durations

of exposure to the agent. The exposure

assessment provides an estimate of

human exposure levels for particular

populations from all potential sources.

In risk characterization (section 6), the

hazard characterization, dose-response

analysis, and exposure assessment for

given populations are combined to

estimate some measure of the risk for

neurotoxicity. As part of risk

characterization, a summary of the

strengths and weaknesses of each

component of the risk assessment is

given, along with major assumptions,

scientific judgments and, to the extent

possible, qualitative and quantitative

estimates of the uncertainties. This

characterization of the health-related

database is always presented in

conjunction with information on the

dose, route, duration, and timing of

exposure as well as the dose-response

analysis including the RfD or RfC. If

human exposure estimates are available,

the exposure basis used for the risk

assessment is clearly described, e.g.,

highly exposed individuals or highly

sensitive or susceptible individuals. The

NOAEL may be compared to the various

estimates of human exposure to

calculate the margin(s) of exposure

(MOE). The considerations for judging

the acceptability of the MOE are similar

to those for determining the appropriate

size of the uncertainty factor for

calculating the RfD or RfC.

The Agency recently issued a policy

statement and associated guidance for

risk characterization (U.S. EPA, 1995b,

1995c), which is currently being

implemented throughout EPA. This

statement is designed to ensure that

critical information from each stage of a

risk assessment is used in forming

conclusions about risk and that this

information is communicated from risk

assessors to risk managers (policy

makers), from middle to upper

management, and from the Agency to

the public. Additionally, the policy

provides a basis for greater clarity,

transparency, reasonableness, and

consistency in risk assessments across

Agency programs.

Final neurotoxicity risk assessment

guidelines may reflect additional

changes in risk characterization

practices resulting from implementation

activities. Risk assessment is just one

component of the regulatory process

and defines the potential adverse health

consequences of exposure to a toxic

agent. The other component, risk

management, combines risk assessment

with statutory directives regarding

socioeconomic, technical, political, and

other considerations in order to decide

whether to control future exposure to

the suspected toxic agent and, if so, the

nature and level of control. One major

objective of these Guidelines is to help

the risk assessor determine whether the

experimental animal or human data

indicate the potential for a neurotoxic

effect. Such information can then be

used to categorize evidence that will

identify and characterize neurotoxic

hazards, as described in section 3.3,

Characterization of the Health-Related

Database, and Table 8 of these

Guidelines. Risk management is not

dealt with directly in these Guidelines

because the basis for decision making

goes beyond scientific considerations

alone, but the use of scientific

information in this process is discussed.

For example, the acceptability of the

MOE is a risk management decision, but

the scientific bases for establishing this

value are discussed here.

1.2. The Role of Environmental Agents

in Neurotoxicity

Chemicals are an integral part of life,

with the capacity to improve as well as

endanger health. The general population

is exposed to chemicals in air, water,

foods, cosmetics, household products,

and drugs used therapeutically or

illicitly. During daily life, a person

experiences a multitude of exposures to

potentially neuroactive substances,

singly and in combination, both

synthetic and natural. Levels of

exposure vary and may or may not pose

a hazard, depending on dose, route, and

duration of exposure.

A link between human exposure to

some chemical substances and

neurotoxicity has been firmly

established (Anger, 1986; OTA, 1990).

Because many natural and synthetic

chemicals are present in today’s

environment, there is growing scientific

and regulatory interest in the potential

for risks to humans from exposure to

neurotoxic agents. If sufficient exposure

occurs, the effects resulting from such

exposures can have a significant adverse

impact on human health. It is not

known how many chemicals may be

neurotoxic in humans (Reiter, 1987).

EPA’s TSCA inventory of chemical

substances manufactured, imported, or

processed in the United States includes

more than 65,000 substances and is

increasing yearly. An overwhelming

majority of the materials in commercial

use have not been tested for neurotoxic

potential (NRC, 1984).

Estimates of the number of chemicals

with neurotoxic properties have been

made for subsets of substances. For

instance, a large percentage of the more

than 500 registered active pesticide

ingredients affect the nervous system of

the target species to varying degrees. Of

588 chemicals listed by the American

Conference of Governmental Industrial

Hygienists, 167 affected the nervous

system or behavior at some exposure

level (Anger, 1984). Anger (1990)

estimated that of the approximately 200

chemicals to which 1 million or more

American workers are exposed, more

than one-third may have adverse effects

on the nervous system if sufficient

exposure occurs. Anger (1984) also

recognized neurotoxic effects as one of

the 10 leading workplace disorders. A

number of therapeutic substances,

including some anticancer and antiviral

agents and abused drugs, can cause

adverse or neurotoxicological side

effects at therapeutic levels (OTA,

1990). The number of chemicals with

neurotoxic potential has been estimated

to range from 3% to 28% of all

chemicals (OTA, 1990). Thus,

estimating the risks of exposure to

chemicals with neurotoxic potential is

of concern with regard to their overall

impact on human health.

1.3. Neurotoxicity Risk Assessment

In addition to its primary role in

psychological functions, the nervous

system controls most, if not all, other

bodily processes. It is sensitive to

perturbation from various sources and

has limited ability to regenerate. There

is evidence that even small anatomical,

biochemical, or physiological insults to

the nervous system may result in
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26929

adverse effects on human health.

Therefore, there is a need for consistent

guidance on how to evaluate data on

neurotoxic substances and assess their

potential to cause transient or persistent

and direct or indirect effects on human

health.

These Guidelines develop principles

and concepts in several areas. They

outline the scientific basis for evaluating

effects due to exposure to

neurotoxicants and discuss principles

and methods for evaluating data from

human and animal studies on behavior,

neurochemistry, neurophysiology, and

neuropathology. They also discuss

adverse effects on neurological

development and function in infants

and children following prenatal and

perinatal exposure to chemical agents.

They outline the methods for

calculating reference doses or reference

concentrations when neurotoxicity is

the critical effect, discuss the

availability of alternative mathematical

approaches to dose-response analyses,

characterize the health-related database

for neurotoxicity risk assessment, and

discuss the integration of exposure

information with results of the doseresponse assessment to characterize

risks. These Guidelines do not advocate

developing reference doses specific for

neurotoxicity, but rather support the use

of neurotoxicity as one possible

endpoint to develop reference doses.

EPA offices have published guidelines

for neurotoxicity testing in animals

(U.S. EPA, 1986, 1987, 1988a, 1991a).

The testing guidelines address the

development of new data for use in risk

assessment.

These neurotoxicity risk assessment

guidelines provide the Agency’s first

comprehensive guidance on the use and

interpretation of neurotoxicity data, and

are part of the Agency’s risk assessment

guidelines development process, which

was initiated in 1984. As part of its

neurotoxicity guidelines development

program, EPA has sponsored or

participated in several conferences on

relevant issues (Tilson, 1990); these and

other sources (see references) provide

the scientific basis for these Guidelines.

This guidance is intended for use by

Agency risk assessors and is separate

and distinct from the recently published

document on principles of neurotoxicity

risk assessment (U.S. EPA, 1994). The

document on principles was prepared

under the auspices of the Subcommittee

on Risk Assessment of the Federal

Coordinating Council for Science,

Engineering, and Technology and was

not intended to provide specific

directives for how neurotoxicity risk

assessment should be performed. It is

expected that, like other EPA risk

assessment guidelines for noncancer

endpoints (U.S. EPA, 1991b), this

document will encourage research and

analysis leading to new risk assessment

methods and data, which in turn would

be used to revise and improve the

Guidelines and better guide Agency risk

assessors.

1.4. Assumptions

There are a number of unknowns in

the extrapolation of data from animal

studies to humans. Therefore, a number

of default assumptions are made that are

generally applied in the absence of data

on the relevance of effects to potential

human risk. Default assumptions should

not be applied indiscriminately. First,

all available mechanistic and

pharmacokinetic data should be

considered. If these data indicate that an

alternative assumption is appropriate or

if they obviate the need for applying an

assumption, such information should be

used in risk assessment. For example,

research in rats may determine that the

neurotoxicity of a chemical is caused by

a metabolite. If subsequent research

finds that the chemical is metabolized to

a lesser degree or not at all in humans,

then this information should be used in

formulating the default assumptions.

The following default assumptions form

the basis of the approaches taken in

these Guidelines:

(1) It is assumed that an agent that

produces detectable adverse neurotoxic

effects in experimental animal studies

will pose a potential hazard to humans.

This assumption is based on the

comparisons of data for known human

neurotoxicants (Anger, 1990; Kimmel et

al., 1990; Spencer and Schaumburg,

1980), which indicate that experimental

animal data are frequently predictive of

a neurotoxic effect in humans.

(2) It is assumed that behavioral,

neurophysiological, neurochemical, and

neuroanatomical manifestations are of

concern. In the past, the tendency has

been to consider only neuropathological

changes as endpoints of concern. Based

on data on agents that are known human

neurotoxicants (Anger, 1990; Kimmel et

al., 1990; Spencer and Schaumberg,

1980), there is usually at least one

experimental species that mimics the

types of effects seen in humans, but in

other species tested, the neurotoxic

effect may be different or absent. For

example, certain organophosphate

compounds produce a delayed-onset

neuropathy in hens similar to that seen

in humans, whereas rodents are

characteristically insensitive to these

compounds. A biologically significant

increase in any of the manifestations is

considered indicative of an agent’s

potential for disrupting the structure or

function of the human nervous system.

(3) It is assumed that the neurotoxic

effects seen in animal studies may not

always be the same as those produced

in humans. Therefore, it may be difficult

to determine the most appropriate

species in terms of predicting specific

effects in humans. The fact that every

species may not react in the same way

is probably due to species-specific

differences in maturation of the nervous

system, differences in timing of

exposure, metabolism, or mechanisms

of action.

(4) It is also assumed that, in the

absence of data to the contrary, the most

sensitive species is used to estimate

human risk. This is based on the

assumption that humans are as sensitive

as the most sensitive animal species

tested. This provides a conservative

estimate of sensitivity for added

protection to the public. As with other

noncancer endpoints, it is assumed that

there is a nonlinear dose-response

relationship for neurotoxicants.

Although there may be a threshold for

neurotoxic effects, these are often

difficult to determine empirically.

Therefore, a nonlinear relationship is

assumed to exist for neurotoxicants.

These assumptions are ‘‘plausibly

conservative’’ (NRC, 1994) in that they

are protective of public health and are

also well founded in scientific

knowledge about the effects of concern.

2. Definitions and Critical Concepts

This section defines the key terms and

concepts that EPA will use in the

identification and evaluation of

neurotoxicity. The various health effects

that fall within the broad classification

of neurotoxicity are described and

examples are provided. Adverse effects

include alterations from baseline or

normal conditions that diminish an

organism’s ability to survive, reproduce,

or adapt to the environment.

Neurotoxicity is an adverse change in

the structure or function of the central

and/or peripheral nervous system

following exposure to a chemical,

physical, or biological agent (Tilson,

1990). Functional neurotoxic effects

include adverse changes in somatic/

autonomic, sensory, motor, and/or

cognitive function. Structural

neurotoxic effects are defined as

neuroanatomical changes occurring at

any level of nervous system

organization; functional changes are

defined as neurochemical,

neurophysiological, or behavioral

effects. Chemicals can also be

categorized into four classes: Those that

act on the central nervous system, the

peripheral nerve fibers, the peripheral
26930 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

nerve endings, or muscles or other

tissues (Albert, 1973). Changes in

function can result from toxicity to

other specific organ systems, and these

indirect changes may be considered

adverse. For example, exposure to a

high dose of a chemical may cause

damage to the liver, resulting in general

sickness and a decrease in a functional

endpoint such as motor activity. In this

case, the change in motor activity could

be considered as adverse, but not

necessarily neurotoxic. A discussion

concerning problems associated with

risk assessment of high doses of

chemicals in the context of drinking

water and health was published by the

National Research Council (1986).

The risk assessor should also know

that there are different levels of concern

based on the magnitude of effect,

duration of exposure, and reversibility

of some neurotoxic effects. Neurotoxic

effects may be irreversible (the organism

cannot return to the state prior to

exposure, resulting in a permanent

change) or reversible (the organism can

return to the pre-exposure condition).

Clear or demonstrable irreversible

change in either the structure or

function of the nervous system causes

greater concern than do reversible

changes. If neurotoxic effects are

observed at some time during the

lifespan of the organism but are slowly

reversible, the concern is also high.

There is lesser concern for effects that

are rapidly reversible or ‘‘transient,’’ i.e.,

measured in minutes, hours, or days,

and that appear to be associated with

the pharmacokinetics of the causal agent

and its presence in the body. Reversible

changes that occur in the occupational

setting or environment, however, may

be of high concern if, for example,

exposure to a short-acting solvent

interferes with operation of heavy

equipment in an industrial plant. The

context of the exposure should be

considered in evaluating reversible

effects. Setting of exposure limits is not

always associated with the

determination of a reference dose,

which is based on chronic dosing. Data

from acute or subacute dosing can be

used for health advisories or in studies

involving developmental exposures.

It should also be noted that the

nervous system is known for its reserve

capacity (Tilson and Mitchell, 1983).

That is, repeated insult to the nervous

system could lead to an adaptation.

There are, however, limits to this

capacity, and when these limits are

exceeded, further exposure could lead

to frank manifestations of neurotoxicity

at the structural or functional level. The

risk assessor should be aware that once

damaged, neurons, particularly in the

central nervous system, have a limited

capacity for regeneration. Reversibility

of effects resulting from cell death or

from the destruction of cell processes

may represent an activation of repair

capacity, decreasing future potential

adaptability. Therefore, even reversible

neurotoxic changes should be of

concern. Evidence of progressive effects

(those that continue to worsen even

after the causal agent has been

removed), delayed-onset effects (those

that occur at a time distant from the last

contact with the causal agent), residual

effects (those that persist beyond a

recovery period), or latent effects (those

that become evident only after an

environmental challenge or aging) have

a high level of concern.

Environmental challenges can include

stress, increased physical or cognitive

workload, pharmacological

manipulations, and nutritional

deficiency or excess. Evidence for

reversibility may depend on the region

of the nervous system affected, the

chemical involved, and organismic

factors such as the age of the exposed

population. Some regions of the nervous

system, such as peripheral nerves, have

a high capacity for regeneration, while

regions in the brain such as the

hippocampus are known for their ability

to compensate or adapt to neurotoxic

insult. For example, compensation is

likely to be seen with solvents (e.g., nhexane) that produce peripheral

neuropathy because of the repair

capacity of the peripheral nerve. In

addition, tolerance to some cholinergic

effects of cholinesterase-inhibiting

compounds may be due to

compensatory down-regulation of

muscarinic receptors. Younger

individuals may have more capacity to

adapt than older individuals, suggesting

that the aged may be at greater risk to

neurotoxic exposure.

Neurotoxic effects can be observed at

various levels of organization of the

nervous system, including

neurochemical, anatomical,

physiological, or behavioral. At the

neurochemical level, for example, an

agent that causes neurotoxicity might

inhibit macromolecule or transmitter

synthesis, alter the flow of ions across

cellular membranes, or prevent release

of neurotransmitter from the nerve

terminals. Anatomical changes may

include alterations of the cell body, the

axon, or the myelin sheath. At the

physiological level, a chemical might

change the thresholds for neural

activation or reduce the speed of

neurotransmission. Behavioral

alterations can include significant

changes in sensations of sight, hearing,

or touch; alterations in simple or

complex reflexes and motor functions;

alterations in cognitive functions such

as learning, memory, or attention; and

changes in mood, such as fear or rage,

disorientation as to person, time, or

place, or distortions of thinking and

feeling, such as delusions and

hallucinations. At present, relatively

few neurotoxic syndromes have been

thoroughly characterized in terms of the

initial neurochemical change, structural

alterations, physiological consequence,

and behavioral effects. Knowledge of

exact mechanisms of action is not,

however, necessary to conclude that a

chemically induced change is a

neurotoxic effect.

Neurotoxic effects can be produced by

chemicals that do not require

metabolism prior to interacting with

their sites in the nervous system

(primary neurotoxic agents) or those

that require metabolism prior to

interacting with their sites (secondary

neurotoxic agents). Chemically induced

neurotoxic effects can be direct (due to

an agent or its metabolites acting

directly on sites in the nervous system)

or indirect (due to agents or metabolites

that produce their effects primarily by

interacting with sites outside the

nervous system). For example,

excitatory amino acids such as domoic

acid damage specific neurons directly

by activating excitatory amino acid

receptors in the nervous system,

whereas carbon monoxide decreases

oxygen availability, which can

indirectly kill neurons. Other examples

of indirect effects include cadmiuminduced spasms in blood vessels

supplying the nervous system,

dichloroacetate-induced perturbation of

metabolic pathways, and chemically

induced alterations in skeletomuscular

function or structure and effects on the

endocrine system. Professional

judgment may be required in making

determinations about direct versus

indirect effects.

The interpretation of data as

indicative of a potential neurotoxic

effect involves the evaluation of the

validity of the database. This approach

and these terms have been adapted from

the literature on human psychological

testing (Sette, 1987; Sette and MacPhail,

1992), where they have long been used

to evaluate the level of confidence in

different measures of intelligence or

other abilities, aptitudes, or feelings.

There are four principal questions that

should be addressed: whether the effects

result from exposure (content validity);

whether the effects are adverse or

toxicologically significant (construct

validity); whether there are correlative

measures among behavioral,

physiological, neurochemical, and
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26931

morphological endpoints (concurrent

validity); and whether the effects are

predictive of what will happen under

various conditions (predictive validity).

Addressing these issues can provide a

useful framework for evaluating either

human or animal studies or the weight

of evidence for a chemical (Sette, 1987;

Sette and MacPhail, 1992). The next

sections indicate the extent to which

chemically induced changes can be

interpreted as providing evidence of

neurotoxicity.

3. Hazard Characterization

3.1. Neurotoxicological Studies:

Endpoints and Their Interpretation

The qualitative characterization of

neurotoxic hazard can be based on

either human or animal data (Anger,

1984; Reiter, 1987; U.S. EPA, 1994).

Such data can result from accidental,

inappropriate, or controlled

experimental exposures. This section

describes many of the general and some

of the specific characteristics of human

studies and reports of neurotoxicity. It

then describes some features of animal

studies of neuroanatomical,

neurochemical, neurophysiological, and

behavioral effects relevant to risk

assessment. The process of

characterizing the sufficiency or

insufficiency of neurotoxic effects for

risk assessment is described in section

3.3. Additional sources of information

relevant to hazard characterization, such

as comparisons of molecular structure

among compounds and in vitro

screening methods, are also discussed.

The hazard characterization should:

a. Identify strengths and limitations of

the database:

• Epidemiological studies (case

reports, cross-sectional, case-control,

cohort, or human laboratory exposure

studies);

• Animal studies (including

structural or neuropathological,

neurochemical, neurophysiological,

behavioral or neurological, or

developmental endpoints).

b. Evaluate the validity of the

database:

• Content validity (effects result from

exposure);

• Construct validity (effects are

adverse or toxicologically significant);

• Concurrent validity (correlative

measures among behavioral,

physiological, neurochemical, or

morphological endpoints);

• Predictive validity (effects are

predictive of what will happen under

various conditions).

c. Identify and describe key

toxicological studies.

d. Describe the type of effects:

• Structural (neuroanatomical

alternations);

• Functional (neurochemical,

neurophysiological, behavioral

alterations).

e. Describe the nature of the effects

(irreversible, reversible, transient,

progressive, delayed, residual, or latent).

f. Describe how much is known about

how (through what biological

mechanism) the chemical produces

adverse effects.

g. Discuss other health endpoints of

concern.

h. Comment on any nonpositive data

in humans or animals.

I. Discuss the dose-response data

(epidemiological or animal) available for

further dose-response analysis.

j. Discuss the route, level, timing, and

duration of exposure in studies

demonstrating neurotoxicity as

compared to expected human

exposures.

k. Summarize the hazard

characterization:

• Confidence in conclusions;

• Alternative conclusions also

supported by the data;

• Significant data gaps; and

• Highlights of major assumptions.

3.1.1. Human Studies

It is well established that information

from the evaluation of human exposure

can identify neurotoxic hazards (Anger

and Johnson, 1985; Anger, 1990).

Prominent among historical episodes of

neurotoxicity in human populations are

the outbreaks of methylmercury

poisoning in Japan and Iraq and the

neurotoxicity seen in miners of metals,

including mercury, manganese, and lead

(Carson et al., 1987; Silbergeld and

Percival, 1987; OTA, 1990). In the past

decade, lead poisoning in children has

been a prominent issue of concern

(Silbergeld and Percival, 1987).

Neurotoxicity in humans has been

studied and reviewed for many

pesticides (Hayes, 1982; NRDC, 1989;

Ecobichon and Joy, 1982; Ecobichon et

al., 1990). Organochlorines,

organophosphates, carbamates,

pyrethroids, certain fungicides, and

some fumigants are all known

neurotoxicants. They may pose

occupational risks to manufacturing and

formulation workers, pesticide

applicators and farm workers, and

consumers through home application or

consumption of residues in foods.

Families of workers may also be

exposed by transport into the home

from workers’ clothing. Data on humans

can come from a number of sources,

including clinical evaluations, case

reports, epidemiologic studies, and

human laboratory exposure studies. A

more extensive description of issues

concerning human neurotoxicology and

risk assessment has been published

elsewhere (U.S. EPA, 1993). A review of

the types of tests used to assess

cognitive and neurological function in

children, in addition to a discussion of

methodological issues in the design of

prospective, longitudinal studies of

developmental neurotoxicity in

humans, has recently been published

(Jacobson and Jacobson, 1996). Stanton

and Spear (1990) reviewed assessment

measures used in developmental

neurotoxicology for their comparability

in humans and laboratory animals and

their ability to detect comparable

adverse effects across species. At the

level of the various functional

assessments for sensory, motivational,

cognitive and motor function, and social

behavior, there was good agreement

across species among the neurotoxic

agents reviewed.

3.1.1.1. Clinical Evaluations

Clinical methods are used extensively

in neurology and neuropsychology to

evaluate patients suspected of having

neurotoxicity. An array of examineradministered and paper-and-pencil

tasks are used to assess sensory, motor,

cognitive, and affective functions and

personality states/traits.

Neurobehavioral data are synthesized

with information from

neurophysiological studies and medical

history to derive a working diagnosis.

Brain functional imaging techniques

based on magnetic resonance imaging or

emission tomography may also be useful

in helping diagnose neurodegenerative

disorders following chemical exposures

in humans (Omerand et al., 1994;

Callender et al., 1994). Clinical

diagnostic approaches have provided a

rich conceptual framework for

understanding the functions (and

malfunctions) of the central and

peripheral nervous systems and have

formed the basis for the development of

methods for measuring the behavioral

expression of nervous system disorders.

Human neurobehavioral toxicology has

borrowed heavily from neurology and

neuropsychology for concepts of

nervous system impairment and

functional assessment methods.

Neurobehavioral toxicology has adopted

the neurologic/neuropsychologic model,

using adverse changes in behavioral

function to assist in identifying

chemical-or drug-induced changes in

nervous system processes.

Neurological and neuropsychological

methods have long been employed to

identify the adverse health effects of

environmental workplace exposures

(Sterman and Schaumburg, 1980).
26932 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

Peripheral neuropathies (with sensory

and motor disturbances),

encephalopathies, organic brain

syndromes, extrapyramidal syndromes,

demyelination, autonomic changes, and

dementia are well-characterized

consequences of acute and chronic

exposure to chemical agents. The range

of exposure conditions that produce

clinical signs of neurotoxicity also has

been defined by these clinical methods.

It is very important to make external/

internal dose measurements in humans

to determine the actual dose(s) that can

cause unwanted effects.

Aspects of the neurological

examination approach limit its

usefulness for neurotoxicological risk

assessment. Information obtained from

the neurological exam is mostly

qualitative and descriptive rather than

quantitative. Estimates of the severity of

functional impairment can be reliably

placed into only three or four categories

(for example, mild, moderate, severe).

Much of the assessment depends on the

subjective judgment of the examiner.

For example, the magnitude and

symmetry of muscle strength are often

judged by having the patient push

against the resistance of the examiner’s

hands. The endpoints are therefore the

absolute and relative amount of muscle

load sensed by the examiner in his or

her arms.

Compared with other methods, the

neurological exam may be less sensitive

in detecting early neurotoxicity in

peripheral sensory and motor nerves.

While clinicians’ judgments are equal in

sensitivity to quantitative methods in

assessing the amplitude of tremor,

tremor frequency is poorly quantified by

clinicians. Thus, important aspects of

the clinical neurologic exam may be

insufficiently quantified and lack

sufficient sensitivity for detecting early

neurobehavioral toxicity produced by

environmental or workplace exposure

conditions. However, a neurological

evaluation of persons with documented

neurobehavioral impairment would be

helpful for identifying nonchemical

causes of neurotoxicity, such as diabetes

and cardiovascular insufficiency.

Administration of a

neuropsychological battery also requires

a trained technician, and interpretation

requires a trained and experienced

neuropsychologist. Depending on the

capabilities of the patient, 2 to 4 hours

may be needed to administer a full

battery; 1 hour may be needed for the

shorter screening versions. These

practical considerations may limit the

usefulness of neuropsychological

assessment in large field studies of

suspected neurotoxicity.

In addition to logistical problems in

administration and interpretation,

neuropsychological batteries and

neurological exams share two

disadvantages with respect to

neurotoxicity risk assessment. First,

neurological exams and

neuropsychological test batteries are

designed to confirm and classify

functional problems in individuals

selected on the basis of signs and

symptoms identified by the patient,

family, or other health professionals.

Their usefulness in detecting low baserate impairment in workers or the

general population is generally thought

to be limited, decreasing the usefulness

of clinical assessment approaches for

epidemiologic risk assessment.

Second, neurological exams and

neuropsychological test batteries were

developed to assess the functional

correlates of the most common forms of

nervous system dysfunction: brain

trauma, focal lesions, and degenerative

conditions. The clinical tests were

validated against these neurological

disease states. With a few notable

exceptions, chemicals are not believed

to produce impairment similar to that

from trauma or lesions; neurotoxic

effects are more similar to the effects of

degenerative disease. There has been

insufficient research to demonstrate

which tests designed to assess

functional expression of neurologic

disease are useful in characterizing the

modes of central nervous system

impairment produced by chemical

agents and drugs.

It should be noted that alternative

approaches are available that avoid

many of the limitations of clinical and

neurological and traditional

neuropsychological methods.

Computerized behavioral assessment

systems designed for field testing of

populations exposed to chemicals in the

community or workplace have been

developed during the past decade. The

most widely used system is the

Neurobehavioral Evaluation System

(NES) developed by Baker et al. (1985).

Advantages of computerized tests

include (1) standardized administration

to eliminate intertester variability and

minimize subject-experimenter

interaction; (2) automated data

collection and scoring, which is faster,

easier, and less error-prone than

traditional methods; and (3) test

administration requires minimal

training and experience. NES tests have

proven sensitive to a variety of solvents,

metals, and pesticides (Otto, 1992).

Computerized systems available for

human neurotoxicity testing are

critically reviewed in Anger et al.

(1996).

3.1.1.2. Case Reports

The first type of human data available

is often the case report or case series,

which can identify cases of a disease

and are reported by clinicians or

discerned through active or passive

surveillance, usually in the workplace.

However, case reports involving a single

neurotoxic agent, although informative,

are rare in the literature; for example,

farmers are likely to be exposed to a

wide variety of potentially neurotoxic

pesticides. Careful case histories assist

in identifying common risk factors,

especially when the association between

the exposure and disease is strong, the

mode of action of the agent is

biologically plausible, and clusters

occur in a limited period of time.

Case reports can be obtained more

quickly than more complex studies.

Case reports of acute high-level

exposure to a toxicant can be useful for

identifying signs and symptoms that

may also apply to lower exposure. Case

reports can also be useful when

corroborating epidemiological data are

available.

3.1.1.3. Epidemiologic Studies

Epidemiology has been defined as

‘‘the study of the distributions and

determinants of disease and injuries in

human populations’’ (Mausner and

Kramer, 1985). Knowing the frequency

of illness in groups and the factors that

influence the distribution is the tool of

epidemiology that allows the evaluation

of causal inference with the goal of

prevention and cure of disease

(Friedlander and Hearn, 1980).

Epidemiologic studies are a useful

means of evaluating the effects of

neurotoxic substances on human

populations, particularly if effects of

exposure are cumulative or exposures

are repeated. Such studies are less

useful in cases of acute exposure, where

the effects are short-term. Frequently,

determining the precise dose or

exposure concentration in

epidemiological studies can be difficult.

3.1.1.3.1. Cross-Sectional Studies.

In cross-sectional studies or surveys,

both the disease and suspected risk

factors are ascertained at the same time,

and the findings are useful in generating

hypotheses. A group of people are

interviewed, examined, and tested at a

single point in time to ascertain a

relationship between a disease and a

neurotoxic exposure. This study design

does not allow the investigator to

determine whether the disease or the

exposure came first, rendering it less

useful in estimating risk. These studies

are intermediate in cost and time
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26933

required to complete compared with

case reports and more complex

analytical studies, but should be

augmented with additional data.

3.1.1.3.2. Case-Control (Retrospective)

Studies.

Last (1986) defines a case-control

study as one that ‘‘starts with the

identification of persons with the

disease (or other outcome variable) of

interest, and a suitable control

population (comparison, reference

group) of persons without the disease.’’

He states that the relationship of an

‘‘attribute’’ to the disease is measured by

comparing the diseased with the

nondiseased with regard to how

frequently the attribute is present in

each of the groups. The cases are

assembled from a population of persons

with and without exposure, and the

comparison group is selected from the

same population; the relative

distribution of the potential risk factor

(exposure) in both groups is evaluated

by computing an odds ratio that serves

as an estimate of the strength of the

association between the disease and the

potential risk factor. The statistical

significance of the ratio is determined

by calculating a p-value and is used to

approximate relative risk.

The case-control approach to the

study of potential neurotoxicants in the

environment provides a great deal of

useful information for the risk assessor.

In his textbook, Valciukas (1991) notes

that the case-control approach is the

strategy of choice when no other

environmental or biological indicator of

neurotoxic exposure is available. He

further states: ‘‘Considering the fact that

for the vast majority of neurotoxic

chemical compounds, no objective

biological indicators of exposure are

available (or if they are, their half-life is

too short to be of any practical value),

the case-control paradigm is a widely

accepted strategy for the assessment of

toxic causation.’’ The case-control study

design, however, can be very

susceptible to bias. The potential

sources of bias are numerous and can be

specific to a particular study. Many of

these biases also can be present in crosssectional studies. For example, recall

bias or faulty recall of information by

study subjects in a questionnaire-based

study can distort the results. Analysis of

the case-comparison study design

assumes that the selected cases are

representative persons with the

disease—either all cases with the

disease or a representative sample of

them have been ascertained. It further

assumes that the control or comparison

group is representative of the

nonexposed population (or that the

prevalence of the characteristic under

study is the same in the control group

as in the general population). Failure to

satisfy these assumptions may result in

selection bias that may invalidate study

results.

An additional source of bias in casecontrol studies is the presence of

confounding variables, i.e., factors

known to be associated with the

exposure and causally related to the

disease under study. These should be

controlled, either in the design of the

study by matching cases to controls on

the basis of the confounding factor, or

in the analysis of the data by using

statistical techniques such as

stratification or regression. Matching

requires time to identify an adequate

number of potential controls to

distinguish those with the proper

characteristics, while statistical control

of confounding factors requires a larger

study.

The definition of exposure is critical

in epidemiologic studies. In

occupational settings, exposure

assessment often is based on the job

assignment of the study subjects, but

can be more precise if detailed company

records allow the development of

exposure profiles. Positive results from

a properly controlled retrospective

study should weigh heavily in the risk

assessment process.

3.1.1.3.3. Cohort (Prospective, 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

dysfunction. Such studies are

invaluable for determining the time

course for development of dysfunction

(e.g., follow-up studies performed in

various cities on the effects of lead on

child development). This approach

allows the direct estimate of risks

attributed to a particular exposure, since

toxic incidence rates in the cohort can

be determined. Prospective study

designs also allow the study of chronic

effects of exposure. One major strength

of the cohort design is that it allows the

calculation of rates to determine the

excess risk associated with an exposure.

Also, biases are reduced by obtaining

information before the disease develops.

This approach, however, can be very

time-consuming and costly.

In cohort studies information bias can

be introduced when individuals provide

distorted information about their health

because they know their exposure status

and may have been told of the expected

health effects of the exposure under

study. More credence should be given to

those studies in which both observer

and subject bias are carefully controlled

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

A special type of cohort study is the

retrospective cohort study, in which the

investigator goes back in time to select

the study groups and traces them over

time, often to the present. The studies

usually involve specially exposed

groups and have provided much

assistance in estimating risks due to

occupational exposures. Occupational

retrospective cohort studies rely on

company records of past and current

employees that include information on

the dates of employment, age at

employment, date of departure, and

whether diseased (or dead in the case of

mortality studies). Workers can then be

classified by duration and degree of

exposure. Positive or negative results

from a properly controlled prospective

study should weigh heavily in the risk

assessment process.

3.1.1.4. Human Laboratory Exposure

Studies

Neurotoxicity assessment has an

advantage not afforded to the evaluation

of other toxic endpoints, such as cancer

or reproductive toxicity, in that the

effects of some chemicals are short in

duration and reversible. This makes it

ethically possible to perform human

laboratory exposure studies and obtain

data relevant to the risk assessment

process. Information from experimental

human exposure studies has been used

to set occupational exposure limits,

mostly for organic solvents that can be

inhaled. Laboratory exposure studies

have contributed to risk assessment and

the setting of exposure limits for several

solvents and other chemicals with acute

reversible effects.

Human exposure studies sometimes

offer advantages over epidemiologic

field studies. Combined with

appropriate sampling of biological

fluids (urine or blood), it is possible to

calculate body concentrations, examine

toxicokinetics, and identify metabolites.

Bioavailability, elimination, doserelated changes in metabolic pathways,

individual variability, time course of

effects, interactions between chemicals,

and interactions between chemical and

environmental/biobehavioral processes

(stressors, workload/respiratory rate) are

factors that are generally easier to

collect under controlled conditions.

Other goals of laboratory studies

include the in-depth characterization of

effects, the development of new

assessment methods, and the

examination of the sensitivity,

specificity, and reliability of

neurobehavioral assessment methods

across chemical classes. The laboratory

is the most appropriate setting for the
26934 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

study of environmental and

biobehavioral variables that affect the

action of chemical agents. The effects of

ambient temperature, task difficulty,

rate of ongoing behavior, conditioning

variables, tolerance/sensitization, sleep

deprivation, motivation, and so forth are

sometimes studied.

From a methodological standpoint,

human laboratory studies can be

divided into two categories: betweensubjects and within-subjects designs. In

the former, the neurobehavioral

performance of exposed volunteers is

compared with that of nonexposed

participants. In the latter, preexposure

performance is compared with

neurobehavioral function under the

influence of the chemical or drug.

Within-subjects designs have the

advantage of requiring fewer

participants, eliminating individual

differences as a source of variability,

and controlling for chronic mediating

variables, such as caffeine use and

educational achievement. A

disadvantage of the within-subjects

design is that neurobehavioral tests

must be administered more than once.

Practice on many neurobehavioral tests

often leads to improved performance

that may confound the effect of the

chemical/drug. There should be a

sufficient number of test sessions in the

pre-exposure phase to allow

performance on all tests to achieve a

relatively stable baseline level.

Participants in laboratory exposure

studies may have been recruited from

populations of persons already exposed

to the chemical/drug or from chemicalnaive populations. Although the use of

exposed volunteers has ethical

advantages, can mitigate against novelty

effects, and allows evaluation of

tolerance/sensitization, finding an

accessible exposed population in

reasonable proximity to the laboratory

can be difficult. Chemical-naive

participants are more easily recruited

but may differ significantly in important

characteristics from a representative

sample of exposed persons. Chemicalnaive volunteers are often younger,

healthier, and better educated than the

populations exposed environmentally,

in the workplace, or

pharmacotherapeutically.

Compared with workplace and

environmental exposures, laboratory

exposure conditions can be controlled

more precisely, but exposure periods are

much shorter. Generally only one or two

relatively pure chemicals are studied for

several hours, whereas the population of

interest may be exposed to multiple

chemicals containing impurities for

months or years. Laboratory studies are

therefore better at identifying and

characterizing effects with acute onset

and the selective effects of pure agents.

In all cases, the potential for participant

bias should be as carefully controlled

for as possible. Even the consent form

can lead to participant bias, as toxic

effects have been reported in some

individuals who were warned of such

effects in an informed consent form. In

addition, double-blind studies have

been shown to provide some control for

observer bias that may occur in singleblind studies. More credence should be

given to those studies in which both

observer and subject bias are carefully

controlled (Benignus, 1993).

A test battery that examines multiple

neurobehavioral functions may be more

useful for screening and the initial

characterization of acute effects.

Selected neurobehavioral tests that

measure a limited number of functions

in multiple ways may be more useful for

elucidating mechanisms or validating

specific effects.

Both chemical and behavioral control

procedures are valuable for examining

the specificity of the effects. A

concordant effect among different

measures of the same neurobehavioral

function (e.g., reaction time) and a lack

of effect on some other measures of

psychomotor function (e.g., untimed

manual dexterity) would increase the

confidence in a selective effect on motor

speed and not on attention or another

nonspecific motor function. Likewise,

finding concordant effects among

similar chemical or drug classes along

with different effects from dissimilar

classes would support the specificity of

chemical effect. For example, finding

that the effects of a solvent were similar

to those of ethanol but not caffeine

would support the specificity of solvent

effects on a given measure of

neurotoxicity.

3.1.2. Animal Studies

This section provides an overview of

the major types of endpoints that may

be evaluated in animal neurotoxicity

studies, describes the kinds of effects

that may be observed and some of the

tests used to detect and quantify these

effects, and provides guidance for

interpreting data. Compared with

human studies, animal studies are more

often available for specific chemicals,

provide more precise exposure

information, and control environmental

factors better (Anger, 1984). For these

reasons, risk assessments tend to rely

heavily on animal studies.

Many tests that can measure some

aspect of neurotoxicity have been used

in the field of neurobiology in the past

50 years. The Office of Prevention,

Pesticides and Toxic Substances

(OPPTS) has published animal testing

guidelines that were developed in

cooperation with the Office of Research

and Development (U.S. EPA, 1991a).

While the test endpoints included in the

1991 document serve as a convenient

focus for this section, there are many

other endpoints for which there are no

current EPA guidelines. The goal of the

current document is to provide a

framework for interpreting data

collected in tests frequently used by

neurotoxicologists.

Five categories of endpoints will be

described: structural or

neuropathological, neurophysiological,

neurochemical, behavioral, and

developmental. Table 1 lists a number

of endpoints in each of these categories.

It is imperative for the risk assessor to

understand that the interpretation of the

indicators listed in Table 1 as

neurotoxic effects is dependent on the

dose at which such changes occur and

the possibility that damage to other

organ systems may contribute to or

cause such changes indirectly.

TABLE 1.—EXAMPLES OF POSSIBLE INDICATORS OF A NEUROTOXIC EFFECT

Structural or neuropathological endpoints:

Gross changes in morphology, including brain weight.

Histologic changes in neurons or glia (neuronopathy, axonopathy, myelinopathy).

Neurochemical endpoints:

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

Alterations in second-messenger-associated signal transduction.

Alterations in membrane-bound enzymes regulating neuronal activity.

Inhibition and aging of neuropathy enzyme.

Increases in glial fibrillary acidic protein in adults.

Neurophysiological endpoints:

Change in velocity, amplitude, or refractory period of nerve conduction.
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26935

TABLE 1.—EXAMPLES OF POSSIBLE INDICATORS OF A NEUROTOXIC EFFECT—Continued

Change in latency or amplitude of sensory-evoked potential.

Change in electroencephalographic pattern.

Behavioral and neurological endpoints:

Increases or decreases in motor activity.

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

Changes in motor coordination, weakness, paralysis, abnormal movement or posture, tremor, ongoing performance.

Absence or decreased occurrence, magnitude, or latency of sensorimotor reflex.

Altered magnitude of neurological measurement, including grip strength, hindlimb splay.

Seizures.

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

Changes in learning, memory, and attention.

Developmental endpoints:

Chemically induced changes in the time of appearance of behaviors during development.

Chemically induced changes in the growth or organization of structural or neurochemical elements.

3.1.2.1. Structural Endpoints of

Neurotoxicity

Structural endpoints are typically

defined as neuropathological changes

evident by gross observation or light

microscopy, although most neurotoxic

changes will be detectable only at the

light microscopic level. Gross changes

in morphology can include discrete or

widespread lesions in nerve tissue. A

change in brain weight is considered to

be a biologically significant effect. This

is true regardless of changes in body

weight, because brain weight is

generally protected during

undernutrition or weight loss, unlike

many other organs or tissues. It is

inappropriate to express brain weight

changes as a ratio of body weight and

thereby dismiss changes in absolute

brain weight. Changes in brain weight

are a more reliable indicator of

alteration in brain structure than are

measurements of length or width in

fresh brain, because there is little

historical data in the toxicology

literature.

Neurons are composed of a neuronal

body, axon, and dendritic processes.

Various types of neuropathological

lesions may be classified according to

the site where they occur (Spencer and

Schaumburg, 1980; WHO, 1986; Krinke,

1989; Griffin, 1990). Neurotoxicantinduced lesions in the central or

peripheral nervous system may be

classified as a neuronopathy (changes in

the neuronal cell body), axonopathy

(changes in the axons), myelinopathy

(changes in the myelin sheaths), or

nerve terminal degeneration. Nerve

terminal degeneration represents a very

subtle change that may not be detected

by routine histopathology, but requires

detection by special procedures such as

silver staining or neurotransmitterspecific immunohistochemistry. For

axonopathies, a more precise location of

the changes may also be described (i.e.,

proximal, central, or distal axonopathy).

In the case of some developmental

exposures, a neurotoxic chemical might

delay or accelerate the differentiation or

proliferation of cells or cell types.

Alteration in the axonal termination site

might also occur with exposure. In an

aged population, exposure to some

neurotoxicants might accelerate the

normal loss of neurons associated with

aging (Reuhl, 1991). In rare cases,

neurotoxic agents have been reported to

produce neuropathic conditions

resembling neurodegenerative disorders,

such as Parkinson’s disease, in humans

(WHO, 1986). Table 2 lists examples of

such neurotoxic chemicals, their

putative site of action, the type of

neuropathology produced, and the

disorder or condition that each typifies.

Inclusion of any chemical in any of the

following tables is for illustrative

purposes, i.e., it has been reported that

the chemical will produce a neurotoxic

effect at some dose; any individual

chemical listed may also adversely

affect other organs at lower doses. It is

important that the severity of each

structural union be graded objectively

and the grading criteria reported.

TABLE 2.—NEUROTOXICANTS AND DISORDERS WITH SPECIFIC NEUROLOGICAL TARGETS

Site of action Neurotoxic change Neurotoxic chemical Corresponding neurodegenerative

disorder

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

Quinolinic acid ............................ Huntington’s disease.

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

Nerve terminal .............................. Terminal destruction ................... 1-Methyl-4-phenyl 1,2, ................

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

pyridine (MPTP) (dopaminergic)

Parkinson’s disease.

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

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

Central axons ............................... Central axonopathy .................... Clioquinol .................................... Subacute myeloopticoneuro-pathy.

Proximal axon ............................... Proximal axonopathy .................. B,B′-Iminodipropionitrile .............. Motor neuron disease.

Alterations in the structure of the

nervous system (i.e., neuronopathy,

axonopathy, myelinopathy, terminal

degeneration) are regarded as evidence

of a neurotoxic effect. The risk assessor

should note that pathological changes in

many cases require time for the

perturbation to become observable,

especially with evaluation at the light

microscopic level. Neuropathological

studies should control for potential

differences in the area(s) and section(s)

of the nervous system sampled; in the

age, sex, and body weight of the subject;

and in fixation artifacts (WHO, 1986).

Concern for the structural integrity of

nervous system tissues derives from
26936 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

their functional specialization and lack

of regenerative capacity.

Within general class of nervous

system structural alteration, there are

various histological changes that can

result after exposure to neurotoxicants.

For example, specific changes in nerve

cell bodies include chromatolysis,

vacuolization, and cell death. Axons can

undergo swelling, degeneration, and

atrophy, while myelin sheath changes

include folding, edematous splitting,

and demyelination. Although terminal

degeneration does occur, it is not

readily detectable by light microscopy.

Many of these changes are a result of

complex effects at specific subcellular

organelles, such as the axonal swelling

that occurs as a result of neurofilament

accumulation in acrylamide toxicity.

Other changes may be associated with

regenerative or adaptive processes that

occur after neurotoxicant exposure.

3.1.2.2. Neurophysiological Endpoints

of Neurotoxicity

Neurophysiological studies measure

the electrical activity of the nervous

system. The term ‘‘neurophysiology’’ is

often used synonymously with

‘‘electrophysiology’’ (Dyer, 1987).

Neurophysiological techniques provide

information on the integrity of defined

portions of the nervous system. Several

neurophysiological procedures are

available for application to

neurotoxicological studies. Examples

are listed in Table 3. They range in scale

from procedures that employ

microelectrodes to study the function of

single nerve cells or restricted portions

of them, to procedures that employ

macroelectrodes to perform

simultaneous recordings of the summed

activity of many cells. Microelectrode

procedures typically are used to study

mechanisms of action and are frequently

performed in vitro. Macroelectrode

procedures are generally used in studies

to detect or characterize the potential

neurotoxic effects of agents of interest

because of potential environmental

exposure. The present discussion

concentrates on macroelectrode

neurophysiological procedures because

it is more likely that they will be the

focus of decisions regarding critical

effects in risk assessment. All of the

procedures described below for use in

animals also have been used in humans

to determine chemically induced

alterations in neurophysiological

function.

TABLE 3.—EXAMPLES OF NEUROPHYSIOLOGICAL MEASURES OF NEUROTOXICITY

System/function Procedure Representative agents

Retina ................................................................ Electroretinography (ERG) ............................... Developmental lead.

Visual pathway .................................................. Flash-evoked potential (FEP) ........................... Carbon disulfide.

Visual function ................................................... Pattern-evoked potential (PEP) (pattern size

and contrast).

Carbon disulfide.

Auditory pathway .............................................. Brain stem auditory evoked potential (BAER)

(clicks).

Aminoglycoside, antibiotics, toluene, styrene.

Auditory function ............................................... BAER (tones) .................................................... Aminoglycoside, antibiotics, toluene, styrene.

Somatosensory pathway ................................... Somatosensory provoked ................................. Acrylamide, n-hexane.

Somatosensory function ................................... Sensory-evoked potential (SEP) (tactile) ......... Acrylamide, n-hexane.

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

Mixed nerve ...................................................... Peripheral nerve compound action potential

(PNAP).

Triethyltin.

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

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

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

General central nervous system/level of arousal.Electroencephalography (EEG) ........................ Toluene.

3.1.2.2.1. Nerve Conduction Studies.

Nerve conduction studies, generally

performed on peripheral nerves, can be

useful in investigations of possible

peripheral neuropathy. Most peripheral

nerves contain mixtures of individual

sensory and motor nerve fibers, which

may or may not be differentially

sensitive to neurotoxicants. It is possible

to distinguish sensory from motor

effects in peripheral nerve studies by

measuring activity in sensory nerves or

by measuring the muscle response

evoked by nerve stimulation to measure

motor effects. While a number of

endpoints can be recorded, the most

critical variables are nerve conduction

velocity, response amplitude, and

refractory period. It is important to

recognize that damage to nerve fibers

may not be reflected in changes in these

endpoints if the damage is not

sufficiently extensive. Thus, the

interpretation of data from such studies

may be enhanced if evaluations such as

nerve pathology and/or other structural

measures are also included.

Nerve conduction measurements are

influenced by a number of factors, the

most important of which is temperature.

An adequate nerve conduction study

will either measure the temperature of

the limb under study and

mathematically adjust the results

according to well-established

temperature factors or will control limb

temperature within narrow limits.

Studies that measure peripheral nerve

function without regard for temperature

are not adequate for risk assessment.

In well-controlled studies, statistically

significant decreases in nerve

conduction velocity are indicative of a

neurotoxic effect. While a decrease in

nerve conduction velocity is indicative

of demyelination, it frequently occurs

later in the course of axonal degradation

because normal conduction velocity

may be maintained for some time in the

face of axonal degeneration. For this

reason, a measurement of normal nerve

conduction velocity does not rule out

peripheral axonal degeneration if other

signs of peripheral nerve dysfunction

are present.

Decreases in response amplitude

reflect a loss of active nerve fibers and

may occur prior to decreases in

conduction velocity in the course of

peripheral neuropathy. Hence, changes

in response amplitude may be more

sensitive measurements of axonal

degeneration than is conduction

velocity. Measurements of response

amplitude, however, can be more

variable and require careful application

of experimental techniques, a larger

sample size, and greater statistical

power than measurements of velocity to

detect changes. The refractory period

refers to the time required after

stimulation before a nerve can fire again

and reflects the functional status of

nerve membrane ion channels.

Chemically induced changes in
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26937

refractory periods in a well-controlled

study indicate a neurotoxic effect.

In summary, alterations in peripheral

nerve response amplitude and refractory

period in studies that are well

controlled for temperature are indicative

of a neurotoxic effect. Alterations in

peripheral nerve function are frequently

associated with clinical signs such as

numbness, tingling, or burning

sensations or with motor impairments

such as weakness. Examples of

compounds that alter peripheral nerve

function in humans or experimental

animals include acrylamide, carbon

disulfide, n-hexane, lead, and some

organophosphates.

3.1.2.2.2. Sensory, Motor, and Other

Evoked Potentials. Evoked potential

studies are electrophysiological

procedures that measure the response

elicited from a defined stimulus such as

a tone, a light, or a brief electrical pulse.

Evoked potentials reflect the function of

the system under study, including

visual, auditory, or somatosensory;

motor, involving motor nerves and

innervated muscles; or other neural

pathways in the central or peripheral

nervous system (Rebert, 1983; Dyer,

1985; Mattsson and Albee, 1988;

Mattsson et al., 1992; Boyes, 1992,

1993). Evoked potential studies should

be interpreted with respect to the

known or presumed neural generators of

the responses, and their likely

relationships with behavioral outcomes,

when such information is available.

Such correlative information

strengthens the confidence in

electrophysiological outcomes. In the

absence of such supportive information,

the extent to which evoked potential

studies provide convincing evidence of

neurotoxicity is a matter of professional

judgment on a case-by-case basis.

Judgments should consider the nature,

magnitude, and duration of such effects,

along with other factors discussed

elsewhere in this document.

Data are in the form of a voltage

record collected over time and can be

quantified in several ways. Commonly,

the latency (time from stimulus onset)

and amplitude (voltage) of the positive

and negative voltage peaks are

identified and measured. Alternative

measurement schemes may involve

substitution of spectral phase or

template shifts for peak latency and

spectral power, spectral amplitude, rootmean-square, or integrated area under

the curve for peak amplitude. Latency

measurements are dependent on both

the velocity of nerve conduction and the

time of synaptic transmission. Both of

these factors depend on temperature, as

discussed in regard to nerve conduction,

and similar caveats apply for sensory

evoked potential studies. In studies that

are well controlled for temperature,

increases in latencies or related

measures can reflect deficits in nerve

conduction, including demyelination or

delayed synaptic transmission, and are

indicators of a neurotoxic effect.

Decreases in peak latencies, like

increases in nerve conduction velocity,

are unusual, but the neural systems

under study in sensory evoked

potentials are complex, and situations

that might cause a peak measurement to

occur earlier are conceivable. Two such

situations are a reduced threshold for

spatial or temporal summation of

afferent neural transmission and a

selective loss of cells responding late in

the peak, thus making the measured

peak occur earlier. Decreases in peak

latency should not be dismissed

outright as experimental or statistical

error, but should be examined carefully

and perhaps replicated to assess

possible neurotoxicity. A decrease in

latency is not conclusive evidence of a

neurotoxic effect.

Changes in peak amplitudes or

equivalent measures reflect changes in

the magnitude of the neural population

responsive to stimulation. Both

increases and decreases in amplitude

are possible following exposure to

chemicals. Whether excitatory or

inhibitory neural activity is translated

into a positive or negative deflection in

the sensory evoked potential is

dependent on the physical orientation

of the electrode with respect to the

tissue generating the response, which is

frequently unknown. Comparisons

should be based on the absolute change

in amplitude. Therefore, either increases

or decreases in amplitude may be

indicative of a neurotoxic effect.

Within any given sensory system, the

neural circuits that generate various

evoked potential peaks differ as a

function of peak latency. In general,

early latency peaks reflect the

transmission of afferent sensory

information. Changes in either the

latency or amplitude of these peaks are

considered convincing evidence of a

neurotoxic effect that is likely to be

reflected in deficits in sensory

perception. The later-latency peaks, in

general, reflect not only the sensory

input but also the more nonspecific

factors such as the behavioral state of

the subject, including such factors as

arousal level, habituation, or

sensitization (Dyer, 1987). Thus,

changes in later-latency evoked

potential peaks should be interpreted in

light of the behavioral status of the

subject and would generally be

considered evidence of a neurotoxic

effect.

3.1.2.2.3. Seizures/Convulsions. Some

neurotoxicants (e.g., lindane,

pyrethroids, trimethyltin,

dichlorodiphenyltrichloroethane [DDT])

produce observable convulsions. When

convulsionlike behaviors are observed,

as described in the behavioral section

on convulsions, neurophysiological

recordings can provide additional

information to help interpret the results.

Recordings of brain electrical activity

that demonstrate seizurelike activity are

indicative of a neurotoxic effect.

In addition to producing seizures

directly, chemicals may also alter the

frequency, severity, duration, or

threshold for eliciting seizures through

other means by a phenomenon known

as ‘‘kindling.’’ Such alterations can

occur after acute exposure or after

repeated exposure to dose levels below

the acute threshold. In experiments

demonstrating changes in sensitivity

following repeated exposures to the test

compound, information regarding

possible changes in the pharmacokinetic

distribution of the compound is

required before the seizure

susceptibility changes can be

interpreted as evidence of neurotoxicity.

Increases in susceptibility to seizures

are considered adverse.

3.1.2.2.4. Electroencephalography

(EEG). EEG analysis is used widely in

clinical settings for the diagnosis of

neurological disorders, and less often

for the detection of subtle toxicantinduced dysfunction (WHO, 1986;

Eccles, 1988). The basis for using EEG

in either setting is the relationship

between specific patterns of EEG

waveforms and specific behavioral

states. Because states of alertness and

stages of sleep are associated with

distinct patterns of electrical activity in

the brain, it is generally thought that

arousal level can be evaluated by

monitoring the EEG. Dissociation of EEG

activity and behavior can, however,

occur after exposure to certain

chemicals. Normal patterns of transition

between sleep stages or between

sleeping and waking states are known to

remain disturbed for prolonged periods

of time after exposure to some

chemicals. Changes in the pattern of the

EEG can be elicited by anesthetic drugs

and stimuli producing arousal (e.g.,

lights, sounds). In studies with

toxicants, changes in EEG pattern can

sometimes precede alterations in other

objective signs of neurotoxicity (Dyer,

1987).

EEG studies should be done under

highly controlled conditions, and the

data should be considered on a case-bycase basis. Chemically induced seizure

activity detected in the EEG pattern is

evidence of a neurotoxic effect.
26938 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

3.1.2.3. Neurochemical Endpoints of

Neurotoxicity

Many different neurochemical

endpoints have been measured in

neurotoxicological studies, and some

have proven useful in advancing the

understanding of mechanisms of action

of neurotoxic chemicals (Bondy, 1986;

Mailman, 1987; Morell and Mailman,

1987; Costa, 1988; Silbergeld, 1993).

Normal functioning of the nervous

system depends on the synthesis and

release of specific neurotransmitters and

activation of their receptors at specific

presynaptic and postsynaptic sites.

Chemicals can interfere with the ionic

balance of a neuron, act as a

cytotoxicant after transport into a nerve

terminal, block reuptake of

neurotransmitters and their precursors,

act as a metabolic poison, overstimulate

receptors, block transmitter release, and

inhibit transmitter synthetic or catabolic

enzymes. Table 4 lists several chemicals

that produce neurotoxic effects at the

neurochemical level (Bondy, 1986;

Mailman, 1987; Morell and Mailman,

1987; Costa, 1988).

TABLE 4.—EXAMPLES OF NEUROTOXICANTS WITH KNOWN NEUROCHEMICAL MECHANISMS

Site of action Examples

Neurotoxicants acting on ionic balance:

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

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

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

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

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

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

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

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

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

Inhibition of transmitter degradation ......................................................... Pesticides of the organophosphate and carbamate classes.

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

As stated previously, any

neurochemical change is potentially

neurotoxic. Persistent or irreversible

chemically induced neurochemical

changes are indicative of neurotoxicity.

Because the ultimate functional

significance of some biochemical

changes is not known at this time,

neurochemical studies should be

interpreted with reference to the

presumed neurotoxic consequence(s) of

the neurochemical changes. For

example, many neuroactive agents can

increase or decrease neurotransmitter

levels, but such changes are not

indicative of a neurotoxic effect. If,

however, these neurochemical changes

may be expected to have

neurophysiological, neuropathological,

or neurobehavioral correlates, then the

neurochemical changes could be

classified as neurotoxic effects.

Some neurotoxicants, such as the

organophosphate and carbamate

pesticides, are known to inhibit the

activity of a specific enzyme,

acetylcholinesterase (for a review see

Costa, 1988), which hydrolyzes the

neurotransmitter acetylcholine.

Inhibition of the enzyme in either the

central or peripheral nervous system

prolongs the action of the acetylcholine

at the neuron’s synaptic receptors and is

thought to be responsible for the range

of effects these chemicals produce,

although it is possible that these

compounds have other modes of action

(Eldefrawi et al., 1992; Greenfield et al.,

1984; Small, 1990).

There is agreement that objective

clinical measures of cholinergic

overstimulation (e.g., salivation,

sweating, muscle weakness, tremor,

blurred vision) can be used to evaluate

dose-response and dose-effect

relationships and define the presence

and absence of effects. A given

depression in peripheral and central

cholinesterase activity may or may not

be accompanied by clinical

manifestations. A depression in RBC

and/or plasma cholinesterase activity

may or may not be accompanied by

clinical manifestations. It should be

noted, however, that reduction in

cholinesterase activity, even if the

anticholinesterase exposure is not

severe enough to precipitate clinical

signs or symptoms, may impair the

organism’s ability to adapt to additional

exposures to anticholinesterase

compounds. Inhibition of RBC and/or

plasma cholinesterase activity is a

biomarker of exposure, as well as a

reflection of cholinesterase inhibition in

other peripheral tissues (e.g.,

neuromuscular junction, peripheral

nerve, or ganglia) (Maxwell et al., 1987;

Nagymajtenyi et al., 1988; Padilla et al.,

1994), thereby contributing to the

overall hazard identification of

cholinesterase-inhibiting compounds.

The risk assessor should also be aware

that tolerance to the cholinergic

overstimulation may be observed

following repeated exposure to

cholinesterase-inhibiting chemicals. It

has been reported, however, that

although tolerance can develop to some

effects of cholinesterase inhibition, the

cellular mechanisms responsible for the

development of tolerance may also lead

to the development of other effects, i.e.,

cognitive dysfunction, not present at the

time of initial exposure (Bushnell et al.,

1991). These adaptive biochemical

changes in the tolerant animal may

render it supersensitive to subsequent

exposure to cholinergically active

compounds (Pope et al., 1992).

In general, the risk assessor should

understand that assessment of

cholinesterase-inhibiting chemicals

should be done on a case-by-case basis

using a weight-of evidence approach in

which all of the available data (e.g.,

brain, blood, and other tissue

cholinesterase activity, as well as the

presence or absence of clinical signs) is

considered in the evaluation. Generally,

the toxic effects of anticholinesterase

compounds are viewed as reversible,

but there is human and experimental

animal evidence indicating that there

may be residual, if not permanent,

effects of exposure to these compounds

(Steenland et al., 1994; Tandon et al.,

1994; Stephens et al., 1995).

A subset of organophosphate agents

also produces organophosphate-induced

delayed neuropathy (OPIDN) after acute

or repeated exposure. Inhibition and

aging of neurotoxic esterase (or

neuropathy enzymes) are associated

with agents that produce OPIDN

(Johnson, 1990; Richardson, 1995). The

conclusion that a chemical may produce

OPIDN should be based on at least two

of three factors: (1) Evidence of a

clinical syndrome, (2) pathological

lesions, and (3) neurotoxic esterase

(NTE) inhibition. NTE inhibition is

necessary, but not sufficient, evidence
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26939

of the potential to produce OPIDN when

there is at least 55%–70% inhibition

after acute exposure (Ehrich et al., 1995)

and at least 45% inhibition following

repeated exposure.

Chemically induced injury to the

central nervous system may be

accompanied by hypertrophy of

astrocytes. In some cases, these

astrocytic changes can be seen light

microscopically with

immunohistochemical stains for glial

fibrillary acidic protein (GFAP), the

major intermediate filament protein in

astrocytes. In addition, GFAP can be

quantified by an immunoassay, which

has been proposed as a marker of

astrocyte reactivity (O’Callaghan, 1988).

Immunohistochemical stains have the

advantage of better localization of GFAP

increases, whereas immunoassay

evaluations are superior at detecting and

quantifying changes in GFAP levels and

establishing dose-response

relationships. The ability to detect and

quantify changes in GFAP by

immunoassay is improved by dissecting

and analyzing multiple brain regions.

The interpretation of a chemicalinduced change in GFAP is facilitated

by corroborative data from the

neuropathology or neuroanatomy

evaluation. A number of chemicals

known to injure the central nervous

system, including trimethyltin,

methylmercury, cadmium, 3-

acetylpyridine, and

methylphenyltetrahydropyridine

(MPTP), have been shown to increase

levels of GFAP. Measures of GFAP are

now included as an optional test in the

Neurotoxicity Screening Battery (U.S.

EPA, 1991a).

Increases in GFAP above control

levels may be seen at dosages below

those necessary to produce damage seen

by standard microscopic or

histopathological techniques. Because

increases in GFAP reflect an astrocyte

response in adults, treatment-related

increases in GFAP are considered to be

evidence that a neurotoxic effect has

occurred. There is less agreement as to

how to interpret decreases in GFAP

relative to an appropriate control group.

The absence of a change in GFAP

following exposure does not mean that

the chemical is devoid of neurotoxic

potential. Known neurotoxicants such

as cholinesterase-inhibiting pesticides,

for example, would not be expected to

increase brain levels of GFAP.

Interpretation of GFAP changes prior to

weaning may be confounded by the

possibility that chemically induced

increases in GFAP could be masked by

changes in the concentration of this

protein associated with maturation of

the central nervous system, and these

data may be difficult to interpret.

3.1.2.4. Behavioral Endpoints of

Neurotoxicity

Behavior reflects the integration of the

various functional components of the

nervous system. Changes in behavior

can arise from a direct effect of a

toxicant on the nervous system, or

indirectly from its effects on other

physiological systems. Understanding

the interrelationship between systemic

toxicity and behavioral changes (e.g.,

the relationship between liver damage

and motor activity) is extremely

important. The presence of systemic

toxicity may complicate, but does not

preclude, interpretation of behavioral

changes as evidence of neurotoxicity. In

addition, a number of behaviors (e.g.,

schedule-controlled behavior) may

require a motivational component for

successful completion of the task. In

such cases, experimental paradigms

designed to assess the motivation of an

animal during behavior might be

necessary to interpret the meaning of

some chemical-induced changes in

behavior.

EPA’s testing guidelines developed

for the Toxic Substances Control Act

and the Federal Insecticide, Fungicide

and Rodenticide Act describe the use of

functional observational batteries (FOB),

motor activity, and schedule-controlled

behavior for assessing neurotoxic

potential (U.S. EPA, 1991a). Examples

of measures obtained in a typical FOB

are presented in Table 5. There are

many other measures of behavior,

including specialized tests of motor and

sensory function and of learning and

memory (Tilson, 1987; Anger, 1984).

TABLE 5.—EXAMPLES OF MEASURES

IN A REPRESENTATIVE FUNCTIONAL

OBSERVATIONAL BATTERY

Home cage

and open field Manipulative Physiological

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

Autonomic

signs.

Click response.Body weight.

Convulsions,

tremors.

Foot splay.

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

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

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

Rearing.

Stereotypy.

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

TABLE 6.—EXAMPLES OF SPECIALIZED BEHAVIORAL TESTS TO MEASURE NEUROTOXICITY

Function Procedure Representative agents

Motor Function

Weakness ......................................................... Grip strength, swimming endurance, suspension rod, discriminative motor function.n-Hexane, methyl.

n-Butylketone, carbaryl.

Incoordination .................................................... Rotorod, gait assessments, righting reflex ....... 3-Acetylpyridine, ethanol.

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

pyrethroids, DDT.

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

Sensory Function

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

Reflex modification.

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

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

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

Olfactory ............................................................ Discrimination conditioning ............................... 3-Methylindole, methylbromide.

Cognitive Function

Habituation ........................................................ Startle reflex ..................................................... Diisopropylfluorophosphate.

Pre/neonatal methylmercury.
26940 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

TABLE 6.—EXAMPLES OF SPECIALIZED BEHAVIORAL TESTS TO MEASURE NEUROTOXICITY—Continued

Function Procedure Representative agents

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

Conditioned flavor ............................................. Carbaryl.

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

Passive avoidance ............................................ Neonatal trimethyltin.

Olfactory conditioning.

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

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

Y-maze avoidance ............................................ Hypervitaminosis A.

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

Morris water maze ............................................ DFP.

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

Delayed matching to sample ............................ DFP.

Repeated acquisition ........................................ Carbaryl.

At the present time, there is no clear

consensus concerning the use of specific

behavioral tests to assess chemicalinduced sensory, motor, or cognitive

dysfunction in animal models. The risk

assessor should also know that the

literature is clear that a number of other

behaviors besides those listed in Tables

1, 5, and 6 could be affected by

chemical exposure. For example,

alterations in food and water intake,

reproduction, sleep, temperature

regulation, and circadian rhythmicity

are controlled by specific regions of the

brain, and chemical-induced alterations

in these behaviors could be indicative of

neurotoxicity. It is reasonable to assume

that an NOAEL or LOAEL could be

based on one or more of these

endpoints.

The following sections describe, in

general, behavioral tests and their uses

and offer guidance on interpreting data.

3.1.2.4.1. Functional Observational

Battery (FOB). An FOB is designed to

detect and quantify major overt

behavioral, physiological, and

neurological signs (Gad, 1982;

O’Donoghue, 1989; Moser, 1989). A

number of batteries have been

developed, each consisting of tests

generally intended to evaluate various

aspects of sensorimotor function (Tilson

and Moser, 1992). Many FOB tests are

essentially clinical neurological

examinations that rate the presence or

absence, and in many cases the severity,

of specific neurological signs. Some

FOBs in animals are similar to clinical

neurological examinations used with

human patients. Most FOBs have

several components or tests. A typical

FOB is summarized in Table 5 and

evaluates several functional domains,

including neuromuscular (i.e.,

weakness, incoordination, gait, and

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

and somatosensory), and autonomic

(i.e., pupil response and salivation)

function.

The relevance of statistically

significant test results from an FOB is

judged according to the number of signs

affected, the dose(s) at which effects are

observed, and the nature, severity, and

persistence of the effects and their

incidence in relation to control animals.

In general, if only a few unrelated

measures in the FOB are affected, or the

effects are unrelated to dose, the results

may not be considered evidence of a

neurotoxic effect. If several neurological

signs are affected, but only at the high

dose and in conjunction with other

overt signs of toxicity, including

systemic toxicity, large decreases in

body weight, decreases in body

temperature, or debilitation, there is less

persuasive evidence of a direct

neurotoxic effect. In cases where several

related measures in a battery of tests are

affected and the effects appear to be

dose dependent, the data are considered

to be evidence of a neurotoxic effect,

especially in the absence of systemic

toxicity. The risk assessor should be

aware of the potential for a number of

false positive statistical findings in these

studies because of the large number of

endpoints customarily included in the

FOB.

FOB data can be grouped into one or

more of several neurobiological

domains, including neuromuscular (i.e.,

weakness, incoordination, abnormal

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

visual, somatosensory), and autonomic

functions (Tilson and Moser, 1992).

This statistical technique may be useful

when separating changes that occur on

the basis of chance or in conjunction

with systemic toxicity from those

treatment-related changes indicative of

neurotoxic effects. In the case of the

developing organism, chemicals may

alter the maturation or appearance of

sensorimotor reflexes. Significant

alterations in or delay of such reflexes

is evidence of a neurotoxic effect.

Examples of chemicals that affect

neuromuscular function are

3-acetylpyridine, acrylamide, and

triethyltin. Organophosphate and

carbamate insecticides produce

autonomic dysfunction, while

organochlorine and pyrethroid

insecticides increase sensorimotor

sensitivity, produce tremors and, in

some cases, cause seizures and

convulsions (Spencer and Schaumburg,

1980).

3.1.2.4.2. Motor Activity. Motor

activity represents a broad class of

behaviors involving coordinated

participation of sensory, motor, and

integrative processes. Assessment of

motor activity is noninvasive and has

been used to evaluate the effects of

acute and repeated exposure to

neurotoxicants (MacPhail et al., 1989).

An organism’s level of activity can,

however, be affected by many different

types of environmental agents,

including non-neurotoxic agents. Motor

activity measurements also have been

used in humans to evaluate disease

states, including disorders of the

nervous system (Goldstein and Stein,

1985).

Motor activity is usually quantified as

the frequency of movements over a

period of time. The total counts

generated during a test period will

depend on the recording mechanism

and the size and configuration of the

testing apparatus. Effects of agents on

motor activity can be expressed as

absolute activity counts or as a

percentage of control values. In some

cases, a transformation (e.g., square root)

may be used to achieve a normal

distribution of the data. In these cases,

the transformed data and not raw data

should be used for risk assessment

purposes. The frequency of motor

activity within a session usually

decreases and is reported as the average

number of counts occurring in each

successive block of time. The EPA’s
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26941

Office of Prevention, Pesticides and

Toxic Substances guidelines (U.S. EPA,

1991a), for example, call for test

sessions of sufficient duration to allow

motor activity to approach steady-state

levels during the last 20 percent of the

session for control animals. A sum of

the counts in each epoch will add up to

the total number of counts per session.

Motor activity can be altered by a

number of experimental factors,

including neurotoxic chemicals.

Decreases in activity could occur

following high doses of non-neurotoxic

agents (Kotsonis and Klaassen, 1977;

Landauer et al., 1984). Examples of

neurotoxic agents that decrease motor

activity include many pesticides (e.g.,

carbamates, chlorinated hydrocarbons,

organophosphates, and pyrethroids),

heavy metals (lead, tin, and mercury),

and other agents (3-acetylpyridine,

acrylamide, and 2,4-dithiobiuret). Some

neurotoxicants (e.g., toluene, xylene,

triadimefon) produce transient increases

in activity by presumably stimulating

neurotransmitter release, while others

(e.g., trimethyltin) produce persistent

increases in motor activity by destroying

specific regions of the brain (e.g.,

hippocampus).

Following developmental exposures,

neurotoxic effects are often observed as

a change in the ontogenetic profile or

maturation of motor activity patterns.

Frequently, developmental exposure to

neurotoxic agents will produce an

increase in motor activity that persists

into adulthood or that results in changes

in other behaviors. This is evidence of

a neurotoxic effect. Like other organ

systems, the nervous system may be

differentially sensitive to toxicants in

groups such as the young. For example,

toxicants introduced to the developing

nervous system may kill stem cells and

thus cause profound effects on adult

structure and function. Moreover,

toxicants may have greater access to the

developing nervous system before the

blood-brain barrier is completely formed

or before metabolic detoxifying systems

are functional.

Motor activity measurements are

typically used with other tests (e.g.,

FOB) to help detect neurotoxic effects.

Agent-induced changes in motor

activity associated with other overt

signs of toxicity (e.g., loss of body

weight, systemic toxicity) or occurring

in non-dose-related fashion are of less

concern than changes that are dose

dependent, are related to structural or

other functional changes in the nervous

system, or occur in the absence of lifethreatening toxicity.

13.1.2.4.3. Schedule-Controlled

Operant Behavior. Schedule-controlled

operant behavior (SCOB) involves the

maintenance of behavior (e.g.,

performance of a lever-press or key-peck

response) by reinforcement. Different

rates and patterns of responding are

controlled by the relationship between

response and subsequent reinforcement.

SCOB provides a measure of

performance of a learned behavior (e.g.,

lever press or key peck) and involves

training and motivational variables that

should be considered in evaluating the

data. Agents may interact with sensory

processing, motor output, motivational

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

training history, and baseline

characteristics (Rice, 1988; CorySlechta, 1989). Qualitatively, rates and

patterns of SCOB display cross-species

generality, but the quantitative measures

of rate and pattern of performance can

vary within and between species.

In laboratory animals, SCOB has been

used to study a wide range of

neurotoxicants, including

methylmercury, many pesticides,

organic and inorganic lead, triethyltin,

and trimethyltin (MacPhail, 1985;

Tilson, 1987; Rice, 1988). The primary

SCOB endpoints for evaluation are

response rate and the temporal pattern

of responding. These endpoints may

vary as a function of the contingency

between responding and reinforcement

presentation (i.e., schedule of

reinforcement). Schedules of

reinforcement that have been used in

toxicology studies include fixed ratio

and fixed interval schedules. Fixed ratio

schedules engender high rates of

responding and a characteristic pause

after delivery of each reinforcement.

Fixed interval schedules engender a

relatively low rate of responding during

the initial portion of the interval and

progressively higher rates near the end

of the interval. For some schedules of

reinforcement, the temporal pattern of

responding may play a more important

role in defining the performance

characteristics than the rate of

responding. For other schedules, the

reverse may be true. For example, the

temporal pattern of responding may be

more important than rate of responding

for defining performance on a fixed

interval schedule. For a fixed ratio

schedule, more importance might be

placed on the rate of responding than on

the post-reinforcement pause.

The overall qualitative patterns are

important properties of the behavior.

Substantial qualitative changes in

operant performance, such as

elimination of characteristic response

patterns, can be evidence of an adverse

effect. Most chemicals, however, can

disrupt operant behavior at some dose,

and such adverse effects may be due

either to neurotoxic or non-neurotoxic

mechanisms. Unlike large qualitative

changes in operant performance, small

quantitative changes are not adverse.

Some changes may actually represent an

improvement, e.g., an increase in the

index of curvature with a decrease in

fixed interval rate of responding.

Assessing the toxicological importance

of these effects requires considerable

professional judgment and evaluation of

converging evidence from other types of

toxicological endpoints. While most

chemicals decrease the efficiency of

responding at some dose, some agents

may increase response efficiency on

schedules requiring high response rates

because of a stimulant effect or an

increase in central nervous system

excitability. Agent-induced changes in

responding between reinforcements

(i.e., the temporal pattern of responding)

may occur independently of changes in

the overall rate of responding.

Chemicals may also affect the reaction

time to respond following presentation

of a stimulus. Agent-induced changes in

response rate or temporal patterning

associated with other overt signs of

toxicity (e.g., body weight loss, systemic

toxicity, or occurring in a non-doserelated fashion) are of less concern than

changes that are dose dependent, related

to structural or other functional changes

in the nervous system, or occur in the

absence of life-threatening toxicity.

3.1.2.4.4. Convulsions. Observable

convulsions in animals are indicative of an

adverse effect. These events can reflect

central nervous system activity comparable

to that of epilepsy in humans and could be

defined as neurotoxicity. Occasionally, other

toxic actions of compounds, such as direct

effects on muscle, might mimic some

convulsionlike behaviors. In some cases,

convulsions or convulsionlike behaviors may

be observed in animals that are otherwise

severely compromised, moribund, or near

death. In such cases, convulsions might

reflect an indirect effect of systemic toxicity

and are less clearly indicative of

neurotoxicity. As discussed in the section on

neurophysiological measures, electrical

recordings of brain activity could be used to

determine specificity of effects on the

nervous system.

3.1.2.4.5. Specialized Tests for

Neurotoxicity. Several procedures have

been developed to measure agentinduced changes in specific

neurobehavioral functions such as

motor, sensory, or cognitive function

(Tilson, 1987; Cory-Slechta, 1989).

Table 6 lists several behavioral tests, the

neurobehavioral functions they were

designed to assess, and agents known to

affect the response. Many of these tests

in animals have been designed to assess

neural functions in humans using

similar testing procedures.

A statistically or biologically

significant chemically induced change
26942 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

in any measure in Table 6 may be

evidence of an adverse effect. However,

judgments of neurotoxicity may involve

not only the analysis of changes seen

but the structure and class of the

chemical and other available

neurochemical, neurophysiological, and

neuropathological evidence. In general,

behavioral changes seen across broader

dose ranges indicate more specific

actions on the systems underlying those

changes, i.e., the nervous system.

Changes that are not dose dependent or

that are confounded with body weight

changes and/or other systemic toxicity

may be more difficult to interpret as

neurotoxic effects.

3.1.2.4.5.1. Motor Function.

Neurotoxicants commonly affect motor

function. These effects can be

categorized generally into (1) weakness

or decreased strength, (2) tremor, (3)

incoordination, and (4) spasms,

myoclonia, or abnormal motor

movements (Tilson, 1987; Cory-Slechta,

1989). Specialized tests used to assess

strength include measures of grip

strength, swimming endurance,

suspension from a hanging rod, and

discriminative motor function. Rotorod

and gait assessments are used to

measure coordination, while rating

scales and spectral analysis techniques

can be used to quantify tremor and other

abnormal movements.

3.1.2.4.5.2. Sensory Function. Gross

perturbations of sensory function can be

observed in simple neurological

assessments such as the hot plate or tail

flick test. However, these tests may not

be sufficiently sensitive to detect subtle

sensory changes. Psychophysical

procedures that study the relationship

between a physical dimension (e.g.,

intensity, frequency) of a stimulus and

behavior may be necessary to quantify

agent-induced alterations in sensory

function. Examples of psychophysical

procedures include discriminated

conditioning and startle reflex

modification.

3.1.2.4.5.3. Cognitive Function.

Alterations in learning and memory in

experimental animals should be inferred

from changes in behavior following

exposure when compared with that seen

prior to exposure or with a nonexposed

control group. Learning is defined as a

relatively lasting change in behavior due

to experience, and memory is defined as

the persistence of a learned behavior

over time. Table 6 lists several examples

of learning and memory tests and

representative neurotoxicants known to

affect these tests. Measurement of

changes in learning and memory should

be separated from other changes in

behavior that do not involve cognitive

or associative processes (i.e., motor

function, sensory capabilities,

motivational factors). In addition, any

apparent toxicant-induced change in

learning or memory should ideally be

demonstrated over a range of stimulus

and response conditions and testing

conditions. In developmental exposures,

it should be shown that the animals

have matured enough to perform the

specified task. Developmental

neurotoxicants can accelerate or delay

the ability to learn a response or may

interfere with cognitive function at the

time of testing. Older animals frequently

perform poorly on some types of tests,

and it should be demonstrated that

control animals in this population are

capable of performing the procedure.

Neurotoxicants might accelerate agerelated dysfunction or alter motivational

variables that are important for learning

to occur. Further, it is not the case that

a decrease in responding on a learning

task is adverse while an increase in

performance on a learning task is not. It

is well known that lesions in certain

regions of the brain can facilitate the

acquisition of certain types of behaviors

by removing preexisting response

tendencies (e.g., inhibitory responses

due to stress) that moderate the rate of

learning under normal circumstances.

Apparent improvement in

performance is not either adverse or

beneficial until demonstrated to be so

by converging evidence with a variety of

experimental methods. Examples of

procedures to assess cognitive function

include simple habituation, classical

conditioning, and operant (or

instrumental) conditioning, including

tests for spatial learning and memory.

3.1.2.4.5.4. Developmental

Neurotoxicity. Although the previous

discussion of various neurotoxicity

endpoints and tests applies to studies in

which developmental exposures are

used, there are particular issues of

importance in the evaluation of

developmental neurotoxicity studies.

This section underscores the importance

of detecting neurotoxic effects following

developmental exposure because an

NRC (1993) report has indicated that

infants and children may be

differentially sensitive to environmental

chemicals such as pesticides. Exposure

to chemicals during development can

result in a spectrum of effects, including

death, structural abnormalities, altered

growth, and functional deficits (U.S.

EPA, 1991b). A number of agents have

been shown to cause developmental

neurotoxicity when exposure occurred

during the period between conception

and sexual maturity (e.g., Riley and

Vorhees, 1986; Vorhees, 1987).

Table 7 lists several examples of

agents known to produce developmental

neurotoxicity in experimental animals.

Animal models of developmental

neurotoxicity have been shown to be

sensitive to several environmental

agents known to produce developmental

neurotoxicity in humans, including

lead, ethanol, x-irradiation,

methylmercury, and polychlorinated

biphenyls (PCBs) (Kimmel et al., 1990;

Needleman, 1990; Jacobson et al., 1985;

Needleman, 1986). In many of these

cases, functional deficits are observed at

dose levels below those at which other

indicators of developmental toxicity are

evident or at minimally toxic doses in

adults. Such effects may be transient,

but generally are considered adverse.

Developmental exposure to a chemical

could result in transient or reversible

effects observed during early

development that could reemerge as the

individual ages (Barone et al., 1995).

TABLE 7.—EXAMPLES OF COMPOUNDS

OR TREATMENTS PRODUCING DEVELOPMENTAL NEUROTOXICITY

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

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

azacytidine.

Insecticides ................ DDT, chlordecone.

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

cadmium.

Polyhalogenated hydrocarbons.PCBs, PBBs.

Testing for developmental

neurotoxicity has not been required

routinely by regulatory agencies in the

United States, but is required by EPA

when other information indicates the

potential for developmental

neurotoxicity (U.S. EPA, 1986, 1988a,

1988b, 1989, 1991a, 1991b). Useful data

for decision making may be derived

from well-conducted adult

neurotoxicity studies, standard

developmental toxicity studies, and

multigeneration studies, although the

dose levels used in the latter may be

lower than those in studies with shorter

term exposure.

Important design issues to be

evaluated for developmental

neurotoxicity studies are similar to

those for standard developmental

toxicity studies (e.g., a dose-response

approach with the highest dose

producing minimal overt maternal or

perinatal toxicity, with number of litters

large enough for adequate statistical

power, with randomization of animals

to dose groups and test groups, with

litter generally considered as the

statistical unit). In addition, the use of

a replicate study design provides added

confidence in the interpretation of data.

A pharmacological/physiological

challenge may also be valuable in
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26943

evaluating neurological function and

‘‘unmasking’’ effects not otherwise

detectable. For example, a challenge

with a psychomotor stimulant such as

d-amphetamine may unmask latent

developmental neurotoxicity (Hughes

and Sparber, 1978; Adams and BuelkeSam, 1981; Buelke-Sam et al., 1985).

Direct extrapolation of developmental

neurotoxicity to humans is limited in

the same way as for other endpoints of

toxicity, i.e., by the lack of knowledge

about underlying toxicological

mechanisms and their significance (U.S.

EPA, 1991b). However, comparisons of

human and animal data for several

agents known to cause developmental

neurotoxicity in humans showed many

similarities in effects (Kimmel et al.,

1990). As evidenced primarily by

observations in laboratory animals,

comparisons at the level of functional

category (sensory, motivational,

cognitive, motor function, and social

behavior) showed close agreement

across species for the agents evaluated,

even though the specific endpoints used

to assess these functions varied

considerably across species (Stanton

and Spear, 1990). Thus, it can be

assumed that developmental

neurotoxicity effects in animal studies

indicate the potential for altered

neurobehavioral development in

humans, although the specific types of

developmental effects seen in

experimental animal studies will not be

the same as those that may be produced

in humans. Therefore, when data

suggesting adverse effects in

developmental neurotoxicity studies are

encountered for particular agents, they

should be considered in the risk

assessment process.

Functional tests with a moderate

degree of background variability (e.g., a

coefficient of variability of 20% or less)

may be more sensitive to the effects of

an agent on behavioral endpoints than

are tests with low variability that may

be impossible to disrupt without using

life-threatening doses. A battery of

functional tests, in contrast to a single

test, is usually needed to evaluate the

full complement of nervous system

functions in an animal. Likewise, a

series of tests conducted in animals in

several age groups may provide more

information about maturational changes

and their persistence than tests

conducted at a single age.

It is a well-established principle that

there are critical developmental periods

for the disruption of functional

competence, which include both the

prenatal and postnatal periods to the

time of sexual maturation, and the effect

of a toxicant is likely to vary depending

on the time and degree of exposure

(Rodier, 1978, 1990). It is also important

to consider the data from studies in

which postnatal exposure is included,

as there may be an interaction of the

agent with maternal behavior, milk

composition, or pup suckling behavior,

as well as possible direct exposure of

pups via dosed food or water (Kimmel

et al., 1992).

Agents that produce developmental

neurotoxicity at a dose that is not toxic

to the maternal animal are of special

concern. However, adverse

developmental effects are often

produced at doses that cause mild

maternal toxicity (e.g., 10%-20%

reduction in weight gain during

gestation and lactation). At doses

causing moderate maternal toxicity (i.e.,

20% or more reduction in weight gain

during gestation and lactation),

interpretation of developmental effects

may be confounded. Current

information is inadequate to assume

that developmental effects at doses

causing minimal maternal toxicity result

only from maternal toxicity; rather, it

may be that the mother and developing

organism are equally sensitive to that

dose level. Moreover, whether

developmental effects are secondary to

maternal toxicity or not, the maternal

effects may be reversible while the

effects on the offspring may be

permanent. These are important

considerations for agents to which

humans may be exposed at minimally

toxic levels either voluntarily or

involuntarily, because several agents

(e.g., alcohol) are known to produce

adverse developmental effects at

minimally toxic doses in adult humans

(Coles et al., 1991).

Although interpretation of

developmental neurotoxicity data may

be limited, it is clear that functional

effects should be evaluated in light of

other toxicity data, including other

forms of developmental toxicity (e.g.,

structural abnormalities, perinatal

death, and growth retardation). For

example, alterations in motor

performance may be due to a skeletal

malformation rather than nervous

system change. Changes in learning

tasks that require a visual cue might be

influenced by structural abnormalities

in the eye. The level of confidence that

an agent produces an adverse effect may

be as important as the type of change

seen, and confidence may be increased

by such factors as reproducibility of the

effect, either in another study of the

same function or by convergence of data

from tests that purport to measure

similar functions. A dose-response

relationship is an extremely important

measure of a chemical’s effect; in the

case of developmental neurotoxicity

both monotonic and biphasic doseresponse curves are likely, depending

on the function being tested. The EPA

Guidelines for Developmental Toxicity

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

be consulted for more information on

interpreting developmental toxicity

studies. The endpoints frequently used

to assess developmental neurotoxicity

in exposed children have been reviewed

by Winneke (1995).

3.1.3. Other Considerations

3.1.3.1. Pharmacokinetics

Extrapolation of test results between

species can be aided considerably by

data on the pharmacokinetics of a

particular agent in the species tested

and, if possible, in humans. Information

on a toxicant’s half-life, metabolism,

absorption, excretion, and distribution

to the peripheral and central nervous

system may be useful in predicting risk.

Of particular importance for the

pharmacokinetics of neurotoxicants is

the blood-brain barrier. The vast

majority of the central nervous system is

served by blood vessels with bloodbrain barrier properties, which exclude

most ionic and nonlipid-soluble

chemicals from the brain and spinal

cord. The brain contains several

structures called circumventricular

organs (CVOs) that are served by blood

vessels lacking blood-brain barrier

properties. Brain regions adjacent to

these CVOs are thus exposed to

relatively high levels of many

neurotoxicants. Pharmacokinetic data

may be helpful in defining the doseresponse curve, developing a more

accurate basis for comparing species

sensitivity (including that of humans),

determining dosimetry at sites, and

comparing pharmacokinetic profiles for

various dosing regimens or routes of

administration. The correlation of

pharmacokinetic parameters and

neurotoxicity data may be useful in

determining the contribution of specific

pharmacokinetic processes to the effects

observed.

3.1.3.2. Comparisons of Molecular

Structure

Comparisons of the chemical or

physical properties of an agent with

those of known neurotoxicants may

provide some indication of the potential

for neurotoxicity. Such information may

be helpful for evaluating potential

toxicity when only minimal data are

available. The structure-activity

relationships (SAR) of some chemical

classes have been studied, including

hexacarbons, organophosphates,

carbamates, and pyrethroids. Therefore,

class relationships or SAR may help
26944 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

predict neurotoxicity or interpret data

from neurotoxicological studies. Under

certain circumstances (e.g., in the case

of new chemicals), this procedure is one

of the primary methods used to evaluate

the potential for toxicity when little or

no empirical toxicity data are available.

It should be recognized, however, that

effects of chemicals in the same class

can vary widely. Moser (1995), for

example, reported that the behavioral

effects of prototypic cholinesteraseinhibiting pesticides differed

qualitatively in a battery of behavioral

tests.

3.1.3.3. Statistical Considerations

Properly designed studies on the

neurotoxic effects of compounds will

include appropriate statistical tests of

significance. In general, the likelihood

of obtaining a significant effect will

depend jointly on the magnitude of the

effect and the variability obtained in

control and treated groups. The risk

assessor should be aware that some

neurotoxicants may induce a greater

variability in biologic response, rather

than a clear shift in mean or other

parameters (Laties and Evans, 1980;

Glowa and MacPhail, 1995). A number

of texts are available on standard

statistical tests (e.g., Siegel, 1956; Winer,

1971; Sokal and Rohlf, 1969; Salsburg,

1986; Gad and Weil, 1988).

Neurotoxicity data present some

unique features that should be

considered in selecting statistical tests

for analysis. Data may involve several

different measurement scales, including

categorical (affected or not), rank (more

or less affected), and interval and ratio

scales of measurement (affected by some

percentage). For example, convulsions

are usually recorded as being present or

absent (categorical), whereas

neuropathological changes are

frequently described in terms of the

degree of damage (rank). Many tests of

neurotoxicity involve interval or ratio

measurements (e.g., frequency of

photocell interruptions or amplitude of

an evoked potential), which are the

most powerful and sensitive scales of

measurement. In addition,

measurements are frequently made

repeatedly in control and treated

subjects, especially in the case of

behavioral and neurophysiological

endpoints. For example, OPPTS

guidelines for FOB assessment call for

evaluations before exposure and at

several times during exposure in a

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

Descriptive data (categorical) and rank

order data can be analyzed using

standard nonparametric techniques

(Siegel, 1956). In some cases, if it is

determined that the data fit the linear

model, the categorical modeling

procedure can be used for weighted

least-squares estimation of parameters

for a wide range of general linear

models, including repeated-measures

analyses. The weighted least-squares

approach to categorical and rank data

allows computation of statistics for

testing the significance of sources of

variation as reflected by the model. In

the case of studies assessing effects in

the same animals at several time points,

univariate analyses can be carried out at

each time point when the overall dose

effect or the dose-by-time interaction is

significant.

Continuous data (e.g., magnitude,

rate, amplitude), if found to be normally

distributed, can be analyzed with

general linear models using a grouping

factor of dose and, if necessary, repeated

measures across time (Winer, 1971).

Univariate analyses of dose, comparing

dose groups to the control group at each

time point, can be performed when

there is a significant overall dose effect

or a dose-by-time interaction. Post hoc

comparisons between control and

treatment groups can be made following

tests for overall significance. In the case

of multiple endpoints within a series of

evaluations, some type of correction for

multiple observations is warranted

(Winer, 1971).

3.1.3.4. In Vitro Data in Neurotoxicology

Methods and procedures that fall

under the general heading of short-term

tests include an array of in vitro tests

that have been proposed as alternatives

to whole-animal tests (Goldberg and

Frazier, 1989). In vitro approaches use

animal or human cells, tissues, or

organs and maintain them in a nutritive

medium. Various types of in vitro

techniques, including primary cell

cultures, cell lines, and cloned cells,

produce data for evaluating potential

and known neurotoxic substances.

While such procedures are important in

studying the mechanism of action of

toxic agents, their use in hazard

identification in human health risk

assessment has not been explored to any

great extent.

Data from in vitro procedures are

generally based on simplified

approaches that require less time to

yield information than do many in vivo

techniques. However, in vitro methods

generally do not take into account the

distribution of the toxicant in the body,

the route of administration, or the

metabolism of the substance. It also is

difficult to extrapolate in vitro data to

animal or human neurotoxicity

endpoints, which include behavioral

changes, motor disorders, sensory and

perceptual disorders, lack of

coordination, and learning deficits. In

addition, data from in vitro tests cannot

duplicate the complex neuronal

circuitry characteristic of the intact

animal.

Many in vitro systems are now being

evaluated for their ability to predict the

neurotoxicity of various agents seen in

intact animals. This validation process

requires considerations in study design,

including defined endpoints of toxicity

and an understanding of how a test

agent would be handled in vitro as

compared to the intact organism.

Demonstrated neurotoxicity in vitro in

the absence of in vivo data is suggestive

but inadequate evidence of a neurotoxic

effect. In vivo data supported by in vitro

data enhance the reliability of the in

vivo results.

3.1.3.5. Neuroendocrine Effects

Neuroendocrine dysfunction may

occur because of a disturbance in the

regulation and modulation of

neuroendocrine feedback systems. One

major indicator of neuroendocrine

function is secretion of hormones from

the pituitary. Hypothalamic control of

anterior pituitary secretions is also

involved in a number of important

bodily functions. Many types of

behaviors (e.g., reproductive behaviors,

sexually dimorphic behaviors in

animals) are dependent on the integrity

of the hypothalamic-pituitary system,

which could represent a potential site of

neurotoxicity. Pituitary secretions arise

from a number of different cell types in

this gland, and neurotoxicants could

affect these cells directly or indirectly.

Morphological changes in cells

mediating neuroendocrine secretions

could be associated with adverse effects

on the pituitary or hypothalamus and

could ultimately affect behavior and the

functioning of the nervous system.

Biochemical changes in the

hypothalamus may also be used as

indicators of potential adverse effects on

neuroendocrine function. Finally, the

development of the nervous system is

intimately associated with the presence

of circulating hormones such as thyroid

hormone (Porterfield, 1994). The nature

of the nervous system deficit, which

could include cognitive dysfunction,

altered neurological development, or

visual deficits, depends on the severity

of the thyroid disturbance and the

specific developmental period when

exposure to the chemical occurred.

3.2. Dose-Response Evaluation

Dose-response evaluation is a critical

part of the qualitative characterization

of a chemical’s potential to produce

neurotoxicity and involves the

description of the dose-response
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26945

relationship in the available data.

Human studies covering a range of

exposures are rarely available, and

therefore animal data are typically used

for estimating exposure levels likely to

produce adverse effects in humans.

Evidence for a dose-response

relationship is an important criterion in

establishing a neurotoxic effect,

although this analysis may be limited

when based on standard studies using

three dose groups or fewer. The

evaluation of dose-response

relationships includes identifying

effective dose levels as well as doses

associated with no increase in adverse

effects when compared with controls.

The lack of a dose-response relationship

in the data may suggest that the effect

is not related to the putative neurotoxic

effect or that the study was not

appropriately controlled. Much of the

focus is on identifying the critical

effect(s) observed at the LOAEL and the

NOAEL associated with that effect. The

NOAEL is defined as the highest dose at

which there is no statistically or

biologically significant increase in the

frequency of an adverse neurotoxic

effect when compared with the

appropriate control group in a database

characterized as having sufficient

evidence for use in a risk assessment

(see section 3.3). The risk assessor

should be aware of possible problems

associated with estimating a NOAEL in

studies involving a small number of test

subjects and that have a poor doseresponse relationship.

In addition to identifying the NOAEL/

LOAEL or BMD, the dose-response

evaluation defines the range of doses

that are neurotoxic for a given agent,

species, route of exposure, and duration

of exposure. In addition to these

considerations, pharmacokinetic factors

and other aspects that might influence

comparisons with human exposure

scenarios should be taken into account.

For example, dose-response curves may

exhibit not only monotonic but also Ushaped 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 doseresponse curve, it should also be

recognized that neurotoxic effects vary

in terms of nature and severity across

dose or exposure level. At high levels of

exposure, frank lesions accompanied by

severe functional impairment may be

observed. Such effects are widely

accepted as adverse. At progressively

lower levels of exposure, however, the

lesions may become less severe and the

impairments less obvious. At levels of

exposure near the NOAEL and LOAEL,

the effects will often be mild, possibly

reversible, and inconsistently found. In

addition, the endpoints showing

responses may be at levels of

organization below the whole organism

(e.g., neurochemical or

electrophysiological endpoints). The

adversity of such effects can be disputed

(e.g., cholinesterase inhibition), yet it is

such effects that are likely to be the

focus of risk assessment decisions. To

the extent possible, this document

provides guidance on determining the

adversity of neurotoxic effects.

However, the identification of a critical

adverse effect often requires

considerable professional judgment and

should consider factors such as the

biological plausibility of the effect, the

evidence of a dose-effect continuum,

and the likelihood for progression of the

effect with continued exposure.

3.3. Characterization of the HealthRelated Database

This section describes a scheme for

characterizing the sufficiency of

evidence for neurotoxic effects. This

scheme defines two broad categories:

sufficient and insufficient (Table 8).

Categorization is aimed at providing

certain criteria for the Agency to use to

define the minimum evidence necessary

to define hazards and to conduct doseresponse analyses. It does not address

the issues related to characterization of

risk, which requires analysis of

potential human exposures and their

relation to potential hazards in order to

estimate the risks of those hazards from

anticipated or estimated exposures.

Several examples using a weight-ofevidence approach similar to that

described in these Guidelines have been

described elsewhere (Tilson et al., 1995;

Tilson et al., 1996).

TABLE 8.—CHARACTERIZATION OF THE HEALTH-RELATED DATABASE

Sufficient evidence ............. The sufficient evidence category includes data that collectively provide enough information to judge whether or not

a human neurotoxic hazard could exist. This category may include both human and experimental animal evidence.

Sufficient human evidence This category includes agents for which there is sufficient evidence from epidemiologic studies, e.g., case control

and cohort studies, to judge that some neurotoxic effect is associated with exposure. A case series in conjunction with other supporting evidence may also be judged ‘‘sufficient evidence.’’ Epidemiologic and clinical case

studies should discuss whether the observed effects can be considered biologically plausible in relation to

chemical exposure. (Historically, often much has been made of the notion of causality in epidemiologic studies.

Causality is a more stringent criterion than association and has become a topic of scientific and philosophical

debate. See Susser [1986], for example, for a discussion of inference in epidemiology.)

Sufficient experimental animal evidence/limited

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 adverse neurotoxic effect in a single appropriate, well-executed study in a single experimental animal species. The minimum evidence needed to judge

that a potential hazard does not exist would include data from an appropriate number of endpoints from more

than one study and two species showing no adverse neurotoxic effects at doses that were minimally toxic in

terms of producing an adverse effect. Information on pharmacokinetics, mechanisms, or known properties of the

chemical class may also strengthen the evidence.
26946 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

TABLE 8.—CHARACTERIZATION OF THE HEALTH-RELATED DATABASE—Continued

Insufficient evidence ........... This category includes agents for which there is less than the minimum evidence sufficient for identifying whether

or not a neurotoxic hazard exists, such as agents for which there are no data on neurotoxicity or agents with

databases from studies in animals or humans that are limited by study design or conduct (e.g., inadequate conduct or report of clinical signs). Many general toxicity studies, for example, are considered insufficient in terms of

the conduct of clinical neurobehavioral observations or the number of samples taken for histopathology of the

nervous system. Thus, a battery of negative toxicity studies with these shortcomings would be regarded as providing insufficient evidence of the lack of a neurotoxic effect of the test material. Further, most screening studies

based on simple observations involving autonomic and motor function provide insufficient evaluation of many

sensory or cognitive functions. Data, which by itself would likely fall in this category, would also include information on SAR or data from in vitro tests. Although such information would be insufficient by itself to proceed further in the assessment it could be used to support the need for additional testing.

Data from all potentially relevant

studies, whether indicative of potential

hazard or not, should be included in

this characterization. The primary

sources of data are human studies and

case reports, experimental animal

studies, other supporting data, and in

vitro and/or SAR data. Because a

complex interrelationship exists among

study design, statistical analysis, and

biological significance of the data, a

great deal of scientific judgment, based

on experience with neurotoxicity data

and with the principles of study design

and statistical analysis, is required to

adequately evaluate the database on

neurotoxicity. In many cases,

interaction with scientists in specific

disciplines either within or outside the

field of neurotoxicology (e.g.,

epidemiology, statistics) may be

appropriate.

The adverse nature of different

neurotoxicity endpoints may be a

complex judgment. In general, most

neuropathological and many

neurobehavioral changes are regarded as

adverse. However, there are adverse

behavioral effects that may not reflect a

direct action on the nervous system.

Neurochemical and electrophysiological

changes may be regarded as adverse

because of their known or presumed

relation to neuropathological and/or

neurobehavioral consequences. In the

absence of supportive information, a

professional judgment should be made

regarding the adversity of such

outcomes, considering factors such as

the nature, magnitude, and duration of

the effects reported. Thus, correlated

measures of neurotoxicity strengthen

the evidence for a hazard. Correlations

between functional and morphological

effects, such as the correlation between

leg weakness and paralysis and

peripheral nerve damage from exposure

to tri-ortho-cresyl phosphate, are the

most common and striking examples of

this form of validity. Correlations

support a coherent and logical link

between behavioral effects and

biochemical mechanisms. Replication of

a finding also strengthens the evidence

for a hazard. Some neurotoxicants cause

similar effects across most species.

Many chemicals shown to produce

neurotoxicity in laboratory animals have

similar effects in humans. Some

neurological effects may be considered

adverse even if they are small in

magnitude, reversible, or the result of

indirect mechanisms.

Because of the inherent difficulty in

‘‘proving any negative,’’ it is more

difficult to document a finding of no

apparent adverse effect than a finding of

an adverse effect. Neurotoxic effects

(and most kinds of toxicity) can be

observed at many different levels, so

only a single endpoint needs to be

found to demonstrate a hazard, but

many endpoints need to be examined to

demonstrate no effect. For example, to

judge that a hazard for neurotoxicity

could exist for a given agent, the

minimum evidence sufficient would be

data on a single adverse endpoint from

a well-conducted study. In contrast, to

judge that an agent is unlikely to pose

a hazard for neurotoxicity, the

minimum evidence would include data

from a host of endpoints that revealed

no neurotoxic effects. This may include

human data from appropriate studies

that could support a conclusion of no

evidence of a neurotoxic effect. With

respect to clinical signs and symptoms,

human exposures can reveal far more

about the absence of effects than animal

studies, which are confined to the signs

examined.

In some cases, it may be that no

individual study is judged sufficient to

establish a hazard, but the total

available data may support such a

conclusion. Pharmacokinetic data and

structure-activity considerations, data

from other toxicity studies, or other

factors may affect the strength of the

evidence in these situations. For

example, given that gamma diketones

are known to cause motor system

neurotoxicity, a marginal data set on a

candidate gamma diketone, e.g., 1/10

animals affected, might be more likely

to be judged sufficient than equivalent

data from a member of a chemical class

about which nothing is known.

A judgment that the toxicology

database is sufficient to indicate a

potential neurotoxic hazard is not the

end of analysis. The circumstances of

expression of the hazard are essential to

describing human hazard potential.

Thus, reporting should contain the

details of the circumstances under

which effects have been observed, e.g.,

‘‘long-term oral exposures of adult

rodents to compound X at levels of

roughly 1 mg/kg have been associated

with ataxia and peripheral nerve

damage.’’

4. Quantitative Dose-Response Analysis

This section describes several

approaches (including the LOAEL/

NOAEL and BMD) for determining the

reference dose (RfD) or reference

concentration (RfC). The NOAEL or

BMD/uncertainty factor approach

results in an RfD or RfC, which is an

estimate (with uncertainty spanning

perhaps an order of magnitude) of a

daily exposure to the human population

(including sensitive subgroups) that is

likely to be without an appreciable risk

of deleterious effects during a lifetime.

The dose-response analysis

characterization should:

• Describe how the RfD/RfC was

calculated;

• Discuss the confidence in the

estimates;

• Describe the assumptions or

uncertainty factors used; and

• Discuss the route and level of

exposure observed, as compared to

expected human exposures.

4.1. LOAEL/NOAEL and BMD

Determination

As indicated earlier, the LOAEL and

NOAEL are determined for endpoints

that are seen at the lowest dose level

(so-called critical effect). Several

limitations in the use of the NOAEL

have been identified and described (e.g.,

Barnes and Dourson, 1988; Crump,

1984). For example, the NOAEL is

derived from a single endpoint from a

single study (the critical study) and
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26947

ignores both the slope of the doseresponse function and baseline

variability in the endpoint of concern.

Because the baseline variability is not

taken into account, the NOAEL from a

study using small group sizes may be

higher than the NOAEL from a similar

study in the same species that uses

larger group sizes. The NOAEL is also

directly dependent on the dose spacing

used in the study. Finally, and perhaps

most importantly, use of the NOAEL

does not allow estimates of risk or

extrapolation of risk to lower dose

levels. Because of these and other

limitations in the NOAEL approach, it

has been proposed that mathematical

curve-fitting techniques (Crump, 1984;

Gaylor and Slikker, 1990; Glowa, 1991;

Glowa and MacPhail, 1995; U.S. EPA,

1995a) be compared with the NOAEL

procedure in calculating the RfD or RfC.

These techniques typically apply a

mathematical function that describes

the dose-response relationship and then

interpolate to a level of exposure

associated with a small increase in

effect over that occurring in the control

group or under baseline conditions. The

BMD has been defined as a lower

confidence limit on the effective dose

associated with some defined level of

effect, e.g., a 5% or 10% increase in

response. These guidelines suggest that

the use of the BMD should be explored

in specific situations. The Agency is

currently developing guidelines for the

use of the BMD in risk assessment.

Many neurotoxic endpoints provide

continuous measures of response, such

as response speed, nerve conduction

velocity, IQ score, degree of enzyme

inhibition, or the accuracy of task

performance. Although it is possible to

impose a dichotomy on a continuous

effects distribution and to classify some

level of response as ‘‘affected’’ and the

remainder as ‘‘unaffected,’’ it may be

very difficult and inappropriate to

establish such clear distinctions,

because such a dichotomy would

misrepresent the true nature of the

neurotoxic response. The risk assessor

should be aware of the importance of

trying to reconcile findings from several

studies that seem to report widely

divergent results. Alternatively,

quantitative models designed to analyze

continuous effect variables may be

preferable. Other techniques that allow

this approach, with transformation of

the information into estimates of the

incidence or frequency of affected

individuals in a population, have been

proposed (Crump, 1984; Gaylor and

Slikker, 1990; Glowa and MacPhail,

1995). Categorical regression analysis

has been proposed because it can

evaluate different types of data and

derive estimates for short-term

exposures (Rees and Hattis, 1994).

Decisions about the most appropriate

approach require professional judgment,

taking into account the biological nature

of the continuous effect variable and its

distribution in the population under

study.

Although dose-response functions in

neurotoxicology are generally linear or

monotonic, curvilinear functions,

especially U-shaped or inverted Ushaped curves, have been reported as

noted earlier (section 3.2). Doseresponse analyses should consider the

uncertainty that U-shaped doseresponse functions might contribute to

the estimate of the NOAEL/LOAEL or

BMD. Typically, estimates of the

NOAEL/LOAEL are taken from the

lowest part of the dose-response curve

associated with impaired function or

adverse effect.

4.2. Determination of the Reference

Dose or Reference Concentration

Since the availability of dose-response

data in humans is limited, extrapolation

of data from animals to humans usually

involves the application of uncertainty

factors to the NOAEL/LOAEL or BMD.

The NOAEL or BMD/uncertainty factor

approach results in an RfD or RfC,

which is an estimate (with uncertainty

spanning perhaps an order of

magnitude) of a daily exposure to the

human population (including sensitive

subgroups) that is likely to be without

an appreciable risk of deleterious effects

during a lifetime. The oral RfD and

inhalation RfC are applicable to chronic

exposure situations and are based on an

evaluation of all the noncancer health

effects, including neurotoxicity data.

RfDs and RfCs in the Integrated Risk

Information System (IRIS–2) database

for several agents are based on

neurotoxicity endpoints and include a

few cases in which the RfD or RfC is

calculated using the BMD approach

(e.g., methylmercury, carbon disulfide).

The size of the final uncertainty factor

used will vary from agent to agent and

will require the exercise of scientific

judgment, taking into account

interspecies differences, the shape of the

dose-response curve, and the

neurotoxicity endpoints observed.

Uncertainty factors are typically

multiples of 10 and are used to

compensate for human variability in

sensitivity, the need to extrapolate from

animals to humans, and the need to

extrapolate from less than lifetime (e.g.,

subchronic) to lifetime exposures. An

additional factor of up to 10 may be

included when only a LOAEL (and not

a NOAEL) is available from a study, or

depending on the completeness of the

database, a modifying factor of up to 10

may be applied, depending on the

confidence one has in the database.

Uncertainty factors of less than 10 can

be used, depending upon the

availability of relevant information.

Barnes and Dourson (1988) provide a

more complete description of the

calculation, use, and significance of

RfDs in setting exposure limits to toxic

agents by the oral route. Jarabek et al.

(1990) provide a more complete

description of the calculation, use, and

significance of RfCs in setting exposure

limits to toxic agents in air.

Neurotoxicity can result from acute,

shorter term exposures, and it may be

appropriate in some cases, e.g., for air

pollutants or water contaminants, to set

shorter term exposure limits for

neurotoxicity as well as for other

noncancer health effects.

5. Exposure Assessment

Exposure assessment describes the

magnitude, duration, frequency, and

routes of exposure to the agent of

interest. This information may come

from hypothetical values, models, or

actual experimental values, including

ambient environmental sampling

results. Guidelines for exposure

assessment have been published

separately (U.S. EPA, 1992) and will,

therefore, be discussed only briefly here.

The exposure assessment should

include an exposure characterization

that:

• Provides a statement of the purpose,

scope, level of detail, and approach

used in the exposure assessment;

• Presents the estimates of exposure

and dose by pathway and route for

individuals, population segments, and

populations in a manner appropriate for

the intended risk characterization;

• Provides an evaluation of the

overall level of confidence in the

estimate of exposure and dose and the

conclusions drawn; and

• Communicates the results of the

exposure assessment to the risk

assessor, who can then use the exposure

characterization, along with the hazard

and dose/response characterizations, to

develop a risk characterization.

A number of considerations are

relevant to exposure assessment for

neurotoxicants. An appropriate

evaluation of exposure should consider

the potential for exposure via ingestion,

inhalation, and dermal penetration from

relevant sources of exposure, including

multiple avenues of intake from the

same source.

In addition, neurotoxic effects may

result from short-term (acute), highconcentration exposures as well as from
26948 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

longer term (subchronic), lower level

exposures. Neurotoxic effects may occur

after a period of time following initial

exposure or be obfuscated by repair

mechanisms or apparent tolerance. The

type and severity of effect may depend

significantly on the pattern of exposure

rather than on the average dose over a

long period of time. For this reason,

exposure assessments for neurotoxicants

may be much more complicated than

those for long-latency effects such as

carcinogenicity. It is rare for sufficient

data to be available to construct such

patterns of exposure or dose, and

professional judgment may be necessary

to evaluate exposure to neurotoxic

agents.

6. Risk Characterization

6.1. Overview

Risk characterization is the

summarization step of the risk

assessment process and consists of an

integrative analysis and a summary. The

integrative analysis (a) involves

integration of the toxicity information

from the hazard characterization and

dose-response analysis with the human

exposure estimates, (b) provides an

evaluation of the overall quality of the

assessment and the degree of confidence

in the estimates of risk and conclusions

drawn, and  describes risk in terms of

the nature and extent of harm. The risk

characterization summary

communicates the results of the risk

assessment to the risk manager in a

complete, informative, and useful

format.

This summary should include, but is

not limited to, a discussion of the

following elements:

• Quality of and confidence in the

available data;

• Uncertainty analysis;

• Justification of defaults or

assumptions;

• Related research recommendations;

• Contentious issues and extent of

scientific consensus;

• Effect of reasonable alternative

assumptions on conclusions and

estimates;

• Highlights of reasonable plausible

ranges;

• Reasonable alternative models; and

• Perspectives through analogy.

The risk manager can then use the

derived risk to make public health

decisions.

An effective risk characterization

should fully, openly, and clearly

characterize risks and disclose the

scientific analyses, uncertainties,

assumptions, and science policies that

underlie decisions throughout the risk

assessment and risk management

processes. The risk characterization

should feature values such as

transparency in the decision-making

process; clarity in communicating with

the scientific community and the public

regarding environmental risk and the

uncertainties associated with

assessments of environmental risk; and

consistency across program offices in

core assumptions and science policies,

which are well grounded in science and

reasonable. The following sections

describe these four aspects of the risk

characterization in more detail.

6.2. Integration of Hazard

Characterization, Dose-Response

Analysis, and Exposure Assessment

In developing the hazard

characterization, dose-response

analysis, and exposure portions of the

risk assessment, the risk assessor should

take into account many judgments

concerning human relevance of the

toxicity data, including the

appropriateness of the various animal

models for which data are available and

the route, timing, and duration of

exposure relative to expected human

exposure. These judgments should be

summarized at each stage of the risk

assessment process (e.g., the biological

relevance of anatomical variations may

be established in the hazard

characterization process, or the

influence of species differences in

metabolic patterns in the dose-response

analysis). In integrating the information

from the assessment, the risk assessor

should determine if some of these

judgments have implications for other

portions of the assessment and whether

the various components of the

assessment are compatible.

The risk characterization should not

only examine the judgments but also

explain the constraints of available data

and the state of knowledge about the

phenomena studied in making them,

including (1) the qualitative conclusions

about the likelihood that the chemical

may pose a specific hazard to human

health, the nature of the observed

effects, under what conditions (route,

dose levels, time, and duration) of

exposure these effects occur, and

whether the health-related data are

sufficient to use in a risk assessment; (2)

a discussion of the dose-response

characteristics of the critical effects,

data such as the shapes and slopes of

the dose-response curves for the various

endpoints, the rationale behind the

determination of the NOAEL and

LOAEL and calculation of the

benchmark dose, and the assumptions

underlying the estimation of the RfD or

RfC; and (3) the estimates of the

magnitude of human exposure; the

route, duration, and pattern of the

exposure; relevant pharmacokinetics;

and the number and characteristics of

the population(s) exposed.

If data to be used in a risk

characterization are from a route of

exposure other than the expected

human exposure, then pharmacokinetic

data should be used, if available, to

make extrapolations across routes of

exposure. If such data are not available,

the Agency makes certain assumptions

concerning the amount of absorption

likely or the applicability of the data

from one route to another (U.S. EPA,

1992).

The level of confidence in the hazard

characterization should be stated to the

extent possible, including the

appropriate category regarding

sufficiency of the health-related data. A

comprehensive risk assessment ideally

includes information on a variety of

endpoints that provide insight into the

full spectrum of potential

neurotoxicological responses. A profile

that integrates both human and test

species data and incorporates a broad

range of potential adverse neurotoxic

effects provides more confidence in a

risk assessment for a given agent.

The ability to describe the nature of

the potential human exposure is

important in order to predict when

certain outcomes can be anticipated and

the likelihood of permanence or

reversibility of the effect. An important

part of this effort is a description of the

nature of the exposed population and

the potential for sensitive, highly

susceptible, or highly exposed

populations. For example, the

consequences of exposure to the

developing individual versus the adult

can differ markedly and can influence

whether the effects are transient or

permanent. Other considerations

relative to human exposures might

include the likelihood of exposures to

other agents, concurrent disease, and

nutritional status.

The presentation of the integrated

results of the assessment should draw

from and highlight key points of the

individual characterizations of

component analyses performed under

these Guidelines. The overall risk

characterization represents the

integration of these component

characterizations. If relevant risk

assessments on the agent or an

analogous agent have been done by EPA

or other Federal agencies, these should

be described and the similarities and

differences discussed.
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26949

6.3. Quality of the Database and Degree

of Confidence in the Assessment

The risk characterization should

summarize the kinds of data brought

together in the analysis and the

reasoning on which the assessment is

based. The description should convey

the major strengths and weaknesses of

the assessment that arise from

availability of data and the current

limits of our understanding of the

mechanisms of toxicity.

A health risk assessment is only as

good as its component parts, i.e., hazard

characterization, dose-response

analysis, and exposure assessment.

Confidence in the results of a risk

assessment is thus a function of

confidence in the results of the analysis

of these elements. Each of these

elements should have its own

characterization as a part of the

assessment. Within each

characterization, the important

uncertainties of the analysis and

interpretation of data should be

explained, and the risk manager should

be given a clear picture of consensus or

lack of consensus that exists about

significant aspects of the assessment.

Whenever more than one view is

supported by the data and choosing

between them is difficult, all views

should be presented. If one has been

selected over the others, the rationale

should be given; if not, then all should

be presented as plausible alternative

results.

6.4. Descriptors of Neurotoxicity Risk

There are a number of ways to

describe risks. Several relevant ways for

neurotoxicity are as follows:

6.4.1. Estimation of the Number of

Individuals

The RfD or RfC is taken to be a

chronic exposure level at or below

which no significant risk occurs.

Therefore, presentation of the

population in terms of those at or below

the RfD or RfC (‘‘not at risk’’) and above

the RfD or RfC (‘‘may be at risk’’) may

be useful information for risk managers.

This method is particularly useful to a

risk manager considering possible

actions to ameliorate risk for a

population. If the number of persons in

the at-risk category can be estimated,

then the number of persons removed

from the at-risk category after a

contemplated action is taken can be

used as an indication of the efficacy of

the action.

6.4.2. Presentation of Specific Scenarios

Presenting specific scenarios in the

form of ‘‘what if?’’ questions is

particularly useful to give perspective to

the risk manager, especially where

criteria, tolerance limits, or media

quality limits are being set. The

question being asked in these cases is,

at this proposed exposure limit, what

would be the resulting risk for

neurotoxicity above the RfD or RfC?

6.4.3. Risk Characterization for Highly

Exposed Individuals

This measure is one example of the

just-discussed descriptor. This measure

describes the magnitude of concern at

the upper end of the exposure

distribution. This allows risk managers

to evaluate whether certain individuals

are at disproportionately high or

unacceptably high risk.

The objective of looking at the upper

end of the exposure distribution is to

derive a realistic estimate of a relatively

highly exposed individual or

individuals. This measure could be

addressed by identifying a specified

upper percentile of exposure in the

population and/or by estimating the

exposure of the highest exposed

individual(s). Whenever possible, it is

important to express the number of

individuals who comprise the selected

highly exposed group and discuss the

potential for exposure at still higher

levels.

If population data are absent, it will

often be possible to describe a scenario

representing high-end exposures using

upper percentile or judgment-based

values for exposure variables. In these

instances caution should be used in

order not to compound a substantial

number of high-end values for variables

if a ‘‘reasonable’’ exposure estimate is to

be achieved.

6.4.4. Risk Characterization for Highly

Sensitive or Susceptible Individuals

This measure identifies populations

sensitive or susceptible to the effect of

concern. Sensitive or susceptible

individuals are those within the

exposed population at increased risk of

expressing the toxic effect. All stages of

nervous system maturation might be

considered highly sensitive or

susceptible, but certain subpopulations

can sometimes be identified because of

critical periods for exposure, for

example, pregnant or lactating women,

infants, or children. The aged

population is considered to be at

particular risk because of the limited

ability of the nervous system to

regenerate or compensate to neurotoxic

insult.

In general, not enough is understood

about the mechanisms of toxicity to

identify sensitive subgroups for all

agents, although factors such as

nutrition (e.g., vitamin B), personal

habits (e.g., smoking, alcohol

consumption, illicit drug abuse), or

preexisting disease (e.g., diabetes,

neurological diseases, sexually

transmitted diseases, polymorphisms for

certain metabolic enzymes) may

predispose some individuals to be more

sensitive to the neurotoxic effects of

specific agents. Gender-related

differences in response to

neurotoxicants have been noted, but

these appear to be related to genderdependent toxicodynamic or

toxicokinetic factors.

In general, it is assumed that an

uncertainty factor of 10 for

intrapopulation variability will be able

to accommodate differences in

sensitivity among various

subpopulations, including children and

the elderly. However, in cases where it

can be demonstrated that a factor of 10

does not afford adequate protection,

another uncertainty factor may be

considered in conducting the risk

assessment.

6.4.5. Other Risk Descriptors

In risk characterization, dose-response

information and the human exposure

estimates may be combined either by

comparing the RfD or RfC and the

human exposure estimate or by

calculating the margin of exposure

(MOE). The MOE is the ratio of the

NOAEL from the most appropriate or

sensitive species to the estimated

human exposure level. If a NOAEL is

not available, a LOAEL may be used in

calculating the MOE. Alternatively, a

benchmark dose may be compared with

the estimated human exposure level to

obtain the MOE. Considerations for the

evaluation of the MOE are similar to

those for the uncertainty factor applied

to the LOAEL/NOAEL or the benchmark

dose. The MOE is presented along with

a discussion of the adequacy of the

database, including the nature and

quality of the hazard and exposure data,

the number of species affected, and the

dose-response information.

The RfD or RfC comparison with the

human exposure estimate and the

calculation of the MOE are conceptually

similar but are used in different

regulatory situations. The choice of

approach depends on several factors,

including the statute involved, the

situation being addressed, the database

used, and the needs of the decision

maker. The RfD or RfC and the MOE are

considered along with other risk

assessment and risk management issues

in making risk management decisions,

but the scientific issues that should be

taken into account in establishing them

have been addressed here.
26950 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

If the MOE is equal to or more than

the uncertainty factor multiplied by any

modifying factor used as a basis for an

RfD or RfC, then the need for regulatory

concern is likely to be small. Although

these methods of describing risk do not

actually estimate risks per se, they give

the risk manager some sense of how

close the exposures are to levels of

concern.

6.5. Communicating Results

Once the risk characterization is

completed, the focus turns to

communicating results to the risk

manager. The risk manager uses the

results of the risk characterization along

with other technological, social, and

economic considerations in reaching a

regulatory decision. Because of the way

in which these risk management factors

may affect different cases, consistent but

not necessarily identical risk

management decisions should be made

on a case-by-case basis. These

Guidelines are not intended to give

guidance on the nonscientific aspects of

risk management decisions.

6.6. Summary and Research Needs

These Guidelines summarize the

procedures that the U.S. Environmental

Protection Agency would use in

evaluating the potential for agents to

cause neurotoxicity. These Guidelines

discuss the general default assumptions

that should be made in risk assessment

for neurotoxicity because of gaps in our

knowledge about underlying biological

processes and how these compare across

species. Research to improve the risk

assessment process is needed in a

number of areas. For example, research

is needed to delineate the mechanisms

of neurotoxicity and pathogenesis,

provide comparative pharmacokinetic

data, examine the validity of short-term

in vivo and in vitro tests, elucidate the

functional modalities that may be

altered, develop improved animal

models to examine the neurotoxic

effects of exposure during the premating

and early postmating periods and in

neonates, further evaluate the

relationship between maternal and

developmental toxicity, provide insight

into the concept of threshold, develop

approaches for improved mathematical

modeling of neurotoxic effects, improve

animal models for examining the effects

of agents given by various routes of

exposure, determine the effects of

recurrent exposures over prolonged

periods of time, and address the

synergistic or antagonistic effects of

mixed exposures and neurotoxic

response. Such research will aid in the

evaluation and interpretation of data on

neurotoxicity and should provide

methods to assess risk more precisely.

Additional research is needed to

determine the most appropriate doseresponse approach to be used in

neurotoxicity risk assessments.

REFERENCES

Adams, J; Buelke-Sam, J. (1981) Behavioral

testing of the postnatal animal: testing and

methods development. In: Kimmel, CA;

Buelke-Sam, J, eds. Developmental

toxicology. New York: Raven Press, pp. 233–

238.

Albert, A. (1973) Selective toxicity: the

physico-chemical basis of therapy. New

York: Wiley, pp. 173–211.

Anger, WK. (1984) Neurobehavioral testing

of chemicals: impact on recommended

standards. Neurobehav Toxicol Teratol

6:147–153.

Anger, WK. (1986) Workplace exposures.

In: Annau, ZA, ed., Neurobehavioral

toxicology. Baltimore: Johns Hopkins

University Press, pp. 331–347.

Anger, WK. (1990) Worksite behavioral

research: results, sensitive methods, test

batteries, and the transition from laboratory

data to human health. Neurotoxicology

11:627–718.

Anger, K; Johnson, BL. (1985) Chemicals

affecting behavior. In: O’Donoghue, J, ed.

Neurotoxicity of industrial and commercial

chemicals. Boca Raton, FL: CRC Press.

Anger, WK; Otto, DA; Letz, R. (Eds) (1996)

Symposium on computerized behavioral

testing of humans in neurotoxicology

research. Neurotoxicol Teratol 18:347–518.

Baker, ER; Letz, R; Fidler, A; Shalat, S;

Plantamura, D; Lyndon, M. (1985) Computerbased neurobehavioral testing for

occupational and environmental

epidemiology: Methodology and validation

studies. Neurobehav Teratol 7:369–377.

Barone, S; Stanton, ME; Mundy, WR.

(1995) Neurotoxic effects of neonatal triethyl

tin (TET) exposure are exacerbated with

aging. Neurobiol Aging 16:723–735.

Barnes, DG; Dourson, M. (1988) Reference

dose (RfD): description and use in health risk

assessments. Regulat Toxicol Pharmacol

8:471–486.

Benignus, VA. (1993) Importance of

experimenter-blind procedure in

neurotoxicology. Neurotoxicol Teratol 15:45–

49.

Bondy, SC. (1986) The biochemical

evaluation of neurotoxic damage. Fundam

Appl Toxicol 6:208–216.

Boyes, WK. (1992) Testing visual system

toxicity using visual evoked potential

technology. In: Isaacson, RL; Jensen, KF, eds.,

The vulnerable brain and environmental

risks, Vol. 1: Malnutrition and hazard

assessment. New York: Plenum, pp. 193–222.

Boyes, WK. (1993) Sensory-evoked

potentials: measures of neurotoxicity. In:

Erinoff, L, ed. Assessing the toxicity of drugs

of abuse. NIDA Research Monograph 136.

National Institute on Drug Abuse, Alcohol,

Drug Abuse and Mental Health

Administration, U.S. Department of Health

and Human Services, pp. 63–100.

Buelke-Sam, J; Kimmel, CA; Adams, J.

(1985) Design considerations in screening for

behavioral teratogens: results of the

collaborative teratology study. Neurobehav

Toxicol Teratol 7:537–589.

Bushnell, P; Padilla, S; Ward, T; Pope, C;

Olszyk, V. (1991) Behavioral and

neurochemical changes in rats dosed

repeatedly with diisopropylfluorophosphate

(DFP). J Pharmacol Exp Therap 256:741–750.

Callender, TJ; Morrow, L; Subramanian, K.

(1994) Evaluation of chronic neurological

sequelae after acute pesticide exposure using

SPECT brain scans. J Toxicol Environ Health

41:275–284.

Carson, BL; Stockton, RA; Wilkinson, RR.

(1987) Organomercury, lead, tin compounds

in the environment and the potential for

human exposure. In: Tilson, HA; Sparber, SB,

eds. Neurotoxicants and neurobiological

function: Effects of organoheavy metals. New

York: J. Wiley, pp. 1–80.

Coles, CD; Brown, RT; Smith, IE; Platzman,

KA; Erickson, S; Falek, A. (1991) Effects of

prenatal alcohol exposure at school age I.

Physical and cognitive development.

Neurotoxicol Teratol 13:357–367.

Cory-Slechta, DA. (1989) Behavioral

measures of neurotoxicity. Neurotoxicology

10:271–296.

Costa, LG. (1988) Interactions of

neurotoxicants with neurotransmitter

systems. Toxicology 49:359–366.

Crump, KS. (1984) A new method for

determining allowable daily intakes. Fundam

Appl Toxicol 4:854–871.

Davis, JM; Svendsgaard, DJ. (1990) Ushaped dose-response curves: their

occurrence and implication for risk

assessment. J Toxicol Environ Health 30:71–

83.

Dyer, RS. (1985) The use of sensory evoked

potentials in toxicology. Fundam Appl

Toxicol 5:24–40.

Dyer, RS. (1987) Macrophysiological

assessment of organometal neurotoxicity. In:

Tilson, HA; Sparber, SB, eds. Neurotoxicants

and neurobiological function effects of

organoheavy metals. New York: J. Wiley, pp.

137–184.

Eccles, CU. (1988) EEG correlates of

neurotoxicity. Neurotoxicol Teratol 10:423–

428.

Ecobichon, DJ; Joy, RM. (1982) Pesticides

and neurological diseases. Boca Raton, FL:

CRC Press, pp. 151–203.

Ecobichon, DJ; Davies, JE; Doull, J; Ehrich,

M; Joy, R; McMillan, D; MacPhail, R; Reiter,

LW; Slikker, W, Jr.; Tilson, H. (1990)

Neurotoxic effects of pesticides. In: Baker,

SR; Wilkinson, CF, eds. The effect of

pesticides on human health. Princeton, NJ:

Princeton Scientific Publishing Co., Inc., pp.

131–199.

Ehrich, M; Jortner, BS; Padilla, S. (1995)

Comparison of the relative inhibition of

acetylcholinesterase and neuropathy esterase

in rats and hens given cholinesterase

inhibitors. Fundam Appl Toxicol 24:94–101.

Eldefrawi, AT; Jett, D; Eldefrawi, ME.

(1992) Direct actions of organophosphorus

anticholinesterases on muscarinic receptors.

In: Chambers, JE; Levi, PE, eds.

Organophosphates: chemistry, fate and

effects. New York, Academic Press, pp. 257–

270.

Friedlander, BR; Hearn, HT. (1980)

Epidemiologic considerations in studying

neurotoxic disease. In: Spencer, PS;
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26951

Schaumberg, HH, eds. Experimental and

clinical neurotoxicology. Baltimore: Williams

and Wilkins, pp. 650–662.

Gad, SC. (1982) A neuromuscular screen

for use in industrial toxicology. J Toxicol

Environ Health 9:691–704.

Gad, S; Weil, CS. (1988) Statistics and

experimental design for toxicologists, 2nd ed.

Caldwell, NJ: Telford Press.

Gaylor, DW; Slikker, W. (1990) Risk

assessment for neurotoxic effects.

Neurotoxicology 11:211–218.

Glowa, JR. (1991) Dose-effect approaches to

risk assessment. Neurosci Biobehav Rev

15:153–158.

Glowa, JR; MacPhail, RC. (1995)

Qualitative approaches to risk assessment in

neurotoxicology. In: Chang, L; Slikker, W,

eds. Neurotoxicology: approaches and

methods. New York, Academic Press, pp

777–787.

Goldberg, AM; Frazier, JM. (1989)

Alternatives to animals in toxicity testing. Sci

Am 261:24–30.

Goldstein, MK; Stein, GH. (1985)

Ambulatory activity in chronic disease. In:

Tryon, WH, ed. Behavioral assessment in

behavioral medicine. New York: Springer

Publishing Co., pp. 160–162.

Greenfield, SA; Chubb, IW; Grunewald,

RA; Henderson, Z; May, J; Protnoy, S;

Weston, J; Wright, MC. (1984) A noncholinergic function for acetylcholinesterase

in substantia nigra: behavioral evidence. Exp

Brain Res 54:513–520.

Griffin, JW. (1990) Basic pathologic

processes in the nervous system. Toxicol

Pathol 18:83–88.

Hayes, WJ. (1982) Pesticides studied in

man. Baltimore: Williams and Wilkins.

Hughes, JA; Sparber, SB. (1978) dAmphetamine unmasks postnatal

consequences of exposure to methylmercury

in utero: methods for studying behavioral

teratogenesis. Pharmacol Biochem Behav

8:365–375.

Jacobson, JL; Jacobson, SW. (1996)

Prospective, longitudinal assessment of

developmental neurotoxicity. Environ Health

Perspect 104: 275–283.

Jacobson, SW; Fein, GG; Jacobson, JL;

Schwartz, PM; Dowler, JK. (1985) The effect

of intrauterine PCB exposure on visual

recognition memory. Child Dev 56:853–860.

Jarabek, AM; Menache, MG; Overton, JH;

Dourson, ML; Miller, FJ. (1990) The U.S.

Environmental Protection Agency’s

inhalation RfD methodology: risk assessment

for air toxics. Toxicol Ind Health 6:279–301.

Johnson, MK. (1990) Organophosphates

and delayed neuropathy: Is NTE alive and

well? Toxicol Appl Pharmacol 102:385–399.

Kimmel, CA; Rees, DC; Francis, EZ, eds.

(1990) Qualitative and quantitative

comparability of human and animal

developmental neurotoxicity. Neurotoxicol

Teratol 12:175–292.

Kimmel, CA; Kavlock, RJ; Francis, EZ.

(1992) Animal models for assessing

developmental toxicity. In: Guzelian, PS;

Henry, CJ; Olin, SS. eds. Similarities and

differences between children and adults:

implications for risk assessment.

Washington, DC: ILSI Press, pp. 43–65.

Kotsonis, FN; Klaassen, CD. (1977)

Toxicity and distributions of cadmium

administered to rats at sublethal doses.

Toxicol Appl Pharmacol 41:667–680.

Krinke, GJ. (1989) Neuropathologic

screening in rodent and other species. J Am

Coll Toxicol 8:141–155.

Landauer, MR; Tomlinson, NT; Balster, RL;

MacPhail, RC. (1984) Some effects of the

formamidine pesticide chlordimeform on the

behavior of mice. Neurotoxicology 5:91–100.

Last, JM. (1986) Epidemiology and health

information. In: Last, JM, ed. Public health

and prevention medicine. New York:

Appleton-Century-Crofts.

Laties, VG; Evans, HL. (1980)

Methylmercury-induced changes in operant

discrimination by the pigeon. J Pharmacol

Exp Therap 214:620–628.

MacPhail, RC. (1985) Effects of pesticides

on schedule-controlled behavior. In: Seiden,

LS; Balster, RL, eds. Behavioral

pharmacology: the current status. New York:

A.R. Liss, pp. 519–535.

MacPhail, RC; Peele, DB; Crofton, KM.

(1989) Motor activity and screening for

neurotoxicity. J Am Coll Toxicol 8:117–125.

Mailman, RB. (1987) Mechanisms of CNS

injury in behavioral dysfunction.

Neurotoxicol Teratol 9:417–426.

Mattsson, JL; Albee, RR. (1992) Sensory

evoked potentials in neurotoxicology.

Neurotoxicol Teratol 10:435–443.

Mattsson, JL; Boyes, WK; Ross, JF. (1992)

Incorporating evoked potentials into

neurotoxicity test schemes. In: Tilson, HA;

Mitchell, CL, eds. Target organ toxicology

series: neurotoxicology. New York: Raven

Press Ltd., pp. 125–145.

Mausner, JS; Kramer, S. (1985)

Epidemiology: an introductory text, 2nd ed.

Philadelphia: WB Saunders.

Maxwell, DM; Lenz, DE; Groff, WA;

Kaminskis, A; Froehlich, HL. (1987) The

effects of blood flow and detoxification on in

vivo cholinesterase inhibition by Soman in

rats. Toxicol Appl Pharmacol 88:66–76.

Morell, P; Mailman, RB. (1987) Selective

and nonselective effects of organometals on

brain neurochemistry. In: Tilson, HA;

Sparber, SB, eds. Neurotoxicants and

neurobiological function: effects of

organoheavy metals. New York: Wiley, pp.

201–230.

Nagymajtenyi, L; Desi, I; Lorencz, R. (1988)

Neurophysiological markers as early signs or

organophosphate neurotoxicity. Neurotoxicol

Teratol 10:429–434.

Moser, VC. (1989) Screening approaches to

neurotoxicity: a functional observational

battery. J Am Coll Toxicol 8:85–93.

Moser, VC. (1995) Comparisons of the

acute effects of cholinesterase inhibitors

using a neurobehavioral screening battery in

rats. Neurotoxicol Teratol 17:617–625.

National Research Council (NRC). (1983)

Risk assessment in the federal government.

Managing the process. Washington, DC:

National Academy of Sciences.

National Research Council (NRC). (1984)

Toxicity testing: strategies to determine

needs and priorities. Washington, DC:

National Academy of Sciences.

National Research Council (NRC). (1986)

Drinking water and health. Washington, DC:

National Academy of Sciences, pp. 173–211.

National Research Council (NRC). (1994)

Science and judgment in risk assessment.

Washington, DC: National Academy of

Sciences.

National Research Council (1993).

Pesticides in the diets of infants and

children. Washington, DC, National Academy

of Sciences.

National Resources Defense Council

(NRDC). (1989) Intolerable risk: pesticides in

our children’s food. New York: Natural

Resources Defense Council.

Needleman, H. (1986) Epidemiological

studies. In: Annau, ZA, ed. Neurobehavioral

toxicology. Baltimore: Johns Hopkins

University Press, pp. 279–287.

Needleman, HL. (1990) Lessons from the

history of childhood plumbism for pediatric

neurotoxicology. In: Johnson, BL; Anger, WK;

Durao, A; Xintaris, C, eds. Advances in

neurobehavioral toxicology: application in

environmental and occupational health.

Chelsea, MI: Lewis Publishers, Inc., pp. 331–

337.

O’Callaghan, JP. (1988) Neurotypic and

gliotypic proteins as biochemical markers of

neurotoxicity. Neurotoxicol Teratol 10:445–

452.

O’Donoghue, JL. (1989) Screening for

neurotoxicity using a neurologically based

examination and neuropathology. J Am Coll

Toxicol 8:97–115.

Office of Technology Assessment (OTA).

(1990) Neurotoxicity: identifying and

controlling poisons of the nervous system.

U.S. Congress, Office of Technology

Assessment (OTA–BA–436). Washington,

DC: U.S. Government Printing Office.

Omerand, IE; Harding, AE; Miller, DH;

Johnson, G; MacManus, D; duBoulay, EPGH;

Kendall, BE; Moseley, IF; McDonald, WI.

(1994) Magnetic resonance imaging in

degenerative atoxic disorders. J Neurol

Neurosurg Psychiatry 57:51–57.

Otto, DA. (1992) Assessment of

neurobehavioral response in humans to a low

level volatile organic compound (VOC)

source. Ann. NY Acad Sci 641–248–260.

Padilla, S; Wilson, VZ; Bushnell, PJ. (1994)

Studies on the correlation between blood

cholinesterase inhibition and ‘‘tissue’’

inhibition in pesticide-treated rats.

Toxicology 92:11–25.

Pope, CN; Chakraborti, TK; Chapman, ML;

Farrar, JD. (1992) Long-term neurochemical

and behavioral effects induced by acute

chlorpyrifos treatment. Pharmacol Biochem

Behav 42:251–256.

Porterfield, S. (1994) Vulnerability of the

developing brain to thyroid abnormalities:

environmental insults to the thyroid system.

Environ Health Perspect 102:125–130.

Rebert, CS. (1983) Multisensory evoked

potentials in experimental and applied

neurotoxicology. Neurobehav Toxicol Teratol

5:659–671.

Reiter, LW. (1987) Neurotoxicology in

regulation and risk assessment. Dev

Pharmacol Ther 10:354–368.

Rees, DC; Hattis, D. (1994) Developing

quantitative strategies for animal to human

extrapolation. In: Hayes, AW, ed. Principles

and methods of toxicology, 3rd ed. New

York: Raven Press, pp. 275–315.

Reuhl, KR. (1991) Delayed expression of

neurotoxicity: the problem of silent damage.

Neurotoxicology 12:341–346.

Rice, DC. (1988) Quantification of operant

behavior. Toxicol Lett 43:361–379.
26952 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

Riley, EP; Vorhees, CV, eds. (1986)

Handbook of behavioral teratology. New

York: Plenum Press.

Richardson, RJ. (1995) Assessment of the

neurotoxic potential of chlorpyrifos relative

to other organophosphorus compounds: a

critical review of the literature. J Toxicol

Environ Health 44: 135–165.

Rodier, PM. (1978) Behavioral teratology.

In: Wilson, JG; Fraser, FC, eds. Handbook of

teratology, vol. 4. New York: Plenum Press,

pp. 397–428.

Rodier, P. (1990) Developmental

neurotoxicology. Toxicol Pathol 18:89–95.

Salsburg, DS. (1986) Statistics for

toxicologists. New York: Marcel Dekker, Inc.

Sette, WF. (1987) Complexity of

neurotoxicological assessment. Neurotoxicol

Teratol 9:411–416.

Sette, WF; MacPhail, RC. (1992)

Qualitative and quantitative issues in

assessment of neurotoxic effects. In: Tilson,

H; Mitchell, C, eds. Target organ toxicity

series: Neurotoxicology, 2nd ed. New York:

Raven Press, pp. 345–361.

Siegel, S. (1956) Nonparametric statistics

for the behavioral sciences. New York:

McGraw-Hill.

Silbergeld, EK. (1987) Neurochemical

approaches to developing markers of

neurotoxicity: Review of current status and

evaluation of future prospects. Environ Res

63:274–286.

Silbergeld, EK; Percival, RV. (1987) The

organometals: impact of accidental exposure

and experimental data on regulatory policies.

In: Tilson, HA; Sparber, SB, eds.

Neurotoxicants and neurobiological function:

effects of organoheavy metals. New York: J.

Wiley, pp. 328–352.

Small, DH. (1990) Non-cholinergic actions

of acetylcholinesterases: proteases regulating

all growth and development? Trends Biol Sci

15:213–216.

Sokal, RR; Rohlf, FJ. (1969) Biometry. San

Francisco: W.H. Freeman and Company.

Spencer, S; Schaumburg, HH. (1980)

Experimental and clinical neurotoxicology.

Baltimore: Williams and Wilkins.

Stanton, ME; Spear, LP. (1990) Workshop

on the qualitative and quantitative

comparability of human and animal

developmental neurotoxicity. Workgroup I

report: Comparability of measures of

developmental neurotoxicity in humans and

laboratory animals. Neurotoxicol Teratol

12:261–267.

Steenland, K; Jenkins, B; Ames, RG;

O’Malley, M; Chrislip, D; Russo, J. (1994)

Chronic neurological sequelae to

organophosphate pesticide poisoning. Am J

Public Health 84:731–736.

Stephens, R; Sprugeon, A; Calvert, IA;

Beach, J; Levy, LS; Berry, H; Harrington, JM.

(1995) Neurophysiological effects of longterm exposure to organophosphates in sheep

dip. The Lancet 345:1135–1139.

Sterman, AB; Schaumberg, HH. (1980) The

neurological examination. In: Spencer, PS;

Schaumberg, HH, eds. Experimental and

clinical neurotoxicology. Baltimore: Williams

and Wilkins, pp. 675–680.

Susser, M. (1986) Rules of inference in

epidemiology. Regulat Toxicol Pharmacol

6:116–128.

Tandon, P; Padilla, S; Barone, S; Pope, CN;

Tilson, HA. (1994) Fenthion produces a

persistent decrease in muscarinic receptor

function in the adult rat retina. Toxicol Appl

Pharmacol 125:271–280.

Tilson, HA. (1987) Behavioral indices of

neurotoxicity: what can be measured?

Neurotoxicol Teratol 9:427–443.

Tilson, HA. (1990) Neurotoxicology in the

1990s. Neurotoxicol Teratol 12:293–300.

Tilson, HA; Moser, VC. (1992) Comparison

of screening approaches. Neurotoxicology

13:1–14.

Tilson, HA; Mitchell, CL. (1983)

Neurotoxicants and adaptive responses of the

nervous system. Fed Proc 42:3189–3190.

Tilson, HA; MacPhail, RC; Crofton, KC.

(1995) Defining neurotoxicity in a decisionmaking context. Neurotoxicology 16:363–

375.

Tilson, HA; MacPhail, RC; Crofton,

KC. (1996) Setting exposure standards: a

decision process. Environ Health

Perspect 104:401–405.

U.S. Environmental Protection

Agency. (1986) Triethylene glycol

monomethyl, monoethyl, and

monobutyl ethers; proposed test rule.

Federal Register 51:17883–17894.

U.S. Environmental Protection

Agency. (1987, May 20) Toxic

Substances Control Act testing

guidelines. 50 FR 39397, September 27,

1985, as amended. 40 CFR 798.6050.

Federal Register 52:19082.

U.S. Environmental Protection

Agency. (1988a) Diethylene glycol butyl

ether and diethylene glycol butyl ether

acetate; final test rule. Federal Register

53:5932–5953.

U.S. Environmental Protection

Agency. (1988b) Proposed guidelines for

assessing male reproductive risk.

Federal Register 53:24850–24869.

U.S. Environmental Protection

Agency. (1989) FIFRA accelerated

reregistration phase 3 technical

guidance, Appendix D. Office of

Prevention, Pesticides and Toxic

Substances, Washington, DC. EPA No.

540/09–90–078. Available from: NTIS,

Springfield, VA. PB–90–161530.

U.S. Environmental Protection

Agency. (1991a) Pesticide assessment

guidelines, subdivision F. Hazard

evaluation: human and domestic

animals. Addendum 10: Neurotoxicity,

series 81, 82, and 83. Office of

Prevention, Pesticides and Toxic

Substances, Washington, DC. EPA 540/

09–91–123. Available from: NTIS,

Springfield, VA. PB91–154617.

U.S. Environmental Protection

Agency. (1991b) Guidelines for

developmental toxicity risk assessment.

Federal Register 56:63798–63826.

U.S. Environmental Protection

Agency. (1992) Guidelines for exposure

assessment. Federal Register 57:22888–

22938.

U.S. Environmental Protection

Agency. (1994) Final report: principles

of neurotoxicology risk assessment.

Federal Register 59:42360–42404.

U.S. Environmental Protection

Agency. (1995a) The use of the

benchmark dose approach in health risk

assessment. Office of Research and

Development, Washington, DC. EPA/

630/R–94/007.

U.S. Environmental Protection

Agency. (1995b) Policy for Risk

Characterization. Office of the

Administrator, Washington, DC.

U.S. Environmental Protection

Agency. (1995c) Guidance for Risk

Characterization. Science Policy

Council, Washington, DC.

U.S. Environmental Protection

Agency. (1996) Guidelines for

Reproductive Toxicity Risk Assessment.

Federal Register 61:56274–56322.

Valciukas, JA. (1991) Foundations of

environmental and occupational

neurotoxicology. New York: Van

Nostrand Reinhold.

Vorhees, CV. (1987) Reliability,

sensitivity and validity of indices of

neurotoxicity. Neurotoxicol Teratol

9:445–464.

Winer, BJ. (1971) Statistical principles

in experimental design. New York:

McGraw-Hill.

Winneke, G. (1995) Endpoints of

developmental neurotoxicity in

environmentally exposed children.

Toxicol Lett 77:127–136.

World Health Organization. (1986)

Principles and methods for the

assessment of neurotoxicity associated

with exposure to chemicals. In:

Environmental Health Criteria

Document 60. Geneva: World Health

Organization.

Part B: Response to Science Advisory

Board and Public Comments

1. Introduction

A notice of availability for public

comments of these Guidelines was

published in the Federal Register in

October 1995. Twenty-five responses

were received. These Guidelines were

presented to the Environmental Health

Committee of the Science Advisory

Board (SAB) on July 18, 1996. The

report of the SAB was provided to the

Agency in April 1997. The SAB and

public comments were diverse and

represented varying perspectives. Many

of the comments were favorable and

expressed agreement with positions

taken in the proposed Guidelines. Some

comments addressed items that were

more pertinent to testing guidance than

risk assessment guidance or were

otherwise beyond the scope of these

Guidelines. Some of the comments

concerned generic points that were not

specific to neurotoxicity issues. Others
Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices 26953

addressed topics that have not been

developed sufficiently and should be

viewed as research issues. There were

conflicting views about the need to

provide additional detailed guidance

about decision making in the evaluation

process as opposed to promoting

extensive use of scientific judgment.

Many public comments provided

specific suggestions for clarification of

details and corrections of factual

material in the Guidelines.

2. Response to Science Advisory Board

Comments

The SAB found the Guidelines

‘‘* * * to be quite successful, and, all

things considered, well suited to its

intended task.’’ However,

recommendations were made to

improve specific areas.

The SAB recommended that EPA

keep hazard identification as an

identifiable qualitative step in the risk

assessment process and that steps

should be taken to decouple the

qualitative step of hazard identification

from the more quantitatively rigorous

steps of exposure evaluation and doseresponse assessment. These Guidelines

now include a hazard characterization

step that clearly describes a qualitative

evaluation of hazard within the context

of the dose, route, timing and duration

of exposure. This step is clearly

differentiated from the quantitative

dose-response analysis, which describes

approaches for determining an RfD or

RfC.

The SAB supported the presumption

that what appears to be reversible

neurotoxicity, especially when arising

from gestational or neonatal exposure

and observed before adulthood, should

not be dismissed as of little practical

consequence. They may be indices of

silent toxicity that emerge later in life or

may suggest more robust and enduring

responses in aged individuals. These

Guidelines explain the concept of

functional reserve and advise caution in

instances where reversibility is seen and

in cases where exposure to a chemical

may result in delayed-onset

neurotoxicity. These Guidelines also

indicate that reversibility may vary with

the region of the nervous system

damaged, the neurotoxic agent involved,

and organismic factors such as age.

The SAB restated previous positions

concerning cholinesterase-inhibiting

chemicals. Agent-induced clinical signs

of cholinergic dysfunction could be

used to evaluate dose-response and

dose-effect relationships and define the

presence and absence of given effects in

risk assessment. The SAB also indicated

that inhibition of RBC and plasma

cholinesterase activity could serve as a

biomarker of exposure to cholinesteraseinhibiting agents and thereby

corroborate observations concerning the

presence of clinical effects associated

with cholinesterase inhibition. The SAB

also indicated that reduced brain

cholinesterase activity should be

assessed in the context of the biological

consequences of the reduction. These

Guidelines indicate that inhibition of

cholinesterase in the nervous system

reduces the organism’s level of

‘‘reserve’’ cholinesterase and, therefore,

limits the subsequent ability to respond

successfully to additional exposures and

that prolonged inhibition could lead to

adverse functional changes associated

with compensatory neurochemical

mechanisms. In general, an attempt was

made to coordinate these Guidelines

with the views of a recently convened

Scientific Advisory Panel regarding the

risk assessment of cholinesteraseinhibiting pesticides (Office of Pesticide

Programs, Science Policy on the Use of

Cholinesterase Inhibition for Risk

Assessments of Organophosphate and

Carbamate Pesticides, 1997).

The SAB indicated that the

Guidelines were inclusive of the major

neurotoxicity endpoints of concern. No

additional neurochemical,

neurophysiological, or structural

endpoints were suggested. Comments

indicated that there was no need to

consider endocrine disruptors

differently from other potential

neurotoxic agents.

The SAB found that the descriptions

of the endpoints used in human and

animal neurotoxicological assessments

were thorough and well documented.

Several sections, particularly

concerning some of the neurochemical

and neurobehavioral measures, were

corrected for factual errors or supported

with more detailed descriptions.

The SAB recommended that the use

of the threshold assumption should

occur after an evaluation of likely

biological mechanisms and available

data to provide evidence that linear

responses would be expected. A strict

threshold is not always clear in the

human population because of the wide

variation in background levels for some

functions. Cumulative

neurotoxicological effects might also

alter the response of some individuals

within a special population, which

might allow the Agency to characterize

the risk to the sensitive population.

Although the SAB did not disagree with

the Guidelines’ assumption of a

threshold as a default for neurotoxic

effects, it was suggested that the term

‘‘nonlinear dose-response curve for most

neurotoxicants’’ be substituted for the

term ‘‘threshold.’’ The Neurotoxicity

Risk Assessment Guidelines have been

amended to harmonize their treatment

of the issue of threshold with the

presentation and position taken with

other guidelines.

The SAB also recommended that the

topic of susceptible populations be

expanded to include the elderly and

other groups. The elderly could be at

increased risk of toxic effects for a

number of reasons, including a decline

in the reserve capacity with aging,

changes in the ability to detoxify or

excrete xenobiotics with age, and the

potential to interact with medicines or

other compounds that could synergize

interactions with toxic chemicals. The

SAB also indicated that other

populations should be considered,

including those with chronic and

debilitating conditions, groups of

workers with potential exposure to

chemicals that may be neurotoxic,

individuals with genetic

polymorphisms that could affect

responsiveness to certain

neurotoxicants, and individuals that

may experience differential exposure

because of their proximity to chemicals

in the environment or diet. The

Guidelines have been modified to

emphasize the possible presence of all

of these susceptible populations. When

specific information on differential risk

is not available, the Agency will

continue to apply a default uncertainty

factor to account for potential

differences in susceptibility.

The SAB recommended that the

benchmark dose (BMD) was not ready

for immediate incorporation into

adjustment-factor-based safety

assessment or to serve as a substitute or

replacement for the more familiar

NOAEL or LOAEL. The SAB also

recommended that research and

development on the BMD should be

aggressively encouraged and actively

supported. The BMD could be a

replacement for the NOAEL or LOAEL

after the appropriate research has been

conducted.

3. Response to Public Comments

In addition to numerous supportive

statements, several issues were

indicated, although each issue was

raised by only a few commentators. The

public comment supported the SAB

recommendation that there was no clear

consensus concerning replacing the

NOAEL approach with the BMD to

calculate RfDs and RfCs for

neurotoxicity endpoints. There was also

support for ensuring that dose-response

and other experimental design

information be considered in

interpreting the results of hazard

identification studies before proceeding
26954 Federal Register / Vol. 63, No. 93 /Thursday, May 14, 1998 / Notices

to quantitative dose-response analysis.

Public comment also supported the

position that reversibility cannot be

ignored in neurotoxicity risk assessment

and that the risk assessor should exert

caution in interpreting reversible effects,

especially where an apparent transient

effect is cited to support evidence for

relatively benign effects. The public

comment also supported the use of

clinical signs in the risk assessment of

cholinesterase-inhibiting compounds

and the finding that inhibition of brain

cholinesterase was an adverse effect.

The Guidelines emphasize the

importance of brain cholinesterase

inhibition, particularly in cases of

repeated exposure. The public comment

agreed with the SAB that RBC and

plasma cholinesterase activity are

biomarkers of exposure. It was

recommended that the Guidelines

incorporate additional information

addressing the neuroendocrine system

as a potential target site, and a section

has been added that defines the

vulnerable components of the

neuroendocrine system and the

behavioral, hormonal, and physiological

endpoints that may be indicative of a

direct or indirect effect on the

neuroendocrine system.

Public comment strongly endorsed

the default assumption that there is a

threshold for neurotoxic effects. The

Guidelines, however, reflect the

argument of the SAB that the term

‘‘nonlinear dose-response curve for most

neurotoxicants’’ be substituted for

‘‘threshold’’ in order to be consistent

with the presentation and positions

taken by other risk assessment

guidelines.

The public comments made a number

of recommendations to improve the

Guidelines with regard to consistency of

language between text and tables,

improve the clarity of some of the

tables, and improve the description of

some of the endpoints used in animal

studies. A number of factual errors were

corrected, including the description of

the blood-brain barrier and the degree of

inhibition of neurotoxic esterase

associated with organophosphateinduced delayed-onset neuropathy.

Therefore, a number of changes have

been made in the Guidelines to clarify

and correct specific passages, but every

effort was made to maintain the original

intent concerning the use and

interpretation of results from various

neurotoxicological endpoints. Finally,

the public comment agreed with the

SAB that factors such as nutrition,

personal habits, age, or preexisting

disease may predispose some

individuals to be differentially sensitive

to neurotoxic chemicals. The risk

characterization section has been

expanded to reflect these potentially

sensitive subpopulations.

[FR Doc. 98–12303 Filed 5–13–98; 8:45 am]

BILLING CODE 6560–50–P