August 17, 1994
Part III


Protection Agency

Final Report: Principles of Neurotoxicity
Risk Assessment; Notice

Federal Register / Vol. 59.  No.  158  /  Wednesday,  August 17,  1994  / Notices


Final Report: Principles of
Neurotoxlcity Risk Assessment

AGENCY: U.S.  Environmental Protection
ACTION: Final Document.

SUMMARY: The U.S. Environmental
Protection Agency is publishing a
document entitled Final Report:
Principles of Neurotoxicity Bisk
Assessment, which was prepared by the
Working Party on Neurotoxicology
under the auspices of the Subcommittee
on Risk Assessment of the Federal
Coordinating  Council for Science,
Engineering, and Technology (FCCSET).
The purpose of this report is to
articulate a view of neurotoxicology that
scientists generally hold in common
today and to draw on this
understanding to generate a  series of
general principles that can be used to
establish guidelines for assessing
neurotoxicity risk. It is not the intent of
this report to provide specific directives
for how neurotoxicity risk assessment
should be performed. The  intent of this
document is to provide the scientific
basis for the development  of a cogent
strategy for neurotoxicity risk
document is the result of the combined
efforts of senior scientists of 13 Federal
agencies comprising the ad hoc
Interagency Committee on
Neurotoxicology, including the Agency
for Toxic Substances and Disease
Registry, Center for Food Safety and
Applied Nutrition, Center  for Biologies
Evaluation and Research, Center for
Drug Evaluation and Research,
Consumer Product Safety Commission,
Department of Agriculture, Department
of Defense, Environmental Protection
Agency, National Center for
Toxicological Research,  National
Institutes of Health, National Institute
for Occupational Safety and  Health, and
National Toxicology Program.
Discussions were held under the
auspices of the Working Party on
Neurotoxicology of the Subcommittee
on Risk Assessment of the  Federal
Coordinating Council for Science,
Engineering, and Technology. The draft
report, a product of the Working Party
on Neurotoxicology, contains six
chapters: an introduction,  an overview
of the discipline of neurotoxicology, a
review of methods for assessing human
neurotoxicity, a review of methods for
assessing animal neurotoxicity, an
                       overview of principles of neurotoxicity
                       risk assessment, and a general summary.
                         The draft report was prepared in view
                       of the decision-making processes
                       currently used by many regulatory
                       agencies relating to neurotoxicity risk
                       assessment. It is intended that the
                       principles reviewed in this document
                       will serve as the basis for consistent
                       regulatory neurotoxicity guidelines to be
                       used by Federal agencies to meet their
                       respective legislative mandates. This
                       document is not meant to be used to
                       perform risk assessment nor does it
                       recommend one approach or strategy.
                       The document reviews the  science of
                       neurotoxicology and attempts to
                       formulate general assumptions and
                       principles that could lead to such
                       approaches or strategies.
                         The draft report has undergone
                       interagency review under the auspices
                       of the Subcommittee on Risk
                       Assessment of FCCSET.  Public
                       comments received were used  in the
                       preparation of the final report by the
                       Working Party on Neurotoxicology.
                         Dated: August 9, 1994.
                       Ken Sexton,
                       Director, Office of Health Research.

                       Final Report: Principles of
                       Neurotoxicology Risk Assessment

                       1. Introduction
                         1.1. Background
                         1.2. Purpose of This Report
                         1.3. Context of This Report
                         1.4. Content of This Report
                       2. Overview of Neurotoxicology
                         2.1. Scope of the Problem
                         2.1.1. Introduction
                         2.1.2. Examples of Neurotoxicity and
                           Incidents of Exposure
                         2.1.3. Federal Response
                Food and Drug Administration
                Occupational Safety  and Health
                National Institute for Occupational
                           Safety and Health
                Environmental Protection Agency
                Consumer Product Safety
                Agency for Toxic Substances and
                           Disease Registry
                         2.2. Basic Toxicological Considerations for
                         2.2.1. Basic Toxicological Principles
                         2.2.2. Basic Neurotoxicological Principles
                         2.3. Basic Neurobiological Principles
                         2.3.1. Structure of the Nervous System
                         2.3.2. Transport Processes
                         2.3.3. Ionic Balance
                         2.3.4. Neurotransmission
                         2.4. Types of Effects on the Nervous
                         2.5. Special Considerations
                         2.5.1. Susceptible Populations
                         2.5.2. Blood-Brain and Blood-Nerve
                         2.5.3. Metabolism
  2.5.4. Limited Regenerative Ability
3. Methods for Assessing Human
  3.1. Introduction
  3.2. Clinical Evaluation
  3.2.1. Neurologic Evaluation
  3.2.2. Neuropsychological Testing
  3.2.3 Applicability of Clinical Methods to
    Neurotoxicology Risk Assessment
  3.3. Current Neurotoxicity Testing Methods
  3.3.1. Neurobehavioral Methods Test Batteries Investigator-Administered Test
    Batteries Computerized Test Batteries
  3.3.2. Neurophysiologic Methods
  3.3.3. Neurochemical Methods
  3.3.4. Imaging Techniques
  3.3.5.'Neuropathologic Methods
  3.3.6. Self-Report Assessment Methods Mood Scales Personality Scales
  3.4. Approaches to Neurotoxicity
  3.4.1. Epidemiologic Studies Case Reports Cross-Sectional Studies Case-Control (Retrospective)
    Studies Prospective (Cohort, Follovvup)
  3.4.2. Human Laboratory Exposure Studies Methodologic Aspects Human Subject Selection Factors Exposure Conditions  and Chemical
    Classes Test Methods Controls Ethical Issues
  3.5. Assessment of Developmental
  3.5.1. Developmental Deficits
  3.5.2. Methodologic Considerations
  3.6. Issues in Human Neurotoxicology Test
  3.6.1. Risk Assessment Criteria for
    Neurobehavioral Test Methods Sensitivity Specificity Reliability and Validity Dose Response Structure-Activity
  3.6.2. Other Considerations in Risk
    Assessment Mechanisms of Action Exposure Duration Time-Dependent Effects Multiple Exposures Generalizability and Individual
    Differences Veracity of Neurobehavioral Test
  3.6.3. Cross-Species Extrapolation
4. Methods to Assess Animal Neurotoxicity
  4.1. Introduction
  4.1.1. Role of Animal Models
  4.1.2. Validity of Animal Models
  4.1.3. Special Considerations  in Animal
    Models Susceptible Populations Dosing Scenario Other Factors Statistical Considerations
  4.2. Tiered Testing in Neurotoxicology
  4.2.1. Type of Test
  4.2.2. Dosing Regimen

                 Federal Register /  Vol. 59,  No. 158 /  Wednesday,  August  17.  1994 / Notices           42361
 4.3. Endpoints of Neurotoxicity
 4.3.1. Introduction
 4.3.2. Behavioral Endpoints Functional Observational Batteries Motor Activity Neuromotor Function Sensory Function Learning and Memory Schedule-Controlled Behavior
 4.3.3. Neurophysiological Endpoints of
   Neurotoxicity Nerve Conduction Studies Sensory Evoked Potentials Convulsions Electroencephalography Electromyography Spinal Reflex Excitability
 4.3.4. Neurochemical Endpoints of
 4.3.5. Structural Endpoints of
 4.3.6. Developmental Neurotoxicity
 4.3.7. Physiological and Neuroendocrine
 4.3.8. Other Considerations Structure-Activity Relationship In Vitro Methods
5. Neurotoxicology Risk Assessment
  5.1. Introduction
  5.2. The Risk Assessment Process
  5.2.1. Hazard Identification Human Studies Animal Studies Special Issues
  5.2.2. Dose-Response Assessment
  5.2.3. Exposure Assessment
  5.2.4. Risk Characterization
  5.3. Generic Assumptions and Uncertainty
6. General Summary
7. References

1-1. Major Regulatory Agencies
1-2. Authorities for Toxicity Testing
2-1. Human Neurotoxic Exposures
3-1. Neurobehavioral Methods
4-1. Examples of Potential Endpoints of
4-2. Examples of Specialized Tests to
    Measure Neurotoxicily
4-3. Summary of Measures in the Functional
    Observational Battery and the Type of
    Data Produced by Each
4—4. Neurotoxicants With Known
    Neurochemical Mechanisms
4-5. Examples of Known Neuropathic Agents
4-6. Partial List of Agents Believed to Have
    Developmental Neurotoxicity
5-1. General Assumptions That Underlie
    Traditional Risk Assessments

1. Introduction

1.1. Background
   Over the years, agencies and programs
have been established to deal with
hazardous substances, with a focus on
deleterious long-term effects,  including
noncancer endpoints such as
neurotoxicity  (Reiter, 1987). Recent
evidence indicates that exposure to
neurotoxic agents may constitute a
significant health problem (WHO, 1986;
OTA, 1990; chapter 2). Table 1-1 lists
the four Federal regulatory agencies
with authority to regulate either
exposure to or use of chemicals and that
require data reporting on assessment  of
hazards. Regulatory bodies vary greatly
in their mandate to require approval of
chemicals prior to entering the
marketplace and to regulate subsequent
exposure (Fisher, 1980) (Table 1-2). The
Occupational Safety and Health
Administration (OSHA) cannot require
chemical testing by the manufacturer
whereas all other agencies can. Only  the
Food and Drug Administration (FDA)
and the Environmental Protection
Agency (EPA) have authority for
premarketing testing of chemicals (i.e.,
FDA for drugs and food additives and
EPA for pesticides). EPA can, under
some circumstances, require premarket
testing of industrial and agricultural
chemicals. The Consumer Product
Safety Commission (CPSC) regulates  a
number of consumer products including
household chemicals and fabric
treatments. Laws administered by CPSC
require cautionary labeling on all
hazardous household products whether
the hazard is based on acute or chronic
effects. These laws also provide the
authority to ban hazardous products  and
to ask for data in support of product
                                      TABLE 1-1.—MAJOR REGULATORY AGENCIES
                                                                                 Statute and sources covered
Food and Drug Administration (FDA)
A unit of the Department of Health and Human Services with authority
  over the regulation of medical and veterinary drugs; foods and food
  additives; cosmetics.
Occupational Safety and Health Administration (OSHA) 	

A unit of the Department of Labor that regulates workplace conditions ..
Environmental Protection Agency (EPA).
Independent agency (i.e.,  not part  of a Cabinet department); admin-
  isters a number of diverse laws concerned with human health and
  the environment.
 Consumer Product Safety Commission (CPSC).
 Regulates a variety of consumer products including household chemi-
   cals and fabric treatments.
                      Food, Drug, and Cosmetics Act for food additives; color in cosmetics;
                        medical devices; animal drugs of medical and feed additives.
                      Occupational Safety  and Health  Act covers toxic chemicals  in the
                      Toxic  Substances Control Act requires premanufacture evaluation of
                        all new  chemicals (other than foods, food additives, drugs, pes-
                        ticides, alcohol, tobacco); allows EPA to regulate existing chemical
                        hazards not sufficiently controlled under other laws.
                      Clean Air Act requires regulation of hazardous air pollutants.
                      Federal Water Pollution Control Act governs toxic water pollutants.
                      Safe Drinking Water Act covers drinking water contaminants.
                      Federal Insecticide, Fungicide, and Rodenticide Act covers pesticides.
                      Resource Conservation and Recovery Act covers hazardous wastes.
                      Marine Protection Research and Sanctuaries Act covers ocean dump-

                      Federal Hazardous Substances Act covers "toxic" household products.
                      Consumer Product Safety Act covers dangerous consumer products.
                      Poison Prevention Packaging Act covers packaging of dangerous chil-
                        dren's products.
                      Lead-Based Paint Poison Prevention Act  covers use of lead paint in
                        federally assisted housing.                  	

Federal Register / Vol. 59, No. 158 / Wednesday. August 17. 1994 / Notices
                                  TABLE  1-2.—AUTHORITIES FOR TOXICITY TESTING
OSHA . .. .

Food, Drug, and Cosmetics Act
Federal Insecticide, Fungicide, and
Rodenticide Act.
Toxic Substances Control Act 	
Clean Air Act 	
Resource Conservation and Recov-
ery Act.
Occupational Safety and Health Act .
Federal Hazardous Substances Act .
Consumer Product Safety Act 	

Food additives and
Industrial chemicals ...
Air pollutants
Industrial waste
Occupational expo-
Consumer products ...
Consumer products ...
Testing by man-
Reporting of
  1 Can require testing based on available data.
 1.2. Purpose of This Report
  The purpose of this document is to:
 (1) articulate a view of neurotoxicity
 that scientists generally hold in
 common today and (2) draw upon this
 understanding to compose, as was done
 here by senior scientists from a number
 of Federal agencies, a series of general
 principles that can be used to establish
 general guidelines for assessing
 neurotoxicity risk. It is not the intent of
 this report to provide specific directives
 to agencies with respect to their own
 approach for neurotoxicity risk
 assessment. This document is intended
 to provide the scientific basis for the
 development of a cogent strategy for
 neurotoxicology risk assessment as
 needed by each agency.
  Because of present gaps in
 understanding, the principles contained
 in this document are based on the best
 judgment of those involved in writing
 this document, as well as statements of
 what is generally accepted as fact. There
has been, however, an attempt to
 distinguish where possible between the
 different types of information presented.
  The principles contained in this
 document can serve as the basis for
 consistent regulatory neurotoxicology
 guidelines that the Federal agencies can
tailor  to meet the requirements of the
 legislative acts they are charged to
 implement. This document should be
viewed broadly as part of an ongoing
 process within the Federal Government
to periodically update and review the
current scientific understanding and
regulatory utility of neurotoxicity risk
  This document is the result of the
combined efforts of senior scientists
 from the following Federal health-
related units, operating under the
 direction of the Office of Science and
Technology Policy (OSTP):
                      Agency for Toxic Substances and
                        Disease Registry (ATSDR)
                      Center for Biologies Evaluation and
                        Research (CBER), FDA
                      Center for Drug Evaluation and Research
                        (CDER), FDA
                      Center for Food Safety and Applied
                        Nutrition (CFSAN), FDA
                      Consumer Product Safety Commission
                      Department of Agriculture (USDA)
                      Department of Defense (DoD)
                      Environmental Protection Agency
                      National Center for Toxicological
                        Research (NCTR), FDA
                      National Institutes of Health (NIH)
                      National Institute for Occupational
                        Safety and  Health
                      National Toxicology Program (NTP)

                      1.3. Context of This Report
                        This document was prepared in light
                      of a decision-making process used by
                      many regulatory agencies pertaining to
                      the assessment of neurotoxicity risks
                      posed by chemical agents. The scientific
                      basis for such assessment can be best
                      understood by examining the decision-
                      making process in some detail.
                        Risk can be thought of as being
                      composed of two aspects, each of which
                      can be addressed by science, i.e., hazard
                      and exposure assessment. Although
                      other definitions have been used
                      historically, this document conforms to
                      present usage. Hazard generally refers to
                      the toxicity of a substance and  is
                      deduced from a wide array of data,
                      including those from epidemiological
                      studies or controlled clinical trials in
                      humans, short- and long-term
                      toxicological  studies in animals, and
                      studies of mechanistic information and
                      structure-activity relationships.
                      Exposure generally refers to the amount
                      of a substance with which people come
                      in contact. The risk in a quantitative risk
                      assessment is estimated by considering
                      the results of the exposure and hazard
assessments. As either the hazard or
exposure approaches zero, the risk also
approaches zero.
  As a first step in assessing the
neurotoxic risk associated with the use
of a particular chemical substance, the
qualitative evidence that a given
chemical substance is likely to be a
human neurotoxicant must be
evaluated. In this step, as in the whole
process, a number of assumptions and
approximations must be made in order
to deal with inherent limitations found
in the existing data bases. Then,
estimates of human exposure and
distribution of exposures  likely to be
encountered in the population are
made. In the absence of dose-response
relationships in humans, one or more
methods for estimating the dose-
response relationship including doses
below those generally used
experimentally must also be evaluated.
Finally, the exposure assessment is
combined with the dose-response
relationship to generate an estimate of
risk. The various ways in  which these
steps are conducted and combined and
their attendant uncertainties constitute
what is generally referred to as
"neurotoxicity risk assessment."
  Some legislation calls for action in the
presence of any risk. Other forms of
legislation use the concept of
unreasonable risk, defined in some acts
as a condition in which the risks
outweigh the benefits. A spectrum of
regulatory responses, from simply
informing the public of a risk through
restricted use to a complete ban, may be
available to bring the risks and benefits
into appropriate balance.
  This document does not perform a
risk assessment nor does it suggest that
one method of neurotoxicology risk
assessment is better than another.
Rather, it attempts to review the science

                Federal Register / Vol.  59,  No. 158  /  Wednesday, August 17, 1994  / Notices
of chemical neurotoxicology and
develops from this review a set of
general principles. It is not a
comprehensive review nor a document
written for the lay public; this document
is a semitechnical review that evaluates
the impact of scientific findings of the
last decade on general assumptions or
principles important to risk assessment.
This is based on the belief that
elucidation of the basic mechanisms
underlying neurotoxicity and the
identification of neurotoxic agents and
conditions, when coupled to research
aimed at identifying and characterizing
the problems caused by such agents,
should provide the best scientific bases
for making sound and reasonable
judgments. These overlapping
approaches to evaluating the problems
of neurotoxicology should form a strong
foundation for decision-making.

1.4. Content of This Report
  Including the Introduction (chapter
1), this document contains six chapters.
Chapter 2 provides an overview of the
discipline of neurotoxicology. It is
important to understand the scope of
the problem as it relates to
neurotoxicology, including: (1)
Definitions of neurotoxicity and adverse
effect, (2) examples of neurotoxicity and
incidents of exposure, and (3) Federal
response to neurotoxicology. Chapter 2
also discusses the basic principles of
toxicology that apply generally to the
evaluation of neurotoxicity. Issues such
as dose, exposure, target site, and the
 intended use of the chemical are
 discussed, as are principles of
 pharmacodynamics, chemical
 interactions, and the concept of
 threshold. Chapter 2 also lays the
 neurobiological basis for understanding
 how and where chemicals can affect the
 nervous system and provides examples
 of such chemical types. Finally, chapter
 2 discusses special considerations for
 neurotoxicology including the issue of
 susceptible populations, the blood brain
 barrier, and the limited capability of the
 nervous system to repair following
 chemical insult.
    Chapter 3 examines methods for
 assessing human neurotoxicity.
 Neurologic evaluations,
 neuropsychological testing, and
 applicability of methods used in clinical
 evaluations  and case studies are
  discussed in this chapter. Epidemiologic
  study designs, endpoints, and methods
  are also discussed, as well as problems
  of causal inference and applications and
  limitations of epidemiologic and field
  study methods for risk assessment.
  Chapter 3 also describes human
  laboratory exposure  studies, including
  methods for assessing neurobehavioral
function, self-report methods for
assessing subjective states, and a
number of other methodological issues.
This chapter also discusses the
comparability of human and animal
laboratory methods and special
considerations in human neurotoxicity
  Chapter 4 assesses methods for
evaluating animal neurotoxicity.
Discussed in this chapter is the role that
animal models play in the assessment of
chemicals for neurotoxicity, the validity
of animal models, and experimental
design considerations in animal
neurotoxicological studies. Also
included in this chapter is a discussion
of tier-testing approaches in chemical
evaluations. Specific endpoints used in
animal neurotoxicological studies are
also discussed, including methods for
neurobehavioral, neurophysiological,
neuroanatomical, and neurochemical
assessments. Developmental
neurotoxicology and in vitro
neurotoxicology are also described in
this chapter.
  Chapter 5 of this document discusses
principles of neurotoxicity risk
assessment. This chapter evaluates the
generic assumptions in neurotoxicity
risk assessment, ending with a
discussion of uncertainty reduction and
identification of knowledge gaps.
  Chapter 6 is a general summary of the
material presented in the first five
2. Overview of Neurotoxicology

2.1. Scope of the Problem

 2.1.1. Introduction
   Chemicals are an integral part  of our
 lives, with the capacity to both improve
 as well as endanger our health. The
 general population is exposed to
 chemicals with neurotoxic properties in
 air, water, foods, cosmetics, household
 products, and drugs used
 therapeutically or illicitly. Naturally
 occurring neurotoxins, such as fish and
 plant toxins, present other hazards.
 During the daily  life of an ordinary
 person, there is a multitude of
 exposures, both voluntary and
 unintentional, to neuroactive
 substances. Under conditions of
 multiple exposures, identifying  the
 substance responsible for an adverse
 response may be difficult. The EPA's
 inventory of toxic chemicals is greater
 than 65,000 and increasing  yearly.
 Concerns have been raised about the
 toxicological data available for many
 compounds used commercially  (NRC,
    It is not known how many chemicals
 are neurotoxic to humans. However,
 estimates have been made for subsets of
substances. A large percentage of the
more than 500 registered active
pesticide ingredients are neurotoxic to
varying degrees. Of 588 chemicals listed
by the American Conference of
Government and Industrial Hygienists
(ACGIH), 167 affected the nervous
system or behavior (Anger, 1984; CDC,
1986). Using a generally broad
definition of neurotoxicity, Anger
(1990a) 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 at
some level of exposure. Anger (1984)
also recognized neurotoxic effects as
one of the ten leading workplace
disorders. In addition, a number of
therapeutic substances, including some
anticancer and antiviral agents and
abused drugs, can cause adverse or
neurotoxicological side effects (OTA,
1990). It has been estimated that there
is inadequate toxicological information
available for more than three-fourths of
the 12,860 chemicals with a production
volume of 1 million pounds or more
(NRC, 1984). It should be noted,
however, that estimates concerning the
number of neurotoxicants vary widely.
O'Donoghue (1989), for example,
reported that of 488 compounds
assessed in his chemical evaluation
process, only 2.7% had effects on the
nervous sytem.
 2.1.2. Examples of Neurotoxicity and
 Incidents of Exposure
   There is a long-standing history
 associating certain neurological and
 psychiatric disorders to exposure to a
 toxin or chemical of an environmental
 origin (OTA, 1990) (Table 2-1). Lead is
 one of the earliest examples of a
 neurotoxic chemical with widespread
 exposure. This metal is widely
 distributed with major sources of
 inorganic lead including industrial
 emissions, lead-based paints, food,
 beverages, and the burning of leaded
 gasolines. Organic lead compounds
 such as tetraethyl lead have been
 reported to produce a toxic psychosis
 (Cassells and Dodds, 1946). If exposure
 occurs at relatively low levels during
 development, lead can cause a variety of
 neurobehavioral problems, learning
 disorders, and altered mental
 development (Bellinger et al., 1987;
 Needleman, 1990). Over the years,
 Federal Government regulations have
 been developed to decrease human
 exposure to lead, and as a goal an
 intervention level of 10 ug/dcl whole
 blood has been recommended (CDC,
  1991). Lead exposure in the United
  States has decreased significantly
  during the last several years.

Federal Register / Vol. 59, No. 158 / Wednesday. August 17, 1994  / Notices
                                  TABLE 2-1 .—HUMAN NEUROTOXIC EXPOSURES
370 B.C 	
1 st century A.D .

United States (New Jer-
United States (South-
United States (Califor-
South Africa 	
Japan (Mina- 	
United States (New
United States 	
United States (Ohio) 	
United States (Virginia) .
United States (Texas) ....
United States (Califor-
United States (Texas) ....
United States
United States 	
United States and Can-
United States

Tetraethyl lead
Tetraethyl lead




Methyl n-butylketone 	
Chlordecone (Keptone) .
Leptophos (Phosvel) 	
Dichloropropene (Telone
(BHMH) (Lucel-7).
pyridine (MPTP).
Toxic oil
Vitamin 85
Domoic acid

Lead toxicity recognized in mining industry.
Vapors recognized as toxic.
Chronic manganese poisoning described.
Workers suffer neurologic symptoms.
Chemical contaminant added to Ginger Jake, an alcoholic beverage
substitute; more than 5,000 paralyzed, 20,000 to 100,000 affected.
Drug containing TOCP causes 60 cases of neuropathy.
Contaminated barley laced with thallium sulfate poisons family, caus-
ing neurologic symptoms.
Paralysis develops after use of contaminated cooking oil.
Neurologic effects observed in people cleaning gasoline tanks.
Fish and shellfish contaminated with mercury are ingested, causing
Medication (Stalinon) containing diethyltin diiodide results in poisoning.
Miners suffer chronic manganese intoxication.
Ingestion of plants associate^ with amyortrophic lateral sclerosis and
Parkinson-like syndrome.
Hexachlorobenzene causes poisoning.
Drug causes neuropathy.
Cooking oil contaminated with lubricating oil causes poisoning.
Mercury-treated seed grain causes neurotoxicity.
Methylmercury neurotoxicity.
Polychlorinated biphenyls are leaked into rice oil, causing
Neuropathy due to n-hexane exposure.
Fungicide-treated grain results in alkyl mercury poisoning.
Hexachlorophene-containing disinfectant is found to be toxic to nerv-
ous system.
Methylmercury used as fungicide to treat seed grain causes poisoning.
Hexachlorophene poisoning of children.
Fabric production plant employees exposed to MnBK solvent suffer
Chemical plant employees exposed to insecticide suffer severe
neurologic problems.
At least nine employees suffer serious neurologic problems after expo-
sure to insecticide.
People hospitalized after exposure to pesticide.
Employees of manufacturing plant experience serious neurologic prob-
Impurity in synthesis of illicit drug causes Parkinson's disease-like ef-
People ingesting toxic substance in oil suffer severe neuropathy.
Excessive intake, causes sensory neuropathy, numbness, parathesia,
and motor dysfunction.
People experience neuromuscular deficits after ingestion of contami-
nated melons.
Ingestion of mussels contaminated with domoic acid causes illnesses.
Ingestion of adulterated rapeseed oil cause polyneuritis.
Ingestion of a chemical contaminant associated with the manufacture
of L-tryptophan results in eosinophilia-myalgia syndrome.
Natural component of gari caused neuropathy associated with optic at-
rophy and ataxia.
  Mercury compounds are potent
neurotoxic substances and have caused
a number of human poisonings, with
symptoms of vision, speech, and
coordination impairments (Chang,
1980). Erethism, a syndrome with such
neurologic features as tremor and
behavioral symptoms as anxiety,
irritability, and pathologic shyness, is
seen in people exposed to elemental
mercury (Bidstrup, 1964). One major
                    incidence of human exposure occurred
                    in the mid-1950's when a chemical
                    plant near Minamata Bay, Japan,
                    discharged mercury as part of waste
                    sludge. An epidemic of mercury
                    poisoning developed when the local
                    inhabitants consumed contaminated
                    fish and shellfish. Congenitally affected
                    children displayed a progressive
                    neurological disturbance resembling
                    cerebral palsy and manifested other
neurological problems as well. In 1971,
an epidemic occurred in Iraq from
methylmercury used as a fungicide to
treat grain (OTA, 1990).
  Manganese is used in metal alloys and
has been proposed to replace lead in
gasoline. It is an essential dietary
substance for normal body functioning
yet parenteral exposure to manganese
can be neurotoxic, producing a
dyskinetic motor syndrome similar to
Parkinson's disease (Cook et al., 1974).

                Federal Register / Vol. 59, No. 158  /  Wednesday,  August 17, 1994  /  Notices          42365
Exposed miners in several countries
have suffered from "manganese
madness" characterized by
hallucinations, emotional instability,
and numerous neurological problems.
Long-term manganese toxicity produces
muscle rigidity and staggering gait
similar to that seen in patients with
Parkinson's disease (Politis et al., 1980).
  A Parkinsonian-like syndrome was
also observed in people who
accidentally ingested l-methyl-4-
pheny 1-1,2,3,6-tetrahy dropyridine
(MPTP) (Langston et al., 1983). MPTP
was a byproduct of a meperidine
derivative sold illicitly as "synthetic
  Organic solvents are encountered
frequently in occupational settings.
Most solvents are volatile,  i.e., they can
be converted from a liquid to a gaseous
state and readily inhaled by the worker.
They are also lipid soluble and readily
accumulate in the fat deposits of the
exposed  organism. An example of a
solvent exposure  in humans is carbon
disulfide. Workers exposed to high
levels of this solvent were  found to have
an increased frequency of  depression
and suicide (Seppalainen and Haltia,
1980). Furthermore, repeated exposure
to organic solvents is suspected of
producing chronic encephalopathy.
Workers exposed to methyl-n-butyl
ketone, a dye solvent and cleaning
agent, displayed peripheral nervous
system neuropathy involving
degeneration of nerve fibers (Spencer
and Schaumburg, 1980). Solvents
including ether, ketones, alcohols, and
 various combinations are commonly
 used in glues, cements, and paints and
 when inhaled can be neurotoxic.
 Repeated abuse of such solvents can
 lead to permanent neurological effects
 due to severe and permanent loss of
 nerve cells (OTA, 1990).
   Pesticides are one of the most
 commonly encountered classes of
 neurotoxic substances. These can
 include  insecticides (used to control
 insects), fungicides (for blight and
 mildew), rodenticides (for rodents such
 as rats, mice, and gophers), and
 herbicides (to control weeds). Active
 ingredients are combined with so-called
 inert substances  to make thousands of
 different pesticide formulations.
 Workers who are overexposed to
 pesticides may display obvious signs  of
 poisoning, including tremors, weakness,
 ataxia, visual disturbances, and short-
 term memory loss (Ecobichon and Joy,
 1982). Chlordecone exposure results in
 nervousness and tremors  (Cannon et al.,
 1978). The organophosphorous
 insecticides have neurotoxic properties
 and account for approximately 40
 percent of registered pesticides. A
delayed neurotoxicity can be seen as a
result of exposure to certain
organophosphate pesticides, producing
irreversible loss of motor function and
an associated neuropathology
(Ecobichon and Joy, 1982).
Organophosphate and carbamate
insecticides are known to interfere with
a specific enzyme, acetylcholinesterase
(AChE) (Davis and Richardson, 1980).
Paralysis has also been reported
following consumption of nonpesticide
organophosphate  products such as tri-o-
cresylphosphate (TOCP).
  Neurotoxicities in humans, domestic
livestock, and poultry associated with
fungal toxins (mycotoxins) have been
well documented (Kurata, 1990; Aibara,
1986; Wyllie and  Morehouse, 1978).
Mycotoxins not only have a negative
economic effect on animal production,
but they also represent a definite threat
to human health.  Mycotoxins occur in
forages, field crops, and grains used for
livestock; they also are incorporated
into cereals, grains, and grain-based
products used for human consumption.
Therefore, human exposure may occur
either through direct consumption of
these products or secondarily through
consumption of meat, milk, or eggs. An
example of human exposure to fungal
toxins is Claviceps purpurea- or C.
paspay/-infected wheat, barley, and oats
used for bread and as a dietary
supplement for livestock. These fungal
toxins are notorious for producing the
gangrenous and convulsive forms of the
disease known as "ergotism" (Bove,
 1970). These fungi are in the family
 Clavicipitaceae and  produce a group of
 compounds known as ergot alkaloids,
 which have neurotropic, uterotonic, and
 vasoconstrictive  activities. They may act
 as dopamine agonists or serotonin
 antagonists, and  also block alpha-
 adrenergic receptors. Since there are
 numerous naturally occurring ergot
 alkaloids, this represents only part of
 their pharmacopoeia (Berde and
 Schield, 1978). These alkaloids are
 highly toxic and cause both acute and
 chronic poisonings. Although
 guidelines now limit the amount of
 C/avi'ceps-contaminated, or "ergot"-
 contaminated, grains, these compounds
 may enter human food sources through
 secondary mechanisms.  Other fungi
 associated with ergot-like syndromes in
 livestock include Acremonium lolii
 (Gallagher et al., 1984) and A.
 coenophialum (Thompson and Porter,
   Cyclopiazonic acid (CPA) is an indole
 tetramic acid produced by Aspergillus
 flavus, A. oryzae, Penicillium
  cyclopium, and  P. camemberti. This
  mycotoxin is suspected  of causing
  "kodua poisoning" in humans who
consumed kodo millet seed in India
(Rao and Husain, 1985). Fusarium
moniliforme is a common fungal
infection in corn (Bacon et al., 1992)
and directly related to neurotoxic
syndrome in horses known as equine
leukoencephalomalaisia (ELEM).
  Natural  plant toxins also represent a
health risk to both livestock and
humans. Movement toward limited uses
of herbicides, fungicides, and no-till
agricultural practices increases the
possibility of noxious weeds and weed
seeds being incorporated into food
products.  Ergot alkaloids also are
produced  by morning glories (Ipomea
violacea) and may be incorporated into
soybeans, corn, peas, etc., during
harvest. Export regulations limit
morning glory-contaminated soybeans
because of the hallucinogenic and other
effects produced by ergot alkaloids.
Jimson weed (Datura stramonium),
another weed incorporated into
agricultural commodities, produced
scopolamine, hyocyamine, and stropine,
all of which have parasympatholytic
(anticholinergic) activities.
   Recently, an outbreak of toxic
encephalopathy caused by eating
mussels contaminated with domoic
acid, an excitotoxin, was reported (Perl
et al., 1990).
2.1.3. Federal Response
   In the United States, several agencies,
including EPA, FDA, OSHA, CPSC,
 N1OSH, and ATSDR, have been given
 the  mandate to regulate or evaluate
 public exposure to toxic chemicals
 (Tilson, 1989). Food and Drug Administration.
   The FDA has the authority to regulate
 the use of food and color additives as
 well as to determine whether or not
 various foods are unsafe  for human
 consumption because of adulteration by
 environmental contaminants. The
 manufacturer must supply adequate
 data to establish the safety of the food
 additives. Before marketing approval,
 the potential toxicity of proposed food
 and color additives is established in a
 battery of animal toxicity studies.
 During all of these studies, clinical signs
 of toxicity, including abnormal
 behavior, are monitored and
 abnormalities recorded. At the
 termination of these studies, tissues
 from all  organs, including the brain, are
 sectioned and evaluated for both gross
 and histopathological changes, in
 addition to being evaluated for their
 clinical chemistry and hematology.
 None of  the routinely required tests is
 specifically designed to assess
 neurotoxicity. If neurotoxic effects are
 detected during any of the standard

Federal  Register  /  Vol. 59. No. 158 / Wednesday, August 17, 1994 / Notices
 toxicity tests, however, they must be
 reported. Specific neurotoxicity testing
 may then be required. The FDA is
 currently revising its guidelines for the
 safety assessment of direct food and
 color additives to include neurotoxicity
 as a routine element in toxicological
   The FDA is also authorized to regulate
 substances in food considered to be
 poisonous or deleterious. Unavoidable
 environmental contaminants in food fall
 into this category. The FDA determines
 a level at which the risks from
 realistically possible intakes are
 negligible or acceptable. Based on this
 risk assessment, an action level or
 tolerance is established. Once the action
 level or tolerance is formally
 established, the FDA may take
 appropriate action to restrict adulterated
 food from the market if these standards
 are exceeded.
   The FDA is responsible for assessing
 the toxicity of human therapeutic
 products. Many products have been
 shown to produce adverse effects on the
 nervous system at standard therapeutic
 doses as well as at higher doses. Before
 marketing approval is given, the toxicity
 of potential new products is assessed. A
 battery of animal toxicity study
 parameters relevant to the nervous
 system, including gross behavioral
 observation and gross and
 histopathological examination of the
 nervous tissue, are evaluated. This
 information is used to help guide the
 surveillance of human subjects for
 adverse effects that are assessed during
 clinical trials. Occupational Safety and Health
  OSHA has been given the
 responsibility to ensure that the working
 environment is a safe and healthy place
 of employment. In the early 1970's,
 OSHA adopted the existing Federal
 standards, most of which were
 developed under the Walsh-Healy Act
 (including the 1968 ACGIH Threshold
 Limit Values), and approximately 20
 consensus standards of the American
 National Standards Institute (ANSI) as
 Permissible Exposure Limits (PELs). Of
 the 393 remaining original PELs, 145
 were set in part to protect the individual
 from neurotoxic effects.
  Since the adoption of the initial
 standards, OSHA has issued new or
 revised health standards or work
 practices for 23 substances. Of these, the
 one concerning lead was based in part
 on nervous system effects. Four other
compounds, inorganic arsenic,
acrylonitrile, ethylene oxide, and 1,2-
dibromo-3-chloropropane, were cited as
causing various disturbances in the
                      nervous system, but the standards for
                      these were based primarily on
                      carcinogenic effects.
                        In 1989, OSHA updated 428 exposure
                      limits for air contaminants. Of these, 25
                      substances were categorized by OSHA
                      as "substances for which limits are
                      based on avoidance of neuropathic
                      effects." In addition, 24 substances were
                      included in the category "substances for
                      which limits are based on avoidance of
                      narcosis." However, OSHA stated that
                      the categorization was intended as a tool
                      to manage the large number of
                      substances being regulated and not to
                      imply that the category selected
                      identified the most sensitive or the
                      exclusive adverse health effects of that

             National Institute for
                      Occupational Safety and Health.
                        The Occupational Safety and Health
                      Act established NIOSH as a Public
                      Health Service  (PHS) agency to develop
                      and  recommend criteria for prevention
                      of disease and hazardous conditions in
                      the workplace.  NIOSH also performs
                      research on occupational health issues
                      and  conducts worksite evaluations of
                      suspected hazards. OSHA and the Mine
                      Safety and Health Administration
                      (MSHA) use NIOSH recommendations
                      in the promulgation of new or revised
                      health and safety standards.
                        In establishing recommended
                      exposure limits (RELs) for chemicals,
                      NIOSH examines all relevant scientific
                      information about a given compound
                      and attempts to identify exposure limits
                      that  will protect nil workers from
                      adverse effects. NIOSH has
                      recommended standards for
                      approximately 644 chemicals or classes
                      of chemicals. For 214 (33 percent) of
                      these, neurotoxicity was cited as a
                      health effect considered when
                      formulating the REL (NIOSH, 1992).
             Environmental Protection
                       The Toxic Substances Control Act
                      (TSCA) and the Federal Insecticide,
                      Fungicide, and  Rodenticide Act (FIFRA)
                      provide the legislative authority for EPA
                      to require data collection for premarket
                      approval of chemicals. Under section 5
                      of TSCA, after a manufacturer has
                      notified EPA of its plans to produce a
                      "new',' chemical that has not yet been
                      listed on the inventory, EPA has the
                      responsibility to assess possible health
                      hazards. Potential neurotoxicity is
                      included in the health hazards
                      assessment. If there are reasons to
                      suspect neurotoxicologic effects (e.g.,
                      from structure-activity analysis,
                      information in the literature, or data
                      submitted by the manufacturer), EPA
 can issue a test rule requiring the
 manufacturer to develop data directed
 toward these effects. At the same time,
 EPA can restrict the chemical or
 prohibit it entirely from entering
 commerce until the required data are
 submitted and reviewed. In addition, for
 "old" chemicals (under section 4 of
 TSCA), if EPA suspects neurotoxicity, a
 test rule would be the mechanism used
 for obtaining the data. Many other
 statutes provide authority to regulate
 chemicals through the setting of
 standards, including the Clean Air Act,
 Clean Water Act, and Safe Drinking
 Water Act.
   Neurotoxicity is recognized as a
 health effect of concern under FIFRA,
 and there are neurotoxicity testing
 requirements for premarketing
 submission  of data to EPA for
 registration  of a pesticide under FIFRA. Consumer Product Safety

   The CPSC is an independent Federal
 regulatory agency with jurisdiction over
 most consumer products. Most chemical
 hazards are  regulated under the Federal
 Hazardous Substances Act (FHSA)
 administered by CPSC. The FHSA
 requires appropriate cautionary labeling
 on all hazardous household products
 (hazards include chronic toxicity such
 as neurotoxicity). While the FHSA does
 not require premarket registration, a
 manufacturer is required to assess the
 hazards of a product prior to marketing
 and assure that it is labeled with all
 necessary cautionary information. Tho
 FHSA also bans children's products that
 are hazardous and provides the CPSC
 with the authority to ban other
 hazardous products.  Agency for Toxic Substances
 and Disease Registry.

  ATSDR has a mission to prevent or
 mitigate adverse effects to both human
 health and the quality of life resulting
 from exposure to hazardous substances
 in the environment. The ATSDR
 publishes a National Priority List (NPL)
 of hazardous substances that are found
 at National Priority Waste Sites. The
 order of priority is based on an
 algorithm, taking into consideration
 frequency with which substances are
 found at NPL sites, toxicity, and
 potential for human exposure; this list
 is reranked on a yearly basis. So far, 129
toxicological profiles have been
 developed for the priority hazardous
substances, and 92 substances have a
profile with  a neurological health effect
endpoint (HAZDAT, 1992).
Neurotoxicity has been selected by the
ATSDR to be one of the seven high-

                 Federal Register  /  Vol. 59, No.  158 / Wednesday, August  17,  1994  /  Notices          42367
priority health conditions resulting from
exposure to environmental toxicants.

2.2. Basic Toxicological Considerations
for Neurotoxicity
2.2.1. Basic Toxicological Principles
  A chemical must enter the body,
reach the tissue target site(s), and be
maintained at a sufficient concentration
for a period of time in order for an
adverse effect to occur. Not all
chemicals have the same level of
toxicity; some may be very toxic in
small amounts while others may have
little effect even at extremely high
amounts. Thus, the dose-response
relationship is a major concept in
determining the toxicity of a specific
substance. Other factors in determining
toxicity include the physical and
chemical properties of the substance,
the route and level of exposure, the
susceptibility of the target tissue, and
the health, gender, and age of the
exposed individual.
   Once the toxic substance has entered
the body, usually through the lungs
(inhalation), the skin (absorption), or the
gastrointestinal tract (ingestion), it is
partitioned into various body tissues
where it can act on its target sites. The
substance is eliminated from the
bloodstream by the process of
accumulation into the various sites in
the body, with the liver and kidney
being major sites of accumulation of
toxic substances. This is thought to be
associated with these organs' large
blood capacity and major role in
elimination of substances from the
body. Lipophilic chemicals accumulate
in lipid-rich areas of the body and
present a significant potential problem
 for the nervous system. The nervous
 system is unique in its high  percentage
 content of lipid (50 percent  of dry
 weight) and may be particularly
 vulnerable to  such chemicals. The site
 or sites of accumulation for  a specific
 toxic substance may or may not be the
 primary sites  of action. Examples
 include two known neurotoxicants,
 carbon monoxide in the red blood cells
 and lead in the bone. It must be noted
 that some substances are not distributed
 throughout the body, partially as a
 function of their insolubility, polarity,
 or molecular weight.
   The effect that a substance has will
 generally depend on the body burden or
 level in the tissue and duration of
 exposure. The time course of the levels
 is determined by several factors,
 including the amount at time of
 exposure, duration of exposure, and
 metabolic fate of the chemical. The
 study of such metabolic processes,
 pharmacokinetics, has demonstrated
complex patterns in the absorption,
distribution, possible biotransformation,
and elimination of various substances
(Klaassen, 1980).
  Many substances are removed by the
kidney and excreted through the urine.
The liver can detoxify substances like
organic lead, which are excreted from
the liver into the bile and then the small
intestines, bypassing the blood and
kidney. Lipophilic toxic substances are
primarily removed from the body
through feces and bile, and water-
soluble metabolites  are removed in the
urine, through the skin, and through
expiration into the air.
Biotransformation is a biochemical
process that converts a substance into a
different chemical compound, allowing
it to be excreted more easily. Substances
are more easily removed if they are
biotransformed into a more hydrophilic
compound. Biotransformation can either
aid in the detoxification of a substance
or produce a more toxic metabolite.
Therefore, the original substance may
not be the substance that is producing
the toxicity on the nervous system or
any other system. Thus, several factors
must be taken into consideration when
evaluating the potential neurotoxicity of
a chemical. They include the
pharmacokinetics of the parent
compound, the target tissue
concentrations of the parent chemical or
its bioactivated proximate toxicant, the
uptake kinetics of the parent chemical
or metabolite into the cell and/or
membrane interactions, and the
interaction of the chemical or metabolite
with presumed receptor sites.
2.2.2. Basic Neurotoxicological
   Neurotoxicity can be manifest as a
structural or functional adverse
response of the nervous system to a
chemical, biological, or physical agent
(Tilson, 1990b).  It is a function of both
the property of the  agent and a property
of the nervous system itself.
Neurotoxicity refers broadly to the
adverse neural responses following
exposure to chemical or physical agents
 (e.g., radiation) (Tilson, 1990b). Adverse
 effects include any change that
 diminishes the ability to survive,
 reproduce, or adapt to the environment.
 Neuroactive substances may also impair
 health indirectly by altering behavior in
 such a way that safety is decreased in
 the performance of numerous activities.
 Toxicity can occur at any time in the life
 cycle, from conception through
 senescence, and its manifestations can
 change with age. The range of responses
 can vary from temporary responses
 following acute exposures to delayed
 responses following acute or chronic
exposure to persistent responses.
Neurotoxicity may or may not be
reversible following cessation of
exposure. The responses may be graded
from transient to fatal and there may be
different responses to the same
neurotoxicant at different dose levels
but similar responses to exposure to
different agents. Displays of a
neurotoxic response may be progressive
in nature, with small deficits occurring
early in exposure and developing to
become more severe over time.
Expression of neurotoxicity can
encompass multiple levels of
organization and complexity including
structural, biochemical, physiological,
and behavioral measurements.
   Caution must be exercised in labeling
a substance neurotoxic. The intended
use and effect of the chemical, the dose,
exposure  scenario and  whether or not
the chemical acts directly or indirectly
on the nervous system, must be taken
into consideration. A substance that
may be neurotoxic at a high
concentration may be safe and
beneficial at a lower concentration. For
example,  vitamin A, vitamin B6, are
required in the diet in trace amounts,
yet all result in neurotoxicity when
consumed in large quantities.
Pharmaceutical agents may also have
adverse effects at high  dose levels or
where the beneficial effects outweigh
the adverse side effects. For example,
antipsychotic drugs have allowed many
people suffering from schizophrenia to
lead relatively normal  lives; however,
chronic prescribed use of some of these
drugs may result in severe tardive
dyskinesia characterized by involuntary
movements of the face, tongue, and
limbs. Other examples include toxic
neuropathies induced  by
chemotherapeutic agents like cis-
platinum, toxic anticholinergic effects of
high doses of tricyclic antidepressants,
disabling movement disorders in
 patients treated with anti-Parkinsonian
 agents and major tranquilizers, and
 hearing loss and balance disruption
 triggered by certain antibacterials
 (Sterman and Schaumburg, 1980). Drugs
 of abuse  such as ethanol also have
 neurotoxic potential. Opiates such as
 heroin may lead to dependence, which
 is considered to be a long-term adverse
 alteration of nervous system
 functioning. Simultaneous exposure to
 drugs or  toxic agents may produce toxic
 interactions either in the environment
 or occupational settings. For example,
 exposure to noise and certain antibiotics
 can exacerbate the loss of hearing
 function (Boottcher et al., 1987; Lim,
 1986; Bhattacharyya and Dayal, 1984).
   The nervous system is a highly
 complex and integrated organ. It is

Federal  Register / Vol. 59, No.  158 / Wednesday, August 17, 1994 / Notices
 possible that nonlinear dose-response
 relationships or a threshold effect could
 exist for some agents. It has been
 hypothesized that the nervous system
 has a reserve capacity that masks subtle
 damage and any exposure that does not
 overcome this reserve capacity may not
 reach the threshold and no observable
 impairment will be evident (Tilson and
 Mitchell, 1983). However, the functional
 reserve may be depleted over time and
 the manifestations of toxicity may be
 delayed in relationship to the exposure.
 The reserve may be depleted by a
 number of factors including aging,
 stress, or chronic exposure to an
 environmental insult, in which case
 functioning will eventually be impaired
 and toxicity will become apparent. If a
 number of events occur simultaneously,
 the response  is progressive in nature, or
 there is a long latency between exposure
 and manifestation of toxicity, the
 identification of a single cause of the
 functional  impairment may not be

 2.3. Basic Neurobiological Principles
 2.3.1. Structure of the Nervous System
  The nervous system is composed of
 two parts: the central nervous system
 (CNS) and the peripheral nervous
 system (PNS) (Spencer and
 Schaumburg, 1980). Within the nervous
 system, there exist predominantly two
 general types of cells—nerve cells
 (neurons) and glial cells. Neurons have
 many of the same structures found in
 every cell of the body; they are unique,
 however, in that they have axons and
 dendrites, extensions of the neuron
 along which nerve impulses travel. The
 structure of the neuron consists of a cell
body, 10  to 100 um in diameter,
 containing  a nucleus and organelles for
 the synthesis of various components
 necessary for the cell's functioning, e.g.,
 proteins and lipids. There are numerous
 branch patterns of elongated processes,
 the dendrites, that emanate from the cell
body and increase the neuronal surface
 area available to receive inputs from
 other sources. Neurons communicate
 with each other by releasing chemical
 signals onto specific surface regions,
 receptors, of the other neuron. The axon
 is a process specialized for the
 conduction of nerve impulses away
 from the  cell toward the terminal
 synapses and eventually toward other
cells (neurons, muscle cells, or gland
  Neurons are responsible for the
reception, integration, transmission, and
storage of information (Raine, 1989).
Certain nerve cells are specialized to
respond to  particular stimuli. For
example, chemoreceptors in the mouth
                      and nose send information about taste
                      and smell to the brain. Cutaneous
                      receptors in the skin are involved in the
                      sensation of pressure, pain, heat, cold,
                      and touch. In the retina, the rods and
                      cones sense light. In general, the length
                      of the axon is tens to thousands of times
                      greater than the cell body diameter. For
                      example, the cell body whose processes
                      innervate the muscles in the human foot
                      is found in the spinal cord at the level
                      of the middle back. The axons of these
                      cells are more than a meter in length.
                      Many, but not all, axons are surrounded
                      by the layers of membrane from the
                      cytoplasmic process of glial cells. These
                      layers are called myelin sheaths and are
                      composed mostly of lipid. In the PNS,
                      the myelin sheaths are formed by
                      Schwann cells, while in the CNS the
                      sheaths are  formed by the
                      oligodendroglia. The myelin sheath
                      formed by one glial cell covers only a
                      short length of the axon. The entire
                      length of the axon is ensheathed in
                      myelin by numerous glial cells.  Between
                      adjacent glial sheaths, a very short
                      length of bare axon exists called the
                      node of Ranvier. In unmyelinated axons,
                      a nerve impulse must travel in a
                      continuous  fashion down the entire
                      length of the nerve. The presence of
                      myelin accelerates the nerve impulse by
                      up'to 100 times by allowing the  impulse
                      to jump from one node to the next in a
                      process called "saltatory conduction."
                        The nerve cells of the PNS are
                      generally found in aggregates called
                      ganglia. The brain and spinal cord make
                      up the CNS  and the neurons are
                      segregated into functionally related
                      aggregates called nuclei. They
                      synthesize and secrete
                      neurotransmitters, which are
                      specialized chemical messengers that
                      interact with receptors of other neurons
                      in the communication process. Various
                      nuclei together with the interconnecting
                      bundles of axonal fibers are functionally
                      related to one another to form higher
                      levels of organization called systems.
                      For example, there is the motor system,
                      the visual system, and the limbic
                      system. At the base of the brain,  several
                      small nuclei in the hypothalamus form
                      the neuroendocrine system, which plays
                      a critical role in the control of the
                      body's endocrine (hormone-secreting)
                      glands. Nerve cells in the hypothalamus
                      secrete chemical messengers into a short
                      loop of blood vessels that carries the
                      messengers to the pituitary gland which,
                      in turn, releases chemical messengers
                      into the general circulation. These
                      pituitary messengers regulate other
                      glands (e.g., the thymus and the
                      gonads). The entire system maintains a
 state of optimal physiological function
 for all of the body's organ systems.
 2.3.2. Transport Processes
  All types of cells must transport
 proteins and other molecular
 components from their site of
 production near the nucleus to the other
 sites in the cell (Hammerschlag and
 Brady, 1989). Neurons are unique in
 that the neuronal cell body must
 maintain not only the functions
 normally associated with its own
 support, but it must also provide
 support to its various processes. This
 support may require transport of
 material over relatively vast distances.
 Delivery of necessary substances by
 intracellular transport down the axon
 (axonal transport) represents a supply
 line that is highly vulnerable to
 interruption by toxic chemicals. In
 addition, the integrity of the function of
 the neuronal cell body is often
 dependent on a supply of trophic factors
 from the cells that it innervates. These
 factors are continually supplied to the
 neural cells by the process of retrograde
 axonal transport, often as a process of
 normal exchange between two or more
 cells. They play a significant factor in
 the normal growth and maintenance of
 the neural cells, and a continual supply
 of certain  trophic factors is necessary for
 cell functioning.
  The majority of axonal transport
 occurs along longitudinally arranged
 fiber tracks called neurofilaments. This
 movement along neurofilaments
 requires energy in the form of oxidative
 metabolism. Toxicants that interfere
 with this metabolism or that disrupt the
 spatial arrangement or production of
 neurofilaments may block axonal
 transport and can produce neuropathy
 (Lowndes and Baker. 1980). This can be
 seen following exposure to many
 substances, such as n-hexane and
 methyl n-butyl ketone as well as the
 drugs vincristine, vinblastine, and taxol.
 Acrylamide produces a dying-back
 axonopathy but by an alternative
 mechanism involving altered axonal

 2.3.3. Ionic Balance
  The axonal membrane is
 semipermeable to positively and
 negatively charged ions (mostly
 potassium, sodium, and chloride)
 within and outside of the axon. There
are several enzyme systems that
 maintain an ionic balance that changes
 following depolarization of the
membrane (Davies, 1968). This is
maintained only by the continual active
transport of ions across the membrane,
which requires an expenditure of
energy. The nerve impulse is a traveling

                Federal Register  / Vol.  59,  No. 158 / Wednesday. August 17. 1994 / Notices
wave of depolarization normally
originating from the cell body; however,
in sensory neurons it originates at the
terminal receptive end of specialized
axons (Davies, 1968). The wave is
continued by openings in the membrane
that allow ions to rush into the axon.
This sudden change in the charge across
the axon's membrane is the nerve
impulse. It is an amplified
depolarization that reaches the
threshold value and spreads  down the
axon from one length to another until
the next length of membrane reaches the
threshold value. It continues in this
fashion until it reaches the synaptic
terminal regions. There are a number of
varieties of membrane channels (e.g.,
calcium) that rapidly open and close
during impulse generation; the common
ones are the sodium and potassium
channels. They are very small and allow
only ions of a certain size to  pass.
Several classes of drugs (e.g., local
anesthetics) and natural toxins (e.g.,
tetrodotoxin) inhibit nerve impulse
conduction by blocking these channels.

2.3.4. Neurotransmission
  The terminal branches of the axon
end in small enlargements called
synaptic "boutons." It is from these
boutons that chemical messengers will
be released in order to communicate
with the target cell at the point of
interaction, the synapse (Hammerschlag
and Brady, 1989). When the nerve
impulse reaches the terminal branches
of the axon, it depolarizes the synaptic
boutons. This depolarization causes the
release of the chemical messengers
 (neurotransmitters and
neuromodulators) stored in vesicles in
 the axon terminal  (Willis and Grossman,
 1973). Classical neurotransmitters
 include serotonin, dopamine,
 acetylcholine, and norepinephrine and
 are typically secreted by one neuron
 into the synaptic cleft where they are on
 the postsynaptic membrane.
 Neuropeptides, however, may travel
 long distances through the bloodstream
 to receptors on distant nerve cells or in
 other tissues. Following depolarization,
 the amount of secretion is dependent on
 the number of nerve impulses that reach
 the synaptic bouton, i.e., the degree of
 depolarization. The chemical
 messengers diffuse across the synaptic
 cleft or into the intraneuronal space and
 bind to receptors on adjacent nerve cells
 or effector organs, thus triggering
 biochemical events that lead to
 electrical excitation or inhibition.
    When information is transmitted from
 nerves to muscle fibers, the point of
 interaction is called the neuromuscular
 junction and the interaction leads to
 contraction or relaxation of the muscle.
When the target is a gland cell, the
interaction leads to secretion. Synaptic
transmission between neurons is
slightly more complicated, but still
dependent on the opening and closing
of ion channels in the membrane. The
binding of the messenger to the receptor
of the receiving cell can lead to either
the excitation or inhibition of the target
cell. At an excitatory synapse, the
neurotransmitter-receptor interaction
leads to an opening in certain ion-
specific channels. The charged ions that
move through these opened chambers
carry a current that serves to depolarize
the cell membranes. At inhibitory
synapses, the interaction leads to an
opening in a different type of ion-
specific channel that produces an
increase in the level of polarization
(hyperpolarization). The sum of all the
depolarizing and hyperpolarizing
currents determines the transmembrane
potential and when a threshold level of
depolarization is reached at the axon's
initial segment, a nerve impulse is
generated and begins to travel down the
  The duration of neurotransmitter
action is primarily a  function of the
length of time it remains in the synaptic
cleft. This duration is very short due to
specialized  enzymes that quickly
remove the  transmitter either by
degrading it or by reuptake systems  that
transport it  back into the synaptic
bouton. A toxic substance may disrupt
this process in several different ways. It
is important that the duration of the
effect of synaptically released chemical
messengers be limited. Some
neurotoxicants, e.g., cholinesterase-
inhibiting organophosphorous
 pesticides, inhibit the enzyme (AChE),
 which serves to terminate the  effect of
 the neurotransmitter (acetylcholine) on
 its target. The result is an
 overstimulation of the target cell. Other
 substances, particularly biological
 toxins, are able to interact with the
 receptor molecule and mimic the action
 of the neurotransmitter. Some toxic
 substances, like neuroactive
 Pharmaceuticals, may interfere with the
 synthesis of a particular
 neurotransmitter, while others may
 block the neurotransmitter's access to its
 receptor molecule.
 2.4. Types of Effects on the Nervous
    The normal activity of the nervous
 system can be altered by many toxic
 substances. A variety of adverse health
 effects can be seen ranging from
 impairment of muscular movement to
 disruption of vision and hearing to
 memory loss and hallucinations (WHO,
 1986; Anger, 1984,1990). Toxic
substances can alter both the structure
and the function of cells in the nervous
system. Structural alterations include
changes in the morphology of the cell
and its subcellular structures. In some
cases, agents produce neuropathic
conditions that resemble naturally
occurring neurodegenerative disorders
in humans (Calne et al.,  1986). Cellular
alterations can include the
accumulation, proliferation, or
rearrangement of structural elements
(e.g., intermediate filaments,
microtubules) or organelles
(mitochondria) as well as the
breakdown of cells. By affecting the
biochemistry and/or physiology of a
cell, a toxic substance can alter the
internal environment of any  neural cell.
Intracellular changes can result from
oxygen deprivation (anoxia)  because
neurons require relatively large
quantities of oxygen due to their high
metabolic rate.
   Many times the response of the
nervous system to a toxic substance can
be a slow degeneration of the nerve cell
body or axon that may result in
permanent neuronal damage.
Substances can act as a cytotoxicant
after having been transported into the
nerve terminal. A complete loss of nerve
cells can  occur following exposure to a
number of toxic substances.  Sensory
nerve cells may be lost following
treatment with megavitamin doses of
vitamin B6; hippocampal neurons
undergo degeneration with trimethyltin
and trimethyl lead poisoning; motor
nerve cells are affected in cycad
toxicity, which has been loosely linked
to Guam-ALS-Parkinsonism dementia.
Acute carbon monoxide poisoning can
 produce a delayed, progressive
 deterioration over a period of weeks  of
 portions  of the nervous system that may
 lead to psychosis and death. Substances
 such as mercury and  lead can cause
 central nervous system dysfunction.  In
 children, mercury intoxication can
 cause degeneration of neurons in the
 cerebellum and can lead to tremors,
 difficulty in walking, visual
 impairment, and even blindness. Lead
 affects the cortex of the immature brain,
 resulting in mental retardation.
   At the cellular level, a substance
 might interfere with cellular processes
 like protein synthesis, leading to a
 reduced  production of
 neurotransmitters and brain dysfunction
 (Bondy,  1985). Nicotine and some
 insecticides mimic the effects of the
 neurotransmitter acetylcholine.
 Organophosphorous  compounds,
 carbamate insecticides, and nerve gases
 act by inhibiting AChE, the enzyme  that
 inactivates the neurotransmitter
 acetylcholine. This results in a buildup

Federal  Register / Vol. 59. No.  158 / Wednesday,  August  17,  1994  /  Notices
 of acetylcholine and can lead to loss of
 appetite, anxiety, muscle twitching, and
 paralysis. Amphetamines stimulate the
 nervous system by releasing and
 blocking reuptake of the
 neurotransmitters norepinephrine and
 dopamine from nerve cells. Cocaine
 affects the release and reuptake of
 norepinephrine, dopamine, and
 serotonin. Both drugs can cause
 paranoia, hyperactivity, aggression, high
 blood pressure, and abnormal heart
 rhythms. Opium-related drugs such as
 morphine and heroin act at specific
 opioid receptors in the brain, producing
 sedation, euphoria, and analgesia. They
 also tend to slow the heart rate and
 cause nausea, convulsions, and slow
 breathing patterns. Other substances can
 alter the synthesis and release of
 specific neurotransmitters and activate
 their receptors  in specific neuronal
 pathways. They may perturb the system
 by overstimulating receptors, blocking
 transmitter release and/or inhibiting
 transmitter degradation, or blocking
 reuptake of neurotransmitter precursors.
   Also at the cellular level, the flow of
 ions such as calcium, sodium, and
 potassium across the cell membrane
 may be changed and the transmission of
 information between nerve cells altered.
 A substance may interfere with the ionic
 balance of a neuron. Organophosphate
 and carbamate insecticides produce
 autonomic dysfunction and
 organochlorine insecticides increase
 sensorimotor sensitivity, produce
 tremors and in some cases cause
 seizures and convulsions (Ecobichon
 and Joy. 1982).  Lindane, DDT,
 pyrethroids, and trimethyltin also
 produce convulsions. Conversely,
 solvents act to raise the threshold for
 eliciting seizures or act to reduce the
 severity or duration of the elicited
  The role of excitatory amino acid
 (EAA)-mediated synaptic activation is
 critical for normal function of the CNS.
 Because endogenous EAA-mediated
 synaptic transmission is a widespread
 excitatory system in the brain and is
 involved in the process of learning and
 memory, the issue of the effects of
 endogenous and exogenous EAA-related
 toxicity has broad implications for both
 CNS morbidity  and mortality in
 humans. Much  of the injury and
 neuronal death  associated with toxicity
 is mediated by receptors for excitatory
 amino acids, especially glutamic acid.
 When applied in sufficient excess from
 either endogenous or exogenous
 sources, EAAs have profound
 neurotoxic effects that can result in the
 destruction of neurons and, as a
consequence, lead to acute phase
confusion, seizures, and generalized
                      weakness or to persistent impairments
                      such as memory loss (Choi, 1988).
                        A final common path in the activation
                      of these receptor classes is an increase
                      in free cytosolic Ca~ that can result in
                      the release and activation of
                      intracellular enzymes (which break
                      down the cytoskeleton) and in further
                      release of glutamate, both of which can
                      be cytotoxic (Choi, 1988). Critical to an
                      understanding of the etiopathology
                      associated with  at least some of the
                      neurotoxic degeneration may be the link
                      that impaired energy metabolism could
                      have with excitotoxic neuronal death. It
                      is likely that reduced oxidative
                      metabolism results in the partial
                      depolarization of resting membrane
                      potential, the activation of ionotropic
                      membrane receptor/channels, and the
                      influx of Ca~ or its release  from
                      intracellular stores.
                        The nervous system is dependent on
                      an extensive system of blood  vessels
                      and capillaries to deliver large
                      quantities of oxygen and nutrients as
                      well as to remove toxic waste products.
                      Damage to the capillaries in the brain
                      can lead to the swelling characteristic of
                      encephalopathy. This can be seen
                      following exposure to higher
                      concentrations of lead. Other metals
                      (e.g., cadmium, thallium, and mercury)
                      and organotin (e.g., trimethyltin) cause
                      rupturing of vessels that can also result
                      in encephalopathy.
                        One large aspect of function that may
                      be affected by neurotoxicants is
                      behavior,  which is the product of
                      various sensory, motor, and associative
                      functions of the  nervous system.
                      Neurotoxic substances can adversely
                      affect sensory or motor functions,
                      disrupt learning and memory processes,
                      or cause detrimental behavioral effects;
                      however,  the underlying mechanisms
                      for these effects  have yet to  be
                      determined. Although changes may be
                      subtle, the assessment of behavior may
                      serve as a robust means of monitoring
                      the well-being of the organism (Tilson
                      and Cabe, 1978).

                      2.5. Special Considerations

                      2.5.1. Susceptible Populations
                       Everyone is at a certain level of risk
                      of being adversely affected by
                      neurotoxic substances. Individuals of
                      certain age groups, health states, and
                      occupations, however, may be at a
                      greater level of risk. Fetuses, children,
                      the elderly, workers in occupations
                      involving exposure to relatively high
                      levels of toxic chemicals, and persons
                      who abuse drugs are among those in
                      high-risk groups. Neurotoxic substances
                      may exacerbate existing neurological  or
                      psychiatric disorders in a population.
 Although controversial (Waddell, 1993),
 recent evidence suggests that there may
 be a subpopulation of people who have
 become sensitive to chemicals and
 experience adverse reactions to low-
 level exposures to environmental
 chemicals (Bell, et al., 1992).
 Confounded in all of these groups is the
 role that nutrition plays in the response
 of the organism to exposure. Both
 general nutritional status and specific
 nutritional deficiencies (for example,
 protein, iron, and calcium) can
 significantly influence the response to a
 toxic substance.
   It is widely accepted that during
 development adverse effects can result
 from exposure to some chemicals at
 lower levels than would be necessary
 for the average adult (Suzuki, 1980). The
 developing nervous system appears to
 be differentially sensitive to  some kinds
 of damage (Cushner, 1981; Pearson and
 Dietrich, 1985; Annau and Eccles, 1986;
 Hill and Tennyson, 1986; Silbergeld,
 1986). During the developmental period,
 the nervous system is actively growing
 and establishing intricate cellular
 networks. Both the blood-brain and
 blood-nerve barriers that will eventually
 protect much of the adult brain, spinal
 cord, and peripheral nerves are
 incomplete. The protective mechanisms
 by which the organism deals with toxic
 substances, such as the detoxification
 systems,  are not fully developed.
 Exposure to chemicals during
 development can result in a range of
 effects. At the highest exposure, effects
 include death, gross structural
 abnormalities, or altered growth. Larger
 populations are generally exposed to
 more moderate  levels resulting in more
 subtle functional impairments. The
 qualitative nature of some injuries
 during development may differ from
 those seen in the adult, such as changes
 in tissue  volume, misplaced  or
 misoriented neurons, or delays or
 acceleration of the appearance of
 functional or structural endpoints
 (Rodier, 1986). In many cases, the
 results of early injuries may become
 evident only as the nervous system
 matures and ages (Rodier, 1990). There
 are several instances in which
 functional alterations have resulted
 from exposure during the period
between conception and sexual maturity
 (Riley and Vorhees, 1986; Vorhees,
  Early exposure to relatively low levels
of lead can result in reduced  scores on
tests of mental development  (Bellinger
et al., 1987; Needleman, 1990). Early
gestational exposure to neurotoxicants
such as cocaine can produce  long-term
neurobehavioral abnormalities
(Anderson-Brown et al., 1990;

                 Federal Register  /  Vol.  59, No. 158 / Wednesday, August  17,  1994  /  Notices          42371
Hutchings et al.. 1989); heavy alcohol
exposure produces craniofacial
abnormalities and mental retardation
(Jones and Smith, 1973), while moderate
levels of alcohol consumption during
gestation can delay motor development
(Little et al., 1989).
  With aging, the level of risk for a
number of health-related factors
increases; it has been hypothesized that
the risk for toxic perturbations to the
nervous system also increases with age
(Weiss, 1990). It is generally believed
that with increasing age  comes a
decreased ability of the nervous system
to respond to adverse events or to
compensate for either biological,
physical, or toxic effects. At the tissue
and cellular level, the aging process can
result in nerve cell loss,  formation of
neurofibrillary tangles (abnormal
accumulation of certain  filamentous
proteins) and neuritic plaques
(abnormal clusters of proteins and other
substances near synapses). As cells die,
the complex neuronal circuitry of the
brain becomes impaired.
Neurotransmitter concentrations and the
enzymes involved in their synthesis
may be altered. Some axons can
gradually lose their myelin sheath,
resulting in a slowed conduction of
nerve impulses along the axon. It has
been postulated that with age, not only
might the nervous system become more
susceptible to new insults, but the
effects of previous exposures also may
become evident, with a  diminished
capacity for compensation  (Weiss,
 1990). The increased incidence of
 multiple drug-taking in  the elderly
 population might also lead to
 interactions, either drug/drug or drug/
 chemical, which can adversely affect the
 nervous system. Nutritionally, the aged
 experience increased incidences of both
 general undernutrition and deficits of
 specific nutrients such as iron or
 calcium, which might influence  the
 response to toxic substances.
   In the geriatric population, the
 clinical manifestation of
 neurodegenerative disorders may have a
 contributing component of past
 exposures to environmental chemical
 agents. Calne et al. (1986) hypothesized
 that various agents contribute to
 Alzheimer's disease, Parkinson's
 disease, or amyotrophic lateral sclerosis
 (ALS, motoneurone disease, or Lou
 Gehrig's disease) by depleting neuronal
 reserves to an extent that perturbations
 become observable in the context of the
 natural aging process. B-N-
 methylamino-L-alanine, from the seed
 of the false sago palm (Cycas circinalis
 L.), has been reported to induce a form
 of amyotrophic lateral sclerosis
 (Spencer et al., 1987). Alzheimer-type
syndromes have been reported in
individuals occupationally exposed to
organic solvents or metal vapors (Freed
and Kandel, 1988). Severe cognitive
dysfunction has been noted in
Alzheimer's disease and aluminum
intoxication (Yokel et al., 1988).
  At any age, preexisting physical as
well as mental disorders of the
individual may play a significant role in
the manifestation of a toxic response
following exposure to a potentially toxic
substance. Both types of disorders
compromise the system in some way so
that either the defense mechanisms of
the organism are not able to deal with
the toxic substance or are not able to
repair themselves quickly. In addition to
the basic altered biology, for individuals
with a physical or mental disorder who
are under some form of medical
intervention, the combination of
therapeutic drugs and toxic substances
may have an interactive effect on the
nervous system. For example, due to the
delicate electrochemical balance of the
nervous system, mental disorders may
be exacerbated by exposure to a toxic
2.5.2. Blood-Brain and Blood-Nerve
   The bioavailability of a specific
chemical to the nervous system is a
function of both the target tissue and the
chemical. The brain, spinal cord, and
peripheral nerves are surrounded by a
series of semipermeable tissues referred
to as the blood-brain and blood-nerve
barriers (Katzman, 1976; Peters et al.,
 1991). In the central nervous system, the
blood-brain barrier is composed of tight
 junctions formed by endothelial cells
 and astrocytes. These tight junctions
 and cellular interactions forming the
barrier restrict the free passage of most
 bloodborne substances. By doing this,
 they create a finely controlled
 extracellular environment for the nerve
 cells. Certain regions of the brain and
 nerves are directly exposed to chemicals
 in the blood because the barrier is not
 present in some areas of the nervous
 system.  For example, it is absent in the
 circumventricular area, around the
 dorsal root ganglion in the peripheral
 nervous system, and around the
 olfactory nerve, which may allow
 chemicals to penetrate directly from the
 nasal region to the frontal cortex.
   The existence of these blood-brain
 and blood-nerve barriers suggests that
 proper functioning of the nervous
 system is dependent on control of the
 substances to which nerve cells are
 exposed. The term "barrier," however,
 is somewhat of a misnomer. Although
 water-soluble and polar compounds
 enter the brain poorly, lipophilic
substances readily cross the barrier. In
addition, a series of specific transport
mechanisms exist through which
required nutrients (hormones, amino
acids, peptides, proteins,  fatty acids,
etc.) reach the brain (Pardridge, 1988). If
toxicants are lipid soluble or if they are
structurally similar to substances that
are normally transported into the brain,
they can achieve high concentrations in
brain tissue. It has been proposed that
one reason why the developing nervous
system may be differentially sensitive to
some toxicants is that the blood-brain
barrier is less effective than in an adult.
The effectiveness of the blood-brain
barrier may also be changed by
chemical-induced physiological events
such as metabolic acidosis and
nutritional deprivation.

2.5.3. Metabolism
   The central nervous system has a very
high metabolic rate and, unlike other
organs, the brain depends almost
entirely on glucose as a source of energy
and raw material for the synthesis of
other molecules (Damstra and Bondy,
1980). The absence of an alternative
energy  source makes the  CNS critically
dependent on an uninterrupted supply
of oxygen as well as the proper
functioning of enzymes that metabolize
glucose. Substances can be toxic to the
nervous system if they perturb neuronal
metabolism. Without glucose, nerve
cells usually begin to die within
minutes. Despite its relatively small
size, the energy demands of the brain
require 14 percent of the heart's output
and consumes about 18 percent of the
oxygen absorbed by the lungs.
 2.5.4. Limited Regenerative Ability
   The nervous system has a
 combination of special features not
 found in other organ systems. It is
 composed of a variety of metabolically
 active neurons and supporting cell types
 that interact through a multitude of
 complex chemical mechanisms. Each
 cell type has its own functions and
 vulnerabilities. At the time of puberty,
 the system is fully developed and
 neurogenesis (the birth of new neurons
 from cell division of precursor cells
 called  neuroblasts) ceases. This is in
 marked and significant contrast to
 almost all other tissues,  where cell
 replacement is continual.
    It is  this loss of neurogenesis that
 limits  the nervous system's ability to
 recover from damage and influences the
 plasticity of the  system. Neurons are
 unable to regenerate following damage;
 therefore, they are no longer able to
 perform their normal functions. Toxic
 damage to the brain or spinal cord that
 results in cell loss is usually permanent.

Federal  Register / Vol. 59, No.  158  /  Wednesday,  August 17, 1994  / Notices
 If nerve cell loss is concentrated in one
 of the CNS's functional subsystems, the
 outcome could be debilitating; for
 example, a relatively small loss of
 neurons that use acetylcholine as their
 neurotransmitter may produce a
 profound disturbance of memory. A
 relatively minor insult concentrated in a
 subsystem that relies on dopamine as its
 neurotransmitter may drastically impair
 motor coordination.  However, in
 response to injury, neurons are able to
 show considerable plasticity both
 during development and after
 maturation. Damage  to the nervous
 system alters connectivity between the
 surviving neurons, permitting
 functional adjustments to occur to
 compensate for the damage. Such
 responsiveness may, in and of itself,
 have profound consequences for
 neurological, behavioral, and related
 body functions.
  After damage to  axons in the
 peripheral nerves, if  the neurons are not
 damaged, the axons have the ability to
 regenerate and to attempt to reach their
 original target site. This is the basis, for
 example, of the eventual return of
 sensation  and muscle control in a
 surgically reattached limb. Neurons in
 the CNS also have  the ability to
 regenerate interrupted axons; however,
 they have a much more difficult task in
 reaching their original targets due to
 both the presence of scar tissue formed
 by proliferating glia and to the increased
 complexity of the connectivity in the

 3. Methods for Assessing Human
 3.1. Introduction
  This chapter outlines and discusses
 current methods for detecting
 neurotoxicity in  humans. In contrast to
 studies of neurotoxicity in animals
 where functional changes readily can be
 correlated with neuroanatornic and
 neurochemical alterations, there are
 ethical and technical  barriers to the
 direct observation of neuronal damage
 in humans. Neurotoxicity in humans is
 most commonly  measured by relatively
 noninvasive neurophysiologic and
 neurobehavioral methods that assess
 cognitive,  affective, sensory, and  motor
 function. The evaluation of human
 neurotoxicity and the relevance to risk
 assessment will be discussed within the
 context of clinical evaluation,
 epidemiologic/worksite studies, and
 human laboratory exposure studies.

 3,2. Clinical Evaluation
  Neurobehavioral assessment methods
are used extensively in clinical
neurology and neuropsychology to
                      evaluate patients suspected of having
                      neurologic disease. An extensive array
                      of examiner-administered and paper
                      and pencil tasks are used to assess
                      sensory, motor, cognitive, and affective
                      functions and personality states/traits.
                      Neurobehavioral data are synthesized
                      with information from neurophysiologic
                      studies, imaging techniques, medical
                      history, etc., to derive a working
                      diagnosis. Clinical diagnostic
                      approaches have provided a rich
                      conceptual framework for
                      understanding the functions (and
                      malfunctions) of the central and
                      peripheral nervous systems and have
                      formed the basis for the development of
                      methods for measuring the behavioral
                      expression of nervous system disorders.
                      Human neurobehavioral toxicology has
                      borrowed heavily from neurology and
                      neuropsychology for concepts of
                      nervous system impairment and
                      functional assessment methods.
                      Neurobehavioral toxicology has adopted
                      the neurologic/neuropsychologic model,
                      using adverse changes in behavioral
                      function to assist in identifying
                      chemically or drug-induced changes in
                      nervous system processes.

                      3.2.1. Neurologic Evaluation

                        Assessment of neurobehavioral
                      function by the clinical examination of
                      a patient has long been used as a
                      primary tool in neurologic diagnosis.
                      The domains of cognitive function,
                      motor function, sensation, reflexes, and
                      cranial nerve function are a standard
                      part of the clinical neurologic exam.
                      Movement and gait, speech fluency and
                      content, verbal memory, deep tendon
                      reflexes, muscle strength, symmetry of
                      movement and strength, ocular
                      movements, sensory function (pressure,
                      vibration, visual, auditory), motor
                      coordination, and logical reasoning are
                      only a few of the functions assessed by
                      neurologists (Denny-Brown et al., 1982).
                       Trained and experienced clinicians
                      gather these data by observation, verbal
                      exchange, and direct examination.
                      Neurologic exams arc sensitive
                      indicators of neurologic disease; the
                      data have predictive value for the
                      diagnosis of underlying nervous system
                      disease, and the methods have been
                      extensively validated against other
                      diagnostic procedures (e.g., imaging,
                      neurophysiologic testing), the course of
                      the illness, and autopsy findings.
                      Examination of the patient in a
                      semistructured procedure can yield a
                      wealth of information and insights
                      about functional impairment and the
                      underlying neuropathology.
 3.2.2. Neuropsychological Testing
   Neuropsychologists have developed
 quantitative methods to supplement
 clinical neurologic exam and laboratory
 data for the diagnosis of neurologic
 disease. Currently, two assessment
 batteries, the Luria-Nebraska and the
 Halstead-Reitan, and shorter versions
 are used in clinical practice. The
 batteries consist of subtests that quantify
 a wide spectrum of cognitive, motor,
 sensory, intellectual, affective, and
 personality functions. The pattern of
 relative performance on the subtests can
 be interpreted along  with historical and
 medical data to suggest the presence or
 absence of neurologic disease and the
 possible anatomic location of any focal
 lesions or degeneration. Clinical
 interpretation of the  data is enhanced by
 data on age-related population norms
 for many subtests and by the systematic
 observation of the patient during testing.
   Several neurotoxicity assessment
 batteries use components of
 neuropsychological tests and have
 adapted and shortened analogs of some
 subtests. Tests derived from the
 Wechsler Adult Intelligence  Scale—
 Revised (WAIS-R) have been used
 frequently to assess neurobehavioral
 impairment from chemical agents, and
 other abbreviated variations of
 neuropsychological battery subtests
 have been incorporated into
 neurobehavioral toxicity batteries and
 used in field and laboratory studies.
 3.2.3. Applicability of Clinical Methods
 to Neurotoxicology Risk Assessment
  Neurologic and neuropsychologic
 methods have long been employed to
 identify the adverse health effects of
 environmental workplace exposures.
 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 using these clinical
 methods. It is very important to make
 external/internal dose measurements in
humans in order to determine the actual
 dose(s) which can cause unwanted
  Aspects of the clinical neurologic
examination approach limit its
usefulness for neurotoxicologic risk
assessment. Information obtained from
the neurologic exam is mostly
qualitative and descriptive rather than
quantitative. Estimates of the severity of
functional impairment can be reliably

                 Federal Register  /  Vol. 59, No.  158 / Wednesday, August  17,  1994  / Notices
placed into only three or four categories
(for example, mild, moderate, severe).
Much of the assessment depends on the
subjective judgment of the examiner; the
magnitude and symmetry of muscle
strength are often judged by having the
patient push against the resistance of
the examiner's hands. The datum is
therefore the absolute and relative
amount of muscle load sensed by the
examiner in his or her arms.
  Compared with other methods, the
clinical neurologic exam may be less
sensitive in detecting early
neurotoxicity in peripheral sensory and
motor nerves. While clinicians'
judgments are equal in sensitivity to
quantitative methods in assessing the
amplitude of tremor, tremor frequency
is poorly quantified by clinicians. Thus,
important aspects of the clinical
neurologic exam  may be insufficiently
quantified and lack sufficient sensitivity
for detecting early neurobehavioral
toxicity produced by environmental or
workplace exposure conditions.
However, a neurologic evaluation of
persons with documented
neurobehavioral  impairment would be
helpful for identifying nonchemical
causes, such as diabetes and
cardiovascular insufficiency.
   Administration of a
 neuropsychological battery also requires
 a trained technician, and interpretation
 requires a trained and experienced
 neuropsychologist. Depending on the
 capabilities of the patient, 2 to 4 hours
 may be needed to administer a full
 battery; 1 hour may be needed for the
 shorter screening versions. These
 practical considerations may limit the
 usefulness of neuropsychological
 assessment in large field studies of
 suspected neurotoxicity.
   In addition to  logistical problems in
 administration and interpretation,
 neuropsychological batteries and
 neurologic exams share two
 disadvantages with respect to
 neurotoxicity risk assessment. First,
 neurologic exams and
 neuropsychological test batteries are
 designed to confirm and classify
 functional problems in individuals
 selected on the basis of signs and
 symptoms identified by the patient,
 family, or other  health professionals.
 Their usefulness in detecting low-base
 rate impairment in workers or the
 general population maybe generally
 thought to be limited, decreasing the
  usefulness of clinical assessment
  approaches for epidemiologic risk
    Second, neurologic exams and
  neuropsychologic test batteries were
  largely 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 primarily validated
against these neurologic disease states.
There has been insufficient research to
demonstrate which tests designed to
assess functional expression of
neurologic disease are most useful in
characterizing the modes of CNS
impairment produced by chemical
agents and drugs. More research is
needed to validate the usefulness of
neuropsychologic test methods in
3.3. Current Neurotoxicity Testing
3.3.1. Neurobehavioral Methods

  Chemical agents directly or indirectly
affect a wide range of nervous system
activities. Many of these chemical
actions are expressed as alterations of
behavior; Anger (1990a) lists 35
neurobehavioral effects of chemical
exposure that illustrate alterations in
sensory, motor, cognitive, affective, and
personality function. Professional
judgment is important in the
interpretation of data from studies using
neurobehavioral methods since some
endpoints can be subjective.
   Dozens of tests of neurobehavioral
 function have been proposed or used in
 field or laboratory studies to assess the
 neurotoxicity of chemical agents. Table
 3-1 lists some frequently used tests of
 motor, sensory, cognitive, and affective
 neurobehavioral function.

                    TABLE 3-1 .—NEUROBEHAVIORAL

Wechsler Memory
Profile of Mood
States© (POMS).
Flicker Fusion.
Lanthony (color
Pursuit Aiming.
Finger Tapping.
Postural Stability.
Reaction Time.
Santa Ana Peg
Benton Visual Re-
Continuous Per-
  formance Task.
Digit Span.
Dual Tasks.
Symbol-Digit Task.
Wechsler Adult In-
  telligence Scale—
  Revised© (Com-
  In contrast to the individual focus in
clinical evaluation, neurobehavioral
tests primarily have been used to
evaluate differences between groups,
comparing unexposed groups with
persons environmentally or
occupationally exposed to a suspected
neurotoxic agent. An ideal evaluation of
groups for quantitative evidence of
chemically induced neurobehavioral
impairment would involve the
assessment of a wide variety of
functions, but testing all possible
neurobehavioral functions that might be
affected in a group of exposed workers,
for example, would be impossible.
Therefore, a testing strategy has been to
use limited  number tests that sample
representative neurobehavioral
functional domains such as dexterity,
visual memory, and reaction time. Test batteries.
   Many field and laboratory studies
have selected neurobehavioral methods
according to available information about
the spectrum of effects of the suspected
neurotoxic agent(s). This focused
strategy is useful for answering specific
questions about known neurotoxins. To
identify unspecified neurotoxic effects
in groups of workers or to characterize
the effects of less well-studied
chemicals or mixtures of chemicals,
 several tests that sample a
 representative range of functional
 domains  have been grouped into test
batteries. The advantage of a
 standardized battery is that data from
 different  study populations and
 chemical classes can be compared, and
 similarities in effects observed (Johnson,
 1987). Standardized batteries can be
 categorized into investigator-
 administered and computer-
 administered types. Investigator-administered test
    The WHO-recommended
 Neurobehavioral Core Test Battery
 (NCTB) (Johnson, 1987), the Finnish
 Institute of Occupational Health (FIOH)
 (Hanninen, 1990), and the Pittsburgh
 Occupational Exposures Test Battery
 (POET) (Ryan et al., 1987) are three
 commonly used batteries. The NCTB is
 frequently used in field studies
 worldwide and can be fit inside a

Federal  Register / Vol. 59. No.  158 / Wednesday. August 17,  1994 / Notices
 medium-sized suitcase for transport.
 The NCTB consists of the following
 tests: simple reaction time task, digit-
 symbol coding task, timed motor
 coordination test (Santa Ana pegboard),
 digit span memory test. Benton Visual
 Retention test, pursuit aiming test, and
 the Profile of Mood States (POMS).
 Based on factor-analytic studies
 (Hooisma et al., 1990). these tests are
 believed to measure the functional
 domains of immediate memory,
 attention, dexterity/hand-eye
 coordination, reaction time, and mood.
 Long-term memory, verbal and language
 functions, auditory sensation, judgment,
 and so forth are not assessed. Computerized test batteries.
  Computerized tests and batteries have
 been developed for field and laboratory
 use. The Neurobehavioral Evaluation
 System (NES) (Baker et al., 1985),
 MicroTox (Eckerman et al., 1985),  the
 SPES (Iregren et al., 1985), and the
 NCTR Operant Battery (Paule et al.,
 1990) are computerized systems
 developed for neurotoxicity assessment.
 Current versions of the NES, for
 example, consist of about 15 different
 neurobehavioral tests, and the battery
 has been used in epidemiologic studies
 of groups exposed to  solvent, pesticide,
 and mercury, and in laboratory studies
 of NCb, ethanol, and toluene (Letz,
  Although many computerized tests
 appear to tap similar neurobehavioral
 domains as noncomputerized batteries,
 the visual mode of presentation, the
 manual mode of response, and the
 emphasis on speed of responding are
believed to have led to significant
 differences in results  obtained from
computerized versus noncomputerized
 forms of similar tests. Attempts to
clarify the differences between
computerized and noncomputerized test
batteries have met with difficulty.
Although some tests are similar in  each
type of battery, size and duration of
stimuli, presentation and response
modality, number of trials, and scoring
vary arbitrarily, preventing direct
comparison. An example is the digit-
symbol test on the NCTB and the
symbol-digit test on the NES. Although
almost identical in task requirements,
procedural and scoring differences
prevent direct comparison of the results
from these two tests.
  Postural stability is an aspect of
integrated sensory and motor function
that increasingly is being evaluated in
clinical, epidemiologic, and laboratory
investigations of effects of pesticides
and solvents, and would be useful  for
assessing therapeutic  drug-induced
movement disorders such as
                      neuroleptics. Measurement of postural
                      stability requires a computer, special
                      software, monitor, and a force
                      transduction platform on which the
                      subjects must stand (Dick et al., 1990).
                      Mechanical and capacitive field
                      methods for assessing the amplitude
                      and frequency of tremor also are seeing
                      more frequent use.
                        An advantage of computerized testing
                      is the standardization of test
                      presentation, but a disadvantage is the
                      need for delicate, expensive computers
                      and measurement devices that require
                      transport for field studies.
                      Noncomputerized test batteries may be
                      less costly to purchase and easier to
                      transport, enhancing their desirability in
                      field studies, but test administrators
                      require training and small differences in
                      test administration may affect the data.

                      3.3.2. Neurophysiologic Methods
                        With improvements in the capabilities
                      and size of equipment, quantitative
                      neurophysiologic measurement of
                      sensory and motor function will be
                      increasingly useful in human
                      neurotoxicity evaluations. A major
                      advantage of these methods for risk
                      assessment is that they can be assessed
                      in both human and animal subjects and
                      the data can be  interpreted in an
                      homologous manner.
                       Electromyographic responses (EMG)
                      and nerve conduction velocity (NCV)
                      have been used in the assessment of
                      peripheral nerve neurotoxicity. Some
                      techniques require that needle
                      electrodes be placed beneath the skin
                      for stimulation and recording and are
                      therefore somewhat uncomfortable for
                      the subject. However, the methods are
                      quantitative, provide multiple
                      endpoints of 1'NS function, and have
                      clinical relevance.
                       The adverse effects of solvents,
                      pesticides, and metals have been
                      identified with EMG/NCV
                      neurophysiologic measures. Although
                      not reduced as a function of duration of
                      employment, maximum  nerve
                      conduction velocity (MCV) has been
                      reported to vary systematically with
                      cumulative exposure to carbon disulfide
                      (Johnson et al.. 1983), suggesting that
                      this measure may be particularly
                      valuable for quantitative risk assessment
                      of some types of peripheral motor nerve
                       Noninvasive neurophysiologic test
                      methods used in neurotoxicity
                      evaluations include the
                      electroencephalogram (EEC), visually
                      evoked response (VER), somatosensory
                      evoked potential (SEP), and the
                      brainstem auditory evoked response
                      (BAER). The EEC is the summed
                      electrical activity of neurons  measured
 with scalp electrodes; voltage and
 frequency are primary measures. Evoked
 methods employ specific eliciting
 stimuli applied to the sense organs to
 measure nervous system electrical
 response. Visual patterns, sounds, and
 cutaneous stimuli are presented to the
 subject, and "evoked" voltage changes
 in the nervous system are  measured
 with skin electrodes.
  While EEGs were developed as a tool
 in the neurologic diagnosis of seizure
 disorders and other brain diseases, dose-
 related EEC changes in chemically
 exposed (especially solvents and
 styrene) individuals have been noted
 (Seppalainen and Harkonen, 1976). EEC
 measurement requires large recording
 devices that can be used in the
 laboratory or clinic, but are difficult to
 use in field studies. However, compact
 computerized recording equipment has
 been developed, and automated spectral
 analyses of EEGs have recently been
 applied to neurotoxicity evaluation
 (Piikivi and Tolonen, 1989).
  In contrast to EEGs, evoked response
 technology is improving, and
 equipment, while expensive, is
 becoming more portable. VERs have
 been used to detect the sensory toxicity
 of solvents and carbon monoxide in
 human subjects, and a relationship has
 been suggested between BAER and
 blood lead levels in children exposed to
 lead-containing dust in the environment
 (Otto and Hudnell, 1990). Evoked
 potentials also may be conditioned,
 allowing the use of sensory methods to
 investigate associative processes.
  Dose-response functions have been
 found with evoked methods. A
 curvilinear relationship was found
 between BAER and blood lead
 concentrations in children (Otto and
 Hudnell, 1990), and a biphasic function
 described visual evoked potential (VEP)
 latency and visual contrast sensitivity
 and perchloroethylene exposure
 concentration in a laboratory study
 (Altmann et al., 1991). In the latter
 study, the direction of the response was
 jointly dependent on dose  and stimulus
 parameters. In addition, changes over
 time in the effect of the solvent on VEP
 were dose and stimulus parameter
  Two important methodologic
considerations are illustrated by BAER
and VEP data. One is that low
concentrations of some chemical agents
may produce effects (shorter latencies in
these examples) that could be
inaccurately interpreted as facilitation
rather than impairment. Changes in
neuronal latencies in either direction
could be a result of a neurotoxic
process. The second is that the detection
of neurotoxic effects is dependent on

                Federal Register / Vol. 59, No. 158  / Wednesday,  August 17,  1994  /  Notices
dose-time-testing parameter
interactions. A thorough understanding
of the effects of testing parameters on
the dose-response relationship and the
time course of chemical effect will be
necessary for interpreting neurotoxicity
  The development of neurophysiologic
methods, such as evoked and
conditioned potentials, for neurotoxicity
risk assessment should be encouraged.
These methods provide relatively
unambiguous quantitative data on
sensory function that may have clear
implications for health, are influenced
by fewer extraneous variables than are
self-report and neurobehavioral
performance tests, and allow relatively
direct extrapolation of effects between
animals and humans.
3.3.3. Neurochemical Methods
  One of the major difficulties in risk
assessment is estimating exposure
parameters and the dose or body burden
actually absorbed by the individual. In
epidemiologic studies, the actual
absorption and bioavailability of a
chemical from an exposure are
frequently unknown.
   Measurement of chemical
concentrations in biologic fluids or
tissues is one way to measure more
precisely the concentration at the site(s)
of toxic effect. In epidemiologic studies,
this has been possible only for chronic
exposure and for acute exposure to
 chemicals with long biologic half-lives
 in the body, such as lead, other metals,
 and bromides. Blood lead levels show
 correlations with neurobehavioral
 impairment, but blood lead levels are
 representative correlates of toxicity only
 for relatively acute doses.  In children,
 for example, the majority of lead-related
 impairment is the result of chronic,
 rather than acute, absorption. The
 cumulative amount of lead sequestered
 in tissues (such as deciduous teeth) may
 be a more representative indicator of the
 area under the time-concentration
   For chemicals with half-lives in the
 body too short for estimating absorbed
 dose, the biochemical products from the
 chemical or from the physiologic effects
 of the chemical may serve as an index
 of exposure. Serum enzyme
 concentrations (cholinesterase) and
 esterases in other tissues (lymphocyte
 target esterase) have been employed in
 field studies to detect pesticide
 exposure, while vanillylmandelic acid
  (product of catecholamine
 neurotransmitter biotransformation)  and
 erythrocyte protoporphyrin
  concentrations have been used with
  varying success in differentiating
 between lead-exposed and control
workers. The addition of similar
"exposure biomarker" measures to
laboratory studies may allow the
development of quantitative estimates of
absorbed dose under various exposure
  The measurement of metabolic
products of neurotoxic agents may be
extremely useful in risk assessment; an
example comes from cancer risk
assessment.  Human data from the early
1970s on saturation of microsomal
methylene chloride biotransformation to
carbon monoxide (Stewart et al., 1972),
along with subsequent animal
carcinogenesis data garnered in the
1980s, provided a quantitative basis for
a physiologically based
pharmacokmetic model of methylene
chloride cancer risk assessment
(Andersen et al., 1991). The information
on human CO pathway kinetics
provided the homologous key that
allowed extrapolation of risk from
animals to humans on a comparative
physiologic basis rather than using
default assumptions.

3.3.4. Imaging Techniques
   A number of recently developed
computerized imaging techniques for
evaluating brain activity and cerebral/
peripheral blood flow have added
valuable information to the neurologic
diagnostic process. These imaging
methods include thermography,
 positron emission tomography, passive
 neuromagnetic imaging
 (magnetoencephalography), magnetic
 resonance imaging, magnetic resonance
 spectroscopy, computerized
 tomography, doppler ultrasonography,
 and computerized EEC recording/
 analysis (brain electrical activity
 mapping). The research application of
 these invasive and noninvasive
 quantitative methods has primarily been
 in neurology, schizophrenia research,
 drug abuse, AIDS research and toxic
 encephalopathy (Hagstadius et al.,
 1989). Although the equipment for brain
 imaging is expensive and not portable,
 neuroimaging techniques promise to be
 valuable clinical and laboratory research
 tools in human neurotoxicology.

 3.3.5. Neuropathologic Methods
   Neuropathologic examination of
 nervous system tissue has been used to
 confirm data from clinical testing and to
 contribute  to the understanding of
 mechanisms of action of neurotoxicity.
 Peripheral nerve biopsies have
 confirmed chemically induced
 peripheral neuropathies and evaluated
 rates of recovery (Fullerton, 1969).
 Postmortem examination of nervous
 tissue also has elucidated the
 neuropathological effects of carbon
disulfide, clioquinol, and doxorubicin
(Spencer and Schaumburg, 1980).
3.3.6. Self-Report Assessment Methods
  Self-report measures relevant to
neurotoxicity risk assessment consist of
histories of symptoms, events,
behaviors, and environmental
conditions. Information is obtained by
face-to-face interviews, structured
interviews (often conducted for
diagnostic purposes), medical histories,
questionnaires, and survey instruments.
  Self-report instruments are the only
means for measuring some symptoms
and all interoceptive states, such as pain
and nausea. Self-reports also are used to
obtain information on behaviors and
events (e.g., exposure conditions)
especially when practical,  legal, or
ethical limitations prevent direct
  Subjective symptoms elucidated from
self-report instruments are responsive to
dose. Hanninen et al. (1979)  found that
subjective symptoms were positively
correlated with blood lead levels in
exposed workers. Subjective pain
estimations are correlated with dose and
type of centrally and peripherally acting
analgesics, and anxiety scores on a
variety of scales are responsive to  the
 size of the anxiolytic dose.
   Symptom checklists are used in
 epidemiologic research to identify the
 pattern of subjective complaints, which
 can be used to guide the selection of
 objective assessment methods. The
 distribution of symptoms can be
 correlated with indices of exposure to
 determine if particular symptoms are
 more  prevalent in exposed persons
 (Sjogren et al., 1990).
   Self-report data are notable for biases
 that may influence them; these biases
 are well known in epidemiology,
 clinical practice, and social science.
 Even  in the most superficial of
 questions, respondents may consciously
 or unknowingly bias the answer to fit
 what  they believe to be the examiner's
 expectations. Details of objective  events
 or subjective states are subject to
 alteration; recall and reporting of
 remembered occurrences  may be  biased
 to fit  interpretations and expectations.
 The socioeconomic status, gender, and
 affiliation of the tester also have been
 identified as biasing variables. Bias
 occurs when information is requested
 about behaviors, beliefs, or feelings
 believed by the respondent to be
 socially undesirable or when
 reinforcement contingencies (e.g.,
 litigation) strongly favor selective
 reporting.  •
    Biases in self-report data can be
 reduced by making the questionnaire
  anonymous or highly confidential;

Federal Register / Vol. 59. No.  158 / Wednesday.  August 17, 1994  / Notices
 objective data can be used to validate
 self-reports. Ethnographic observations,
 objective measurement of behavior,
 biologic samples, and the observations
 of significant others are employed to
 validate self-report data. Consistent
 descriptions of events by several
 persons lend credence to the reliability
 of the report. Many clinical interviews
 and self-report assessment instruments
 include some mechanisms for detecting
 self-report bias, either by looking for
 endorsement of improbable behaviors,
 or by examining the consistency of
 information gathered in several ways or
 from several sources. Concordance
 among biologic indices, observations,
 and physical examinations increases the
 judged validity of self-reports. Mood scales.
   Changes in mood and emotionality
 can be  consequences of neurotoxicity.
 For example, case reports have
 identified mood changes from exposure
 to mercury, lead, solvents, and
 organophosphate insecticides. The
 Taylor  Manifest Anxiety Scale and the
 Profile  of Mood States (POMS) are
 standardized self-report assessment
 instruments for which there is some
 evidence of sensitivity to chemical
   The POMS, a component of the
 Neurobehavioral Core Test Battery, is a
 self-report measure that asks
 respondents to use a 5-point scale to
 rate the magnitude of 65 subjective
 states, such as "tense," "relaxed,"
 "hopeless," "guilty," etc., that they have
 experienced within the past week. The
 responses are scored according to six
 mood factors, and a Total Mood
 Disturbance Score also may be
 calculated. Liang et al.  (1990) used the
 POMS to evaluate lead-exposed workers
 (mean blood lead concentration of 41
 ug/dL) from a battery plant and a control
 group from a fabric-weaving
 manufacturer. Exposed workers were
 significantly higher on tension,
 depression, anger, fatigue, and
 confusion scales.
  Mood scales were developed to aid in
 assessment of psychological disorders,
 such as  depression, and to track
 treatment response. In addition, mood  is
 modulated by metabolic and endocrine
 variables in health and disease and can
 change rapidly in response to
 interpersonal, workplace, and
 environmental events. The large number
 of nonchemical variables and the
 lability  of mood make inclusion of
carefully selected controls essential in
using affect as an endpoint in
neurotoxicity research.
  The validity of mood scales may be
limited to the specific populations in
                      which the validity studies were
                      performed. As characterizations of
                      internal states, the meaning of the
                      descriptors in the POMS established for
                      one culture may not be the same as the
                      meaning of that concept or term in other
                      cultures or in other language systems.
                      There may be variations in
                      interpretation of the terms by
                      respondents across English-speaking
                      subcultures, perhaps as a function of
                      education  or the size of the verbal
                      community. While these differences
                      may not impede a global clinical
                      interpretation, the reduction in
                      generalizability across study
                      populations may be sufficient to
                      decrease the usefulness of subjective
                      scales in quantitative neurotoxicity risk

             Personality scales.
                       The Minnesota Multiphasic
                      Personality Inventory (MMPI), the
                      Cattell 16 PF, and the Eysenck
                      Personality Inventory have occasionally
                      been used in neurotoxicity research.
                      Exposed and nonexposed groups have
                      differed on several scales derived  from
                      these standardized questionnaires. The
                      diagnostic power of the MMPI, for
                      example, is not in the individual scales
                      but in the pattern of scores on the  10
                      clinical and 3 validity scales. Because
                      interpretation of the MMPI requires a
                      trained diagnostician with experience in
                      the population of interest, it  is less
                      likely to be useful in quantitative
                     neurotoxicity assessment.

                     3.4. Approaches to Neurotoxicity

                     3.4.1. 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.
                     Epidemiologic studies are a means of
                     evaluating the effects of neurotoxic
                     substances in human populations, but
                     such studies are limited because they
                     must be performed shortly after
                     exposure if the effect is acute. Most
                     often these effects are suspected to be a
                     result of occupational exposures due to
                     the increased opportunity for exposure
                     to industrial and other chemicals.
            Case reports.
                       The first type of human study
                     undertaken  is 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.
 For example, the neurological hazards
 of exposure to Kepone,
 dimethylaminopropionitrile, and
 methyl-n-butyl ketone were first
 reported as case studies by physicians
 who noted an unusual cluster of
 diseases in persons later found to have
 been exposed to these chemicals (Cone
 et al., 1987). However, case histories
 where exposure involved a single
 neurotoxic agent, though  informative,
 are rare in the literature; for example,
 farmers are exposed to a wide variety of
 potentially neurotoxic pesticides.
 Careful case histories assist in
 identifying common risk factors,
 especially when the association between
 the exposure and disease  is strong, the
 mode of action of the agent is
 biologically plausible, and clusters
 occur in a  limited period of time.
   Case reports are inexpensive
 compared  with other types of
 epidemiologic studies and can be
 obtained more quickly than more
 complex studies. They provide little
 information about disease frequency or
 population at risk, but their importance
 has been clearly demonstrated,
 particularly in accidental poisoning or
 acute exposure to high levels of
 toxicant. They remain an important
 source of index cases of new diseases
 and for surveillance in occupational
 settings. These studies require
 confirmation by additional
 epidemiologic research employing other
 study design. 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 is
 interviewed, examined, and tested at a
 single point in time to ascertain a
 relationship between a disease and a
 neurotoxic exposure. This study design
 does not allow the investigator to
 determine whether the disease or the
 exposure came first, rendering it less
 useful in estimating risk. These studies
 are intermediate in cost and time
 required to complete compared with
 case reports and more complex
 analytical studies. Case-control (retrospective)
  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)

                 Federal Register  / Vol. 59, No.  158  /  Wednesday. August 17, 1994 / Notices
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 neurotoxins in the
environment has provided a great deal
of information. In his recent text,
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, and will
 be discussed only briefly here. Many of
 these biases also can be present in cross-
 sectional studies. For example, recall
 bias or faulty recall of information by
 study subjects in a questionnaire-based
 study can distort the results of the
 study. Analysis of the case-comparison
 study design assumes that the selected
 cases are representative persons with
 the disease—either all cases with the
 disease or a representative sample of
 them have been ascertained. It further
 assumes that the control or comparison
 group is representative of the
 nondiseased population (or that the
 prevalence of the characteristic under
 study is the same in the control group
 as in general population). Failure to
 satisfy these assumptions may result in
 selection bias, but violation of
  assumptions does not necessarily
  invalidate the study results.
    An additional source of bias in case-
  control studies is the presence of
  confounding variables, i.e., factors
  known to be associated with the
  exposure and causally related to the
disease under study. These must be
controlled either in the design of the
study by matching cases to controls on
the basis of the confounding factor or in
the analysis of the data by using
statistical techniques such as
stratification or regression. Matching
requires time to identify an  adequate
number of potential controls to
distinguish those with the proper
characteristics, while statistical control
of confounding requires a larger study.
  The definition of exposure is critical
in epidemiologic studies. In
occupational settings, exposure
assessment 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. Prospective (cohort, followup)
   In a prospective study design, a
healthy group of people is assembled
and followed forward in time and
observed for the development of
disease. Such studies are invaluable for
determining the time course for
development of disease (e.g., followup
studies performed in various cities on
the effects of lead on child
 development). This approach allows the
 direct estimate of risks attributed to a
 particular exposure since disease
 incidence rates in the cohort are
 determined and allows 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
   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 clue 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. A retrospective cohort study
was performed in which a cohort of
1,790 bricklayers and 2,601 men
exposed to paint solvents was
retrospectively identified and, if a
disability pension had been awarded,
the subjects were examined for evidence
of presenile dementia. This study found
a rate ratio of 3.4 for presenile dementia
among the painters as compared with
the bricklayers (Johnson, 1987).

3.4.2. Human Laboratory Exposure
   Neurotoxicity assessment has an
advantage not afforded 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. Under certain
circumstances, it is 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. These chemicals
 include methylene chloride,
 perchloroethylene, trichloroethylene,
 and p-xylene (Dick and Johnson, 1986).
   Human exposure studies offer
 advantages over epidemiologic field
 studies. Combined with appropriate
 biological sampling (breath or blood), it
 is possible to calculate body
 concentrations, to examine
 toxicokinetics, and identify metabolites.
 Bioavailability, elimination, dose-
 related changes in metabolic pathways,
 individual variability, time course of
 effects, interactions between chemicals,
 interactions between chemical and
 environmental/biobehavioral factors
 (stressors, workload/respiratory rate) are
 some processes that can be evaluated in
 laboratory studies.
    Other goals of laboratory studies
  include the indepth characterization of
  effects, the development of new
  assessment methods, and the
  examination of the sensitivity,
  specificity, and reliability of
  neurobehavioral assessment methods
  across chemical classes.
    The laboratory is the most appropriate
  setting for the study of environmental
  and biobehavioral variables that affect
  the action of chemical agents. The
  effects of ambient temperature, task
  difficulty, the rate  of ongoing behavior,
  conditioning variables, tolerance/

Federal Register / Vol. 59, No.  158  /  Wednesday. August 17, 1994 / Notices
 sensitization, sleep deprivation,
 motivation, etc., can be studied. Methodologic aspects.
   From a methodologic standpoint,
 human laboratory studies can be
 divided into two categories—between-
 subjects and within-subjects designs. In
 the former, the neurobehavioral
 performance of exposed volunteers is
 compared with that of nonexposed
 participants. In the latter, preexposure
 performance is compared with
 neurobehavioral function under the
 influence of the chemical or drug.
 Within-subjects designs have the
 advantage of requiring fewer
 participants, eliminating individual
 differences as a source of variability,
 and controlling for chronic mediating
 variables, such as caffeine use and
 educational achievement. A
 disadvantage of the within-subjects
 design is that neurobehavioral tests
 must be administered more than once.
 Practice on many neurobehavioral  tests
 often leads to improved performance
 that may confound the effect of the
 chemical/drug. It is important to allow
 a sufficient number of test sessions in
 the preexposure phase of the study to
 allow performance on all tests to
 achieve a relatively stable baseline level. Human subject selection factors.
  Participants in laboratory exposure
 studies may be recruited from
 populations of persons already exposed
 to the chemical/drug or from naive
 populations. Although the use of
 exposed volunteers has ethical
 advantages, can militate against novelty
 effects, and allows evaluation of
 tolerance/sensitization, finding an
 accessible exposed population in
 reasonable proximity to the laboratory is
 difficult. Naive participants are more
 easily recruited, but may differ
 significantly in important characteristics
 from a representative sample of exposed
 persons. Naive volunteers are often
 younger, healthier, and better educated
 than the populations exposed
 environmentally, in the workplace, or
 pharmacotherapeutically. For example,
 phase I drug trial data from relatively
 young and healthy volunteers may not
 adequately predict the incidence of
 neurotoxic side effects in older persons
 with chronic health problems. Exposure conditions and
 chemical classes.
  Compared with workplace and
environmental exposures, laboratory
exposure conditions can be controlled
more precisely, but exposure periods are
much shorter. Generally only one or two
relatively pure chemicals are studied for
                      several hours while the population of
                      interest may be exposed to multiple
                      chemicals containing impurities for
                      months or years. Laboratory studies are
                      therefore better at identifying and
                      characterizing effects with acute onset
                      and the selective effects of pure agents.
                        Most laboratory studies of
                      neurobehavioral function have
                      employed individual solvents,
                      combinations of two solvents, or very
                      low concentrations of chemicals
                      released from household and office
                      materials (volatile organic compounds).
                      This selection is primarily because
                      solvent effects are reversible, because
                      there are wide margins of safety for
                      acute effects of solvents, because
                      solvents can be administered via
                      inhalation methods that allow
                      calculation of body concentrations by
                      breath sampling methods that do not
                      require needle sticks, because over 1
                      million workers may have occupational
                      solvent exposure, and because of the
                      extensive use of solvents in household
                      products. Chemicals studied in the
                      laboratory over the past 40 years have
                      included ozone, NO2, CO, styrene,  lead,
                      anesthetic gases, pesticides, irritants,
                      chlorofluorocarbon compounds, and
                      propylene glycol dinitrite. Caffeine,
                      diazepam, and ethanol have been used
                      in laboratory studies as positive control

             Test methods.
                       Neurobehavioral test methods may be
                      selected according to several strategies.
                      A test battery that examines multiple
                      neurobehavioral functions may  be more
                      useful for screening and the initial
                      characterization of acute effects.
                      Selected neurobehavioral tests that
                      measure a more limited number of
                      functions in multiple ways may be  more
                      useful for elucidating mechanisms or
                      validating specific effects.
                       Both chemical and behavioral control
                      procedures are valuable for examining
                      the specificity of the effects. A
                      concordant effect among different
                      measures of the same neurobehavioral
                      function (e.g., reaction time) and a lack
                      of effect on some other measures of
                      psychomotor function (e.g., untimed
                      manual dexterity) would increase the
                      confidence in a selective effect on motor
                      speed and not on attention or on
                      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. Ethical issues.

   Most human exposure studies in the
 laboratory have been justified on the
 basis of data indicating that the
 chemical or drug exposure produces
 only temporary and reversible
 functional effects. The use of
 occupationally, environmentally, or
 therapeutically exposed populations as
 a source of participants also makes the
 risks from research exposure small
 relative to nonlaboratory sources of risk.
 Protection of human subjects is also
 provided by the informed consent
 process; the health risks (known and
 unknown) and benefits of the research
 are thoroughly explained to each
 participant, who may terminate
 participation in the study at any time.
  Despite safeguards, several chemicals
 and drugs thought at the time of the
 exposure study to produce only
 temporary neurobehavioral effects are
 now (20 years later) suspected of being
 potential  human carcinogens on the
 basis of animal and human data (e.g.,
 methylene chloride, perchloroethylene).
 Other chemicals, however, are now
 thought to be less carcinogenic or
 otherwise less toxic in humans than
 once believed. Rapid advances in all
 areas of toxicology make it difficult to
 communicate, to potential subjects,
 reliable information about the
 likelihood of long-term, latent, or
 delayed adverse effects on health
 subsequent to the study. The
 communication of uncertainty about
 potential long-term effects to research
 participants is essential if human
 exposure studies are to be conducted
 ethically and are to continue their
 contributions to neurotoxicology and
 risk  assessment.

 3.5. Assessment of Developmental

 3.5.1. Developmental Deficits

  While adult neurotoxicology
 evaluates  the effects of chemical
 exposure on relatively stable nervous
 system structure and function,
 developmental neurotoxicology
 addresses the special vulnerabilities of
 the young and the old. Neurobehavioral
 assessment of chemical neurotoxicity is
 complicated by having to measure
 functional impairment within a
 sequential progression of emergence,
maturation, and gradual decline of
nervous system capabilities. Methods in
developmental neurotoxicity assessment
must reflect the diversity of

                Federal Register / Vol.  59,  No. 158 / Wednesday. August 17, 1994 / Notices
neurobehavioral functions, from
neonates to the elderly.
  Exposure of pregnant women to
alcohol, drugs of abuse, therapeutic
drugs, nicotine, and environmental
chemicals may result in the immediate
or delayed appearance of
neurobehavioral impairment in children
(Kimmel, 1988; Nelson, 1991a).
Postnatal exposure of children to
chemical agents in the environment,
such as lead, also may impair IQ and
other indices of neurobehavioral
function (Needleman et al., 1979).
Neurotoxic effects may impair speech
and language, attention, general
intelligence, "state" regulation and
responsiveness to external stimulation,
learning and memory, sensory and
motor skills, visuospatial processing,
affect and temperament, and
responsiveness to nonverbal social
stimuli. Chemical neurotoxicity may be
manifested as decreases in functional
capabilities or delays in normative
developmental progression.
   Neurotoxic effects are not limited to
direct exposure of the fetus or child to
the chemical. Animal studies suggest
that altered neurobehavioral
development in offspring may result
from exposure of males (Joffe and
 Soyka, 1981) and females to chemical
 substances prior  to conception. In this
 case, altered postnatal development may
 reflect chemical influences on
 mechanisms of inheritance, copulatory
 behavior, nutritional status, hormonal
 status, or the uterine environment. In
 animals and humans, chemical
 exposure of parents may indirectly
 impair postnatal development through
 changes in  milk composition, parenting
 behaviors, and other aspects of the
    In older adults the normal aging
 process alters the response to
 neurotoxicants. Both pharmacodynamic
 and pharmacokinetic changes may
 underlie altered sensitivities to the
 neurotoxic effects of drugs and
 chemicals. An example well known in
 geriatric medicine is the apparent
 increase in sensitivity of the elderly to
 the toxic effects  of anxiolytics (Salzman,
 1981). Decreases in biotransformation
 rate and renal elimination of parent
 drug and active metabolites, not related
 to disease processes, may partially
 account for the increased vulnerability
 (Friedel, 1978). Chronic disease states in
 older persons may result in decreased
 functional capabilities and increased
 vulnerability to  neurotoxic effects.
 Chronic diseases also may prompt
 pharmacotherapy that may impair
 neurobehavioral function.
 Cardiovascular, psychopharmacologic,
  and antineoplastic medications may
result in patterns of neurobehavioral
impairment not typically seen in
younger individuals.
3.5.2. Methodologic Considerations
  Standardized methods are being
developed for pediatric neurotoxicity
assessment. Neurobehavioral functions
emerge during developmental phases
from neonatal stage through secondary
school, and nervous system insult may
be reflected not only in impairment of
emergent functions, but also as delays in
the appearance of new functions. Both
the severity and type of deficit are
affected by the dose and duration of
exposure (Nelson,  1991b), and different
sensitivities to chemical effects may be
exhibited at different stages of nervous
system development. Early episodes of
exposure may produce structural
damage to the nervous system that may
not be developmentally expressed in
behavior for several months or years.
  The selection of appropriate testing
methods and conditions is more
important when assessing children
because of shorter attention spans and
increased dependence on parental and
environmental supports. In addition,
because of the increasing complexity of
 functional capabilities during early
 development, only a few tests
 appropriate for infants can be validly
 readministered to older children. Given
 the complexity of these variables, the
 task of devising sensitive, reliable, and
 valid assessment instruments or
 batteries for pediatric populations will
 be challenging.
   Assessment methods in older adults
 must be capable of distinguishing
 chemical and drug effects from the
 effects of aging processes and chronic
 disease states (Crook et al., 1983).
 Assessment methods must be valid and
 reliable with repeated administration
 across a significant portion of the
 lifespan, and take into consideration the
 time (days, months, or years) that may
 intervene between exposure/insult  and
 the expression of neurotoxicity as
 functional impairment. Research on
 nonexposed populations to develop age-
 appropriate normative scores for
 neurobehavioral functions will be
 important for the interpretation of
 assessment instruments.
    Environmental exposure to neurotoxic
 chemicals and drugs is correlated with
 socioeconomic and ethnic status.
 Assessment methods will therefore have
 to be adapted  to diverse ethnic, cultural,
 and language groups. While  gender
 differences in early development have
 been noted, differential responses of
  males and females to neurotoxicants
 have been less well explored and should
  receive attention.
3.6. Issues in Human Neurotoxicology
Test Methods
3.6.1. Risk Assessment Criteria for
Neurobehavioral Test Methods
  The value of human neurobehavioral
test methods for quantitative risk
assessment is related to the number of
the following criteria that can be met:
  a. Demonstrate sensitivity to the kinds
of neurobehavioral impairment
produced by chemicals; that is, able to
detect a difference between exposed and
nonexposed populations in field studies
or between exposure and nonexposure
periods in human laboratory research or
within exposed populations  over time.
  b! Show specificity for neurotoxic
chemical effects and not be unduly
responsive to a host of other
nonchemical factors, and show
specificity for the neurobehavioral
function believed to be measured by the
test method.
  c. Demonstrate adequate reliability
(consistency of measurement over time)
and validity (concordance with other
behavioral, physiologic, biochemical, or
anatomic measures of neurotoxicity).
  d. Show graded amounts of
neurobehavioral change as a function of
exposure parameter, absorbed dose, or
body burden along some ordinal or
continuous metric (dose response).
   e. For representative classes or
 subclasses of CNS/PNS-active
 chemicals, identify single effects or
 patterns of impairment across several
 tests or functional domains  that are
 reasonably consistent from study to
 study (structure-activity).
   f. Be amenable to the development of
 a procedurally similar counterpart that
 can be used to assess homologous
 behaviors in animals.
   g. Whenever it is relevant, care must
 be taken to insure to the extent possible
 that subjects are blind to the variate of
 interest (Benignus, 1993). Sensitivity.
    Individual neurobehavioral tests and
 test batteries have detected  differences
 between exposed and  nonexposed
 populations in epidemiologic studies
 and in laboratory studies. Effects have
 been detected by neurobehavioral
 methods at concentrations thought by
 other kinds of evaluation not to produce
 neurotoxicity. Workplace exposure
 limits to many chemicals have been set
 on the basis of neurobehavioral studies.
 While the overall sensitivity of
 neurobehavioral methods is sufficient to
 be useful in neurotoxicology risk
 assessment, some methods are notably
 insensitive across several chemical
 classes while the sensitivity of other
 neurobehavioral tests varies according

Federal  Register / Vol. 59, No. 158  /  Wednesday. August 17, 1994 / Notices
 to the spectrum of neurotoxic effects of
 the chemical or drug.
   Sensitivity is sometimes negatively
 correlated with reliability; selecting for
 tests that show little change over time
 may  also select for tests that are not
 sensitive to neurotoxic insult.
   Having more control over the testing
 environment and using a repeated
 measures design may decrease
 variability and increase statistical
 power, but these tactics may introduce
 other problems. There is some
 suggestion that experience in highly
 structured laboratory environments with
 explicit stimulus conditions may reduce
 the sensitivity  of humans and animals to
 the effects of drugs and chemicals, and
 the sensitivity  of neurobehavioral
 measures to impairment by a chemical
 or drug may  depend on neurobehavioral
 training history (Terrace, 1963;  Brady
 and Barrett, 1986). Sensitivity may also
 be decreased if baseline behaviors are
 stable and well practiced or an escape/
 avoidance procedure is employed.
   The systematic introduction of
 stimulus or response changes to induce
 transitional behaviors, such as in a
 transitional state or repeated learning
 paradigms, may be one way to retain the
 advantage of a stable baseline, have
 sufficient sensitivity, and avoid practice
 effects (Anger and Setzer, 1979). Specificity.

   There are two kinds of specificity in
 neurobehavioral assessment of chemical
 or drug neurotoxicity. Chemical
 specificity refers to the ability of a test
 to reflect chemical or drug effects and to
 be relatively resistant to the influence of
 nonchemical variables. The second type
 of specificity refers to the ability of a
 test method to measure changes in a
 single neurobehavioral function (e.g.,
 dexterity) or a restricted number of
 functions, rather than a broad range of
 functions (attention, reasoning,
 dexterity, and vision).
  The neurobehavioral expression of
 neurotoxic chemical or drug effects is a
 function of the  joint interaction  of
 ongoing nervous system processes with
 the chemical substance and with
 biopsychosocial variables that also
 influence nervous system activity. In
 laboratory exposure studies numerous
 environmental,  behavioral,  and biologic
 variables can influence the type or
 magnitude of neurotoxic effects  of
 chemical agents and drugs (MacPhail,
 1990). These  variables include ambient
 temperature,  physical workload, task
 difficulty, the social and tangible reward
characteristics of the laboratory  setting,
redundancy of stimuli, the rate and form
of the behavioral response, conditioning
                      factors, and the interoceptive stimulus
                      properties of the chemicals.
                        Tne laboratory research participant's
                      history and habits outside the laboratory
                      also may affect chemical-
                      neurobehavioral interactions by
                      influencing the baseline level of
                      performance on neurobehavioral tests or
                      directly affecting the response of the
                      CNS to the exposure. Age, gender,
                      educational level, intellectual
                      functioning, economic status, acute and
                      chronic health conditions (including
                      developmental or current neurologic
                      conditions), alcohol/drug/tobacco
                      effects or withdrawal, emotional status
                      or significant life events, sleep
                      deprivation, fatigue, and cultural factors
                      are only a few of the variables that may
                      affect performance in laboratory studies
                      (Williamson, 1991; Cassitto et al., 1990).
                        The influence of these selection and
                      biopsychosocial variables on the
                      neurobehavioral effects of workplace
                      chemicals is poorly understood,
                      although their effects on drug-behavior
                      interactions have been more thoroughly
                      explored. Controlling or understanding
                      chemical and nonchemical variables
                      will be important for ensuring adequate
                      specificity for risk assessment purposes.
             Reliability and validity.
                        Reliability refers to the ability of a
                      given test to produce closely similar
                      results when administered more than
                      once over a period of time or in similar
                      populations. Reliability is meaningful
                      only with respect to the measurement of
                      functions that would not be expected to
                      change significantly over the time
                      period. Test-retest reliability coefficients
                      are between 0.6 and 0.9 (Beaumont,
                      1990) for most of the tests in the NCTB.
                      With notable exceptions, other
                      neurobehavioral tests have similar
                      reliabilities. Reliabilities in the 0.8 to
                      0.9 range are usually thought
                      acceptable.  As reliability decreases,
                      measurement error is more likely to
                      mask neurotoxic chemical effects.
                        The validity of a given neurotoxicity
                      test relies on evidence that it adequately
                      measures the domain of interest and is
                      not highly correlated with tests that are
                      believed to measure unrelated
                      functions. These convergent and
                      divergent aspects of validity are
                      frequently divided into construct,
                      content, and criterion subcategories.
                      Construct validity refers to the ability of
                      a given test  to measure  the intended
                      function or construct (e.g., attention),
                      content to how  well the test measures
                      the major aspects of the function, and
                      criterion to how highly the test
                      correlates with  other tests of the same
                      function or predicts neurotoxic
                      impairment after similar insult.
   Many neurobehavioral tests purport to
 measure the same or similar cognitive,
 sensory, or motor functions, but
 correlations between these tests under
 chemical exposure or control conditions
 can be disappointingly low. This is not
 surprising given the procedural
 differences that exist among
 neurobehavioral tests.  Tests intended to
 measure the same function often have
 different presentation and response
 modalities (visual, verbal, manual), have
 differing numbers of trials or a different
 time limit, and have different methods
 for scoring the results.  Many tests have
 such large procedural differences that
 direct  comparison is difficult.
 Assessment of validity for
 neurobehavioral tests of specific
 constructs, such as attention, is further
 complicated in that sensory input, other
 cognitive processes, and motor
 responses are unavoidable contributors
 to the test result. Dose response.
  Dose in this discussion refers to the
 measurement of chemical or metabolite
 concentrations in the body and to
 estimations of exposure. Both exposure
 assessment and biologic concentrations
 should be measured whenever possible.
 Dose-response relationships have been
 observed both in field and laboratory
 studies. Two recent human solvent
 exposure studies used lower exposure
 concentration that resulted in mucosal
 membrane effects reported by subjects
 as odors or irritation (Dick et al., 1992;
 Hjelm et al., 1990). Neurobehavioral
 impairment was not detected in these
 studies. A review of over 50 organic
 solvent human exposure experiments
 found that neurobehavioral impairment
 generally occurred at mean
 concentrations higher than those
 associated with irritation, although
 there was often overlap among the
 irritant and impairment concentration
 ranges (Dick, 1988). Defining neurotoxic
 dose-response relationships in humans
 decreases the uncertainties of
 extrapolation from animal data and
 allows a more accurate risk assessment.
  Recent human solvent exposure
 studies have employed low
 concentrations under which
 neurobehavioral impairment was not
 observed. Rather, these studies have
 primarily detected the effects of solvents
 on mucosal membranes reported by
subjects as odors or irritation (Dick,
unpublished observation). While these
data may be relevant to setting
workplace and environmental exposure
limits,  they can be expected to provide
little information about the
neurobehavioral impairment that occurs
at higher concentrations. The

relationship between irritant/odor
concentration-effect functions and
neurobehavioral impairment
concentration-effect functions is not
known, but it is probably not linear.
Dose-dependent mechanisms of toxic
effect can be expected to complicate risk
extrapolation across the dose-response
range in humans.
  A further complication in dose-
response extrapolation is that low
concentrations of chemicals may appear
to improve performance as measured by
neurobehavioral tests, while higher
doses are more likely  to impair
performance. Improved performance
does not necessarily indicate  the
absence of neurotoxicity; both increases
and decreases in neurobehavioral
 performance may result from
 deleterious chemical  interactions with
 neurons. Dose-response extrapolation is
 further complicated by the observation
 that facilitative or impairment effects
 within a given dosage range may occur
 at some parameters of the test stimulus
 or aspects of the response (response
 rate-dependent) but not at others
 (Altmann et al., 1991). Therefore, dose
 extrapolations are more difficult when
 there is uncertainty about the shape of
 the dose-response function (biphasic,
 linear, etc.) at the relevant test stimulus
 and response parameters.
    The risk assessment process with
 animal  data involves extrapolation from
  the effects of high doses in animals to
  predict the effects of chronic low-dose
  exposure in humans. With data from
  laboratory studies of hurnans in a risk
  assessment, however, the extrapolation
  is in the other direction,  from very low-
  dose laboratory exposure to predict the
  effects  of chronic exposure at higher
  (but still low) concentrations in the
  environment and workplace. Low- to
  high-dose extrapolation within the same
  species may require different
  assumptions and risk assessment
  procedures. Although high-dose human
  exposures have occurred in accidents,
  those data are primarily descriptive in
  nature and cannot easily be plugged into
  a quantitative risk extrapolation process.
   Low dose laboratory data may be
   combined with data from epidemiologic
   studies of persons exposed to higher
   concentrations. Structure-activity.
     Structure-activity relationships for
   well-known chemicals have largely been
   established by clinical methods (and
   animal studies) and verified by
   neurobehavioral and neurophysiologic
   testing. Although an area of active
   research, neurobehavioral  testing of
   humans has not yet been able to identify
   reliable patterns of impairment among
chemical classes. This endeavor has
been hampered by most laboratory
research having been limited to the
evaluation of low concentrations of
solvents and a few other reversible
toxicants and by the exposure
uncertainties, biases, and confounding
variables found in cross-sectional or
cohort field studies.
 3.6.2. Other Considerations in Risk
 Assessment Mechanisms of action
   Uncovering behavioral and
 neurophysiologic mechanisms of action
 is a potential contribution of human
 laboratory exposure studies to
 neurotoxicity risk assessment. For
 example, Stewart et al. (1972)
 demonstrated that methylene chloride
 was metabolized to carbon monoxide in
 humans, and further studies (Putz et al.,
 1979) found that CO production could
 account for some of the neurobehavioral
 impairment observed with that
 chemical. Recent human laboratory
 studies of solvents employed low
 concentrations that produced mucosal
 irritation and strong odor, but little
 neurobehavioral impairment (Dick,
 unpublished observation). The
 mechanisms of action that produce
 mucosal irritation and the neurotoxic
 mechanisms that are expressed in
 neurobehavioral impairment may be
  quite different. Data on mucosal
  irritation and odor may therefore
  provide limited information for a
  neurotoxicity risk assessment. Exposure duration
    A criticism of extrapolation from
  animal studies to human exposure
  conditions is that the effects of short-
  term exposure (months to 1-2 years) in
  animals may not accurately predict the
  effects of chronic exposure (>10 years)
  in humans. Laboratory studies rarely
  expose human subjects to solvents for
  more than 4-6 hours per day for 2-5
  days while environmental and
  workplace exposures of concern involve
  6-8 hours of exposure per day for years.
  The uncertainties of extrapolating from
   relatively acute exposures to predict the
   risks from chronic exposure will not be
   eliminated by using human laboratory
   exposure data in risk assessment. Time-dependent effects
     The acute exposures that are possible
   in human laboratory studies may
   provide little information on chronic
   time-dependent neurobehavioral effects.
   The effects of initial exposure may
   remain the same, decrease (tolerance),
   or increase (sensitization) with
   continued or repeated exposure to the
   chemical. All effects will not change in
unison; tolerance and sensitization may
be observed simultaneously on different
measures of neurobehavioral function.
The multiple toxicodynamic effects of
chemical exposure (neurobehavioral
and other) seem to follow individual
time courses suggestive of multiple
mechanisms of action. In addition, the
processes of tolerance and sensitization
can be influenced by testing conditions
and the nature of the behavioral task.
   One also must be concerned about
latent effects that do not appear for
some time after a brief exposure and
"silent" cumulative neurotoxic effects
that are not observable in acute human
studies. Latent and silent effects not
 only bring up the possibility of
 unknown risks for human subjects, but
 also make more difficult the
 extrapolation of chronic neurotoxic
 risks on the basis of acute exposures.
   Therefore, the acute exposure
 conditions possible in human laboratory
 studies may provide us with very
 limited information about the long-term
 effects of chronic exposure. Multiple exposures
    In the environment and the
 workplace, persons are seldom exposed
 to only a single chemical. Rather, they
 are most often exposed to complex
 mixtures of chemicals, the relative
 concentrations of which may vary over
  time. For example, one farmer had more
  than 50 different chemical products
  (pesticides, herbicides,  solvents, metals,
  gases) with nervous system effects that
  he used, prepared, or stored in his work
  shed. Chemicals used in industrial
  processes may also contain impurities
  or contaminants that may produce
  neurotoxic effects or alter the
  neurotoxicity of the more abundant
  chemical species. Chemical mixtures
  may have additive or potentiating
   effects not predictable from studies of
   single chemicals (Strong and Garruto,
   1991). Human laboratory exposure
   studies traditionally have employed one
   highly purified chemical or
   combinations of two chemicals (usually
   solvents) and thus may produce a
   spectrum of neurotoxic effects different
   from environmental and occupational
     Recently volatile organic compounds
   (VOCs) have been used in human
   exposure studies (Otto and Hudnell,
   1991). VOCs consist of multiple volatile
   compounds administered at
   concentrations commonly found in
   indoor air from emissions by laminates,
   carpet, plastics, and other building and
    decorating materials. Although VOCs
    are thought to produce primarily
    mucosal irritation and odors, reports of
    "sick building syndrome" and

Federal  Register / Vol. 59. No.  158 /  Wednesday.  August 17. 1994  / Notices
   individual sensitivity to indoor air
   contaminants suggest that other
   neurobehavioral mechanisms also may
   be operating. Generalizability and individual
     The results of field studies and
   laboratory exposure studies are most
   valuable when they can be extrapolated
   to the general population. Studies
   conducted in male workers or in young,
   healthy volunteers may have limited
   applicability to women or to people in
   other age ranges. It therefore is
  important to conduct studies that
  include males and females of different
  ages and ethnic heritage. Culture-
  sensitive neurobehavioral test methods
  are being developed and validated in
  the United States and other countries.
    While it is important to increase the
  generalizability of results, it is equally
  important to know when results cannot
  be generalized. Studies should be
  specifically directed toward identifying
  subsets of individuals who are more or
  less sensitive to neurotoxic insult or
  differ in mode of expression. There are
  many examples of individual
  differences that alter response to
  chemicals and drugs: phenylketonurics
  are more sensitive to dietary tyramine
  and persons with variants of plasma
  pseudocholinesterase are more affected
 by some neuromuscular blocking agents. Veracity of neurobehavioral test
   In most epidemiologic and human
 laboratory studies, research volunteers
 are highly motivated to perform well on
 tests of neurobehavioral function. Under
 voluntary conditions, actual
 neurobehavioral performance may serve
 as a reasonable index of nervous system
 capabilities. Some studies, however, are
 conducted in response to complaints of
 symptoms thought to be related to
 workplace, environmental, or
 therapeutic exposure to chemicals and
 drugs. The performance of research
 participants with symptoms and
 complaints may be significantly affected
 (consciously or unconsciously) by
 monetary rewards, emotional relief, or
 social gains from the validation of their
 complaints. Under these conditions,
 performance may or may not accurately
 reflect the capabilities of the nervous
 system and may lead to inaccurate
 conclusions about the magnitude of
 nervous system dysfunction or about
 putative chemical or drug etiologies.
  In addition to suboptimal
 performance engendered by potential
reinforcers or rewards, research
participants involved in disputes over
suspected neurotoxic exposures or in
                      litigation for monetary damages are
                      likely to be experiencing significant
                      emotional and behavioral reactions from
                      situational sources that can alter the
                      outcome of neurobehavioral assessment.
                      Anxiety, depression, sleep disturbances,
                      fatigue, worry, obsessive thoughts, and
                      distractibility may contribute to less
                      than optimal performance on motor and
                      cognitive neurobehavioral tasks,
                      especially where speed and sustained
                      concentration are important. Under
                      stressful conditions, it may be extremely
                      difficult to differentiate between
                      neurotoxic and situational sources of
                      observed functional impairment.
                      Functional neurobehavioral tests are not
                      well equipped to distinguish between
                      impairment from neurotoxicity and
                      from nonchemical variables. The use of
                      functional tests in symptomatic
                      populations requires great care in
                     interpretation. The development of
                     validity scales and other control
                     procedures for assessing nonchemical
                     influences on performance is greatly
   across species is of paramount
   importance for neurotoxicity risk
   4. Methods to Assess Animal
   4.1. Introduction
   4.1.1. Role of Animal Models
                     3.6.3. Cross-Species Extrapolation
                       Many neurobehavioral tests were
                     developed according to constructs of
                     human cognitive processes. The diverse
                     measures of cognitive, sensory,  and
                     motor performance in humans are
                     therefore not easily compared with
                     neurobehavioral function in animals.
                     While it may be possible to
                     conceptually relate some animal and
                     human neurobehavioral tests {e.g., grip
                     strength or signal detection), many
                     procedural differences prevent direct
                     comparison between species.
                      A more direct extrapolation from
                     animals to man might  be possible if the
                     tests were chosen on the basis of
                     procedural similarity rather than on a
                     conceptual basis (Anger, 1991). Stebbins
                     and colleagues (1975) were successful in
                     developing homologous procedures in
                    nonhuman primates for the
                    psychophysical evaluation of antibiotic
                    ototoxicity. Efforts to develop
                    comparable tests of memory and other
                    neurobehavioral functions in animals
                    and humans are under way (Stanton and
                    Spear, 1990, Paule et al., 1990), and
                    such efforts may aid in cross-species
                    extrapolation. Other procedurally
                    defined methods, such as Pavlovian
                    conditioning (Solomon and Pendlebury,
                    1988), operant conditioning (Cory-
                    Slechta,  1990), signal detection, and
                    psychophysical scaling techniques
                    (Stebbins and Coombs, 1975), could also
                    be used to facilitate interspecies risk
                    extrapolation. Deriving comparable
                    neurobehavioral assessment methods in
                    animals and humans that will allow a
                    more straightforward extrapolation
    Determining the risk posed to human
  health from chemicals requires
  information about the potential
  lexicological hazards and the expected
  levels of exposure. Some toxicological
  data can be derived directly from
  humans. Sources of such information
  include  accidental exposures to
  industrial chemicals, cases of food-
  related poisoning, epidemiological
  studies,  as well as clinical
  investigations. While human data are
  available from clinical trials for
  therapeutics and they provide the most
  direct means of determining effects of
  potentially toxic substances, for other
  categories of substances, it is generally
  difficult, expensive, and, in some cases,
  unethical to develop this type of
  information. Quite often, the nature and
  extent of available human toxicological
  data are too incomplete  to serve as the
  basis for  an adequate assessment of
  potential health hazards. Furthermore,
  for a majority of chemical substances
  human toxicological data are simply not
  available. Consequently, for most
  toxicological assessments it is necessary
 to rely on information derived from
 animal models, usually rats or mice.
 One of the primary functions of animal
 studies is to predict human toxicity
 prior to human exposure. In some cases,
 species phylogenetically more similar to
 human, such as monkeys or baboons,
 are used in neurotoxicological studies.
   Biologically, animals resemble
 humans in many ways and can serve as
 adequate  models for toxicity studies
 (Russell, 1991). This is particularly true
 with regard to the assessment of adverse
 effects to  the nervous system, whereby
 animal models provide a variety of
 useful information that helps minimize
 exposure  of humans to the risk of
 neurotoxicity. There are many
 approaches to testing for neurotoxicity,
 including whole animal (in vivo) testing
 and tissue/cell culture (in vitro) testing.
  At present, in vivo animal studies
 currently  serve as the principal
 approach  to detect and characterize
 neurotoxic hazard and to help identify
 factors affecting susceptibility to
 neurotoxicity. Data from animal studies
 are used to supplement or clarify
 limited information obtained from
clinical or epidemiological studies in
humans, as well as provide specific
types of information not readily

                 Federal Register  /  Vol. 59, No.  158  /  Wednesday, August 17, 1994 / Notices
obtainable from humans due to ethical
considerations. Frequently, results from
animal studies are used to guide the
design of lexicological studies in
  In vitro tests have been proposed as
a means of complementing whole
animal tests, which could ultimately
reduce the number of animals used in
routine toxicity testing. It also has been
proposed that in vitro testing, when
properly developed, may be less time-
consuming and more cost-effective than
in vivo assessments (Goldberg and
Frazier, 1989; Atterwill and Walum,
1989). By understanding the biological
structures or functions affected by toxic
substances in vitro, it also may be
possible to predict neurotoxicological
effects in the whole animal. An added
advantage of in vitro testing is the
growing availability of human cell lines
that could be used for directly assessing
potential neurotoxic effects on human
tissue. The currently available strategies
for in vitro testing have  certain
limitations, including the inability to
model neurobehavioral  effects such as
loss of memory or sensory dysfunction
or to evaluate effectively the influence
of organ system interactions (e.g.,
neuronal, endocrinological, and
immunological) on the development
and expression of neurotoxicity.
   In using animal models to predict
neurotoxic risk in humans, it is
important to understand that the
biochemical and physiological
mechanisms that underlie human
biological processes, particularly those
 involving neurological and
 psychological functions, are very
 complex and are sometimes difficult, if
 not impossible, to model exactly in a
 lower species. While this caveat does
 not preclude extrapolating the results of
 animal studies to humans, it does
 highlight the importance of using valid
 animal models in well-designed
 experimental studies.
 4.1.2. Validity of Animal Models
   Whether animal tests or methods
 actually measure what  they are intended
 to measure,  whether the data from such
 tests can be  obtained reliably, and
 whether such data can  be logically
 extrapolated to humans are problems for
 most disciplines in toxicology. Various
 proposals have been made for the
 standardization and validation of
 methods used in neurotoxicological
 research. It is generally agreed that
 validation is an ongoing process that
 establishes the  credibility of a test,
 building an increasing  level of
 confidence in the effective utility of any
 model of evaluation. The credibility of
 a method, as it applies to testing, is
usually discussed within several
different contexts, including construct
validity, criterion validity, predictive
validity, and detection accuracy.
  Construct validity concerns the ability
of a method to measure selectively a
particular biological function and not
other dimensions. Construct validity is
frequently established empirically. For
example, sensory dysfunction such as
hearing loss is reported by humans
exposed to some chemicals, and tests
are designed to detect and quantify
those changes. Such tests are designed
to measure changes in auditory
function, while other sensations are
unaffected (Tilson, 1987; Moser, 1990).
  Criterion validity refers to the ability
of a method to measure a characteristic
relative to some standard.  For example,
Horvath and Frantik (1973) noted that
the significance of a test measurement
as an index of an actual treatment effect
should be validated relative to the
effects of a defined reference substance
or positive control. Furthermore, each
specific test or type of effect may require
an appropriate reference substance for
which the given type of effect is a
determining factor of the toxicity. Use of
reference agents has obvious advantages
in the assessment of unknown
  Predictive validity refers to the ability
of a method to predict effects from an
incomplete or partial data set. An
animal model of neurotoxicity with
good predictive validity would reliably
predict neurotoxicity in humans, i.e.,
the animal to human extrapolation
would be good. There are several
 examples in neurotoxicology where
 animal models have been developed
 based on neurotoxicological reports
 from humans. Presumably, the
 predictive validity of such models
 would enable detecting similar kinds of
 effects produced by uncharacterized
 chemicals having a similar mechanism
 of action.
   It has been proposed (Tilson and
 Cabe, 1978) that the most logical
 approach to validate animal methods in
 neurotoxicology is to evaluate chemicals
 with and without known neurotoxicity
 in humans in tests designed for animals
 (predictive validity).  By using such an
 approach, it is possible to generate a
 profile of effects characteristic of each
 type of neurotoxicant (criterion
 validity). This profile could then be
 used to assess the construct validity of
 various tests. That is, procedures
 assumed to measure the same
 neurobiological dimension should show
 similar effects; measures  designed to
 detect changes in other functions should
 not be affected. This approach to test
 validation has been described as the
multitrait-multimethod process of
validation (Campbell and Fiske, 1959).
  Of particular importance in
establishing the credibility of a method
is the accuracy of detecting a treatment-
related effect (Gad, 1989). Accuracy is a
function of two interacting elements,
specificity and sensitivity. Specificity is
the ability of a test to respond positively
only when the toxic endpoint of interest
is present. Sensitivity is the ability to
detect a change when present. This
aspect depends on the inherent design
of the procedure and experiment.
Increasing the specificity of a test may
reduce the possibility of classifying a
chemical as neurotoxic when, in fact, it
is not (false positive), but it may
increase the probability of missing a
true neurotoxicant (false negative).
Increasing sensitivity of a test may
reduce the possibility of false negatives,
but may increase the probability of false
4.1.3. Special Considerations in Animal
Models Susceptible populations.
   Like most other measures of
lexicological effect, neurotoxic
endpoints are subject to a number of
experimental variables that may affect
susceptibility to the biological effects of
toxicants. In this regard, genetic
 variation (Festing, 1991) is a particularly
 important issue in neurotoxicology. For
 example, most neurotoxicological
 assessments are carried out with only
 one or two species. This may pose
 problems, however, since species may
 differ in sensitivity to neurotoxicants.
 For example, nonhuman primates are
 more sensitive than rats (Boyce et al.,
 1984) or mice (Heikkila et al., 1984) to
 the neurodegenerative effects of MPTP,
 a byproduct in the illicit synthesis of a
 meperidine analog (Langston et al.,
 1983). In the assessment of delayed
 neuropathology produced by some
 cholinesterase inhibitors, it is well
 known that hens are much more
 sensitive than rodents (Cavanagh, 1954;
 Abou-Donia, 1981,1983). In addition,
 rat strains also may be differentially
 sensitive to some neurotoxicants (Moser
 et al., 1991). Although it is preferred
 that more than one species be tested, the
 cost required for routine multispecies
 testing must be considered. Whenever
 possible, the choice of animal models
  should take into account differences in
  species with regard to
  pharmacodynamic, genetic composition
  and sensitivity to neurotoxic agents.
    In addition to species, other factors
  such as gender of the test animal must
  be taken into consideration. Some toxic
  substances may have a greater

Federal  Register / Vol. 59, No.  158  /  Wednesday. August 17. 1994 / Notices
  neurotoxicological effect in one gender
  (Squibb et al.( 1981; Matthews et al.,
  1990). Thus, screening evaluations
  frequently require both male and female
  animals. Another important variable is
  the age of the animal (Veronesi et al.,
  1990). Whether a chemical produces
  neurotoxicity may depend on the
  maturations! stage of the organism
  (Rodier, 1986). Most preliminary
  assessments are designed to provide
  information on adults, which have the
  greatest probability of being exposed.
  However, populations undergoing rapid
 maturation or aged individuals may be
 especially vulnerable to neurotoxic
 agents. Longitudinal studies that assess
 both genders at any stage of
 development address many of the
 problems associated with differentially
 sensitive populations. Dosing scenario.
   The dosing strategy used in
 experimental studies is an important
 variable in the development and
 expression of neurotoxicity (WHO,
 1986). Some neurotoxicants can
 produce neurotoxicity following a single
 exposure, while others require repeated
 dosing. Repeated dosing represents the
 typical pattern of human exposure to
 many chemical substances. Significant
 differences in response  may occur when
 an acutely toxic quantity of material is
 administered over different exposure
 periods. For some neurotoxicants the
 onset of neurotoxicity can occur
 immediately after dosing, while others
 may require time after exposure for the
 toxicity to develop. Effects of repeated
 exposure may result in a progressive
 alteration in nervous system function or
 structure, while latent or residual effects
 may be discovered only in association
 with age-related changes or after
 suitable environmental or
 pharmacological challenge (Zenick,
 1983; MacPhail et al., 1983). To ensure
 adequate assessment of neurotoxicity,
 study designs should include multiple
 dosing regimens, e.g., repeated
 exposure, with  appropriate dose-to-
 response intervals of testing. Conduct of
 neurotoxicological evaluations in
 studies utilizing excessively toxic doses
 should be avoided. Other factors.
  There are a number of other factors
 that should be considered in the design
 and interpretation of studies using
 animal models (WHO, 1986). Design
 factors include such issues as using
 properly trained personnel to conduct
the studies, the use of appropriate
numbers of animals per group to
achieve reliable statistical significance,
and controlling the time-of-day
                      variability. Time of testing relative to
                      exposure is also important for assessing
                      neurotoxic endpoints such as behavior,
                      and experiments should be designed to
                      generate a time course of effects,
                      including recovery of function, if any.
                      Housing is an important environmental
                      design factor, because animals housed
                      individually and animals housed in
                      groups can respond differently to toxic
                      agents. Temperature, as an experimental
                      variable, may also affect the outcome of
                      neurotoxicological studies. The
                      responsiveness to some chemicals (e.g.,
                      triethyltin, methamphetamine) varies
                      with ambient temperature (Dyer and
                      Howell,  1982; Bowyer et al., 1992).
                      Some neurobiological endpoints, such
                      as sensory evoked potentials, can be
                      influenced by the endogenous
                      temperature of the animal (Dyer, 1987).
                      Therefore, changes in body temperature,
                      whether due to fluctuations in ambient
                      temperature or to some chemically
                      induced effect such as inhibition of
                      sweating, can confound the
                      interpretation of measures such as
                      evoked responses unless proper controls
                      are included in the experimental design.
                       Because a variety of other
                      physiological changes can influence
                      neuronal functions, it is important to
                      recognize that chemical-related
                      neurotoxicity could result  from
                      treatment-induced physiological
                      changes, such as altered nutritional state
                      (WHO, 1986). As part of a
                      neurotoxicological profile, correlative
                      measures, such as relative and absolute
                      organ weights, food and water
                      consumption, and body weight and
                      weight gain, may be signs of
                      physiological change associated with
                      systemic toxicity and may be useful in
                     determining the relative contribution of
                     general toxicity.

            Statistical considerations.
                       Experimental designs for
                     neurotoxicological studies are
                     frequently complex, with two or more
                     major variables (e.g., gender, time of
                     testing) varying in any single
                     experiment. In addition, such studies
                     typically generate varying types of data,
                     including continuous, dichotomous,
                     and rank-order data. Knowledge and
                     experience in experimental design and
                     statistical analyses are important. There
                     are several key statistical concepts that
                     should be understood in
                     neurotoxicological studies (WHO, 1986;
                     Gad, 1989). The power, or probability,
                     of a study to detect a true effect is
                     dependent on the size of the study
                     group, the frequency of the outcome
                     variable in the general population, and
                     the magnitude of effect to be identified.
                     Statistical evaluation of a treatment-
 related effect involves the consideration
 of two factors or types of errors to be
 avoided. A Type I error refers to the
 attribution of an exposure-related
 neurotoxicological effect when none has
 occurred (false positive), while a Type
 II error refers to the failure to attribute
 an effect when an exposure-related
 effect has actually occurred (false
 negative). In general, the probability of
 a Type I error should not exceed 5
 percent and the probability of a Type II
 error should not exceed 20 percent.
 Power is defined as one minus the
 probability of a Type II error.
   Determination of power also requires
 knowledge of the difference in
 magnitude of outcome measures
 observed between exposed and control
 groups and the variability of the
 outcome measure among subjects. The
 sample size required to achieve a given
 level of statistical power increases as
 variability increases or the difference
 between groups decreases.
  Continuous data (i.e., magnitude, rate,
 amplitude), if found to be normally
 distributed, can be analyzed with a
 general linear model using a grouping
 factor of dose and, if necessary, repeated
 measures across time. Post hoc
 comparisons between control and other
 treatment groups can be made following
 tests for overall significance. In the case
 of multiple endpoints within a series of
 evaluations, correction for multiple
 observations (e.g., Bonferroni's) might
 be necessary.
  Descriptive data (categorical) and rank
 data can be analyzed using standard
 nonparametric techniques. In some
 cases,  if it is believed that the data fit
 the linear model, the categorical data
 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

 4.2. Tiered Testing in Neurotoxicology
  The  utility of tiered testing as an
 efficient and cost-effective approach to
 evaluate chemical toxicity, including
 neurotoxicity, has been recognized
 (NRC, 1975). Briefly, first-tier tests are
 designed to determine the presence or
absence of neurotoxicity, while second-
tier tests characterize the neurotoxic
effect (NRC, 1992). There are at least
two aspects of tiered testing, one
involving the type of test used (Tilson,
 1990a) and the other involving the
dosing regimen (Goldberg and Frazier

                 Federal Register  /  Vol. 59, No.  158  /  Wednesday, August  17. 1994 / Notices
4.2.1. Type of Test
  Tests designed to measure the
presence or absence of an effect are
usually different from those used to
assess the degree of toxicity or the
lowest exposure level required to
produce an effect (Tilson, 1990a).
Screening procedures are first-tier tests
that typically permit the testing of many
groups of animals. Such procedures may
not require extensive resources and are
usually simple to perform. However,
these techniques may be labor intensive,
provide subjective measures, yield
semiquantitative data, and may not be
as sensitive to subtle effects as those
designed to characterize neurotoxic
effects or second-tier tests. Specialized
tests  are usually more sensitive and
employed in studies concerning
mechanisms of action or the estimation
of the lowest effective dose. Such testing
procedures are usually referred to as
secondary tests and may require special
equipment and more extensive
resources. Secondary tests are usually
quantitative and yield graded or
continuous data amenable to routine
 parametric statistical analyses.
   Testing at the first tier is used to
 determine if a chemical might produce
 neurotoxicity following exposure, i.e.,
 hazard detection. In this case, there may
 be little existing information concerning
 the neurotoxic potential of an agent.
 Examples of first-tier tests include
 functional observational batteries (FOB),
 including an evaluation of motor
 activity and routine
 neurohistopathology. For some
 chemicals or types of chemicals, there
 may be a  specific interest in screening
 for a particular presumed mechanism of
 toxicity (e.g., inhibition of
 cholinesterase or neurotoxic esterase) or
 neurobiological response (e.g., a site-
 specific neuronal degeneration). In these
 cases, specific neurochemical or
 neuropathological endpoints can be
 used in conjunction with first-tier tests.
 It is desirable that tests selected for use
 in hazard detection provide a suitable
 level of sensitivity using the smallest
 number of animals necessary.
    A decision to test at the next tier is
 based on data suggesting that an agent
  produces neurotoxicity. The
  information used to make a decision to
  test a chemical at the secondary level
  can come from a variety of sources,
  including neurotoxicological data
  already in the literature, structure-
  activity relationships, data from first-tier
  testing, or following reports of specific
  neurotoxic effects in humans exposed to
  the agent. Testing at the secondary level
  includes detailed neuropathological
  evaluation as well as specific behavioral
tests, e.g., procedures to assess learning
and memory, or sensory function. Tests
at the second tier usually measure the
most sensitive endpoints of
neurotoxicity, and are the most suitable
for determining the no observable
adverse effect level or benchmark dose.
At this stage of testing, the use of a
second species is considered to address
the issue of cross-species extrapolation.
At the present time, tiered testing
approaches in neurotoxicology rely
heavily on functional endpoints. It is
possible that future testing protocols
will employ a different strategy as more
information concerning neurotoxic
mechanisms of action become available
and biologically based dose-response
models are developed.
 4.2.2. Dosing Regimen
   Goldberg and Frazier (1989) have
 indicated that first-tier evaluations
 identify effects of substances following
 acute or repeated exposure over a wide
 range of doses. Measures are simple,
 focused on detection of effects, and
 results are used to help establish
 parameters for the second tier of testing.
 The subsequent stage(s) of tier testing
 are designed to characterize more fully
 the toxicity of repeated dosing. In this
 case, animals are exposed repeatedly or
 continuously to define the scope of
 toxicity, including latent or delayed
 effects, development of tolerance, and
 the reversibility of adverse effects. The
 subsequent stage(s) of testing also
 provide  information about specific
 effects or study mechanisms of
 neurotoxicity. This tier uses methods
 appropriate to characterize the effects
 observed in the first tier of testing.

 4.3. Endpoints of Neurotoxicity

 4.3.1. Introduction
    As applied to the safety assessment of
 chemical substances, neurotoxicity is
 any adverse change in the development,
 structure, or function of the central and
  peripheral  nervous system following
 exposure to a chemical agent (Tilson,
  1990b).  Measures used in animal
  neurotoxicological studies are designed
  to assess these changes. Neurotoxicity
  can be described at multiple levels of
  organization, including chemical,
  anatomical, physiological, or behavioral
  levels. At the chemical level, for
  example, a neurotoxic substance might
  inhibit  protein or transmitter synthesis,
  alter the flow of ions across cellular
  membranes, or prevent release of
  neurotransmitter from nerve terminals.
  Anatomical changes may include
  destruction of the neuron, axon, or
  myelin sheath. At the physiological
  level, neuronal responsiveness to
stimulation might be enhanced by a
decrease of inhibitory thresholds in the
nervous system. Chemical-induced
effects at the behavioral level can
involve a variety of alterations in motor,
sensory, or cognitive function, including
increases or decreases in frequency or
accuracy of responding. Although
behavioral and neurophysiological
endpoints  may be very sensitive
indicators  of neurotoxicity, they can be
influenced by other factors. The
uncertainties associated with data from
functional endpoints can be reduced if
interpreted within the context of other
neurotoxicological measures
(neurochemical or neuropathological)
and systemic toxicity endpoints,
particularly if such measures are taken
concurrently. Behavioral effects that
reflect an indirect effect secondary to
systemic toxicities may also be
considered adverse. Table 4-1 provides
examples  of potential endpoints of
 neurotoxicity at the behavioral,
 physiological, chemical, and structural

 Behavioral Endpoints:
   Absence or altered occurrence, magnitude,
     or latency of sensorimotor reflex
   Altered magnitude of neurological meas-
     urements,  such as  grip  strength  or
     hindlimb splay
   Increases or decreases in motor activity
   Changes in rate or temporal patterning of
     schedule-controlled behavior
   Changes in motor coordination, weakness,
     paralysis, abnormal  movement or pos-
     ture, tremor, ongoing performance
   Changes in touch, sight, sound,  taste, or
     smell sensations
   Changes in learning and memory
   Occurrence of seizures
   Altered temporal development of behaviors
     or reflex responses
   Autonomic signs
  Neurophysiological Endpoints:
   Change in velocity, amplitude, or refractory
     period of nerve conduction
    Change in latency or amplitude of sensory-
     evoked potential
    Change in EEG pattern or power spectrum
  Neurochemical Endpoints:
    Alterations  in  synthesis,  release, uptake,
      degradation of neurotransmitters
    Alterations  in  second  messenger associ-
      ated signal transduction
    Alterations  in  membrane-bound  enzymes
      regulating neuronal activity
    Decreases in brain AChE
    Inhibition of NTE
    Altered developmental patterns  of neuro-
      chemical systems
    Altered proteins (c fos, substance P)
   Structural Endpoints:

Federal Register / Vol.  59. No.  158  / Wednesday, August  17,  1994 / Notices
   Accumulation,  proliferation, or rearrange-
     ment of structural elements
   Breakdown of cells
   GFAP increases (adult)
   Gross changes in morphology,  including
     brain weight
   Discoloration of nerve tissue
   Hemorrhage in nerve tissue
                       4.3.2. Behavioral Endpoints

                          Neurotoxicants produce a wide array
                       of functional deficits, including motor,
                       sensory, and learning or memory
                       dysfunction (WHO, 1986; Tilson and
                       Mitchell, 1984). Many procedures have
                       been devised to assess overt as well as
                       relatively subtle changes in those
                       functions; hence their applicability to
                       the detection of neurotoxicity and to
                       hazard characterization. Many of the
                       behavioral tests have been developed
 and validated with well-characterized
 neurotoxicants. Behavioral tests and
 agents that affect them have been
 reviewed recently (WHO, 1986; Cory-
 Slechta, 1989). Examples of such tests,
 the nervous system function being
 measured, and neurotoxicants known to
 affect these measures are listed in Table
   Grip strength; swimming endurance; suspension from  rod; discriminative motor
     function; hindlimb splay.
   Rotorod, gait measurements 	
   Rating scale, spectral analysis	.".'"!.'"!"!".'"".'.'."""."".'	
                                                                                                       methyl   butylketone,
 Myoclonia, spasms ....
 Visual toxicity 	
 Somatosensory tox-
 Pain sensitivity 	
 Olfactory toxicity	
 Classical conditioning
Operant or instrumen-
  tal conditioning.
   Rating scale, spectral analysis
   Discriminated conditioning Reflex modification
   Discriminated conditioning	
   Discriminated conditioning	
   Discriminated conditioning (titration); functional observational battery
   Discriminated conditioning	
   Startle reflex	
   Nictitating membrane	
   Conditioned flavor aversion	
   Passive avoidance	I.""!!!""!!!!!"!'
   Olfactory conditioning  	
   One-way avoidance 	""".""!
                     Two-way avoidance 	
                     Y-maze avoidance	
                     Biel water maze 	
                     Morris water maze 	
                     Radial arm maze 	
                     Delayed matching to sample .
                     Repeated acquisition 	
                     Visual discrimination learning
          3-acetylpyridine, ethanol.
          Chlordecone,  Type I  pyrethroids,
          DDT, Type II pyrethroids.

          Toluene, trimethyltin.
          Methyl mercury.

          3-methylindole methylbromide.

          Diisopropyl-flurophosphate (DFP).
          Trimethyltin, IDPN.
          Neonatal trimethyltin.

          Neonatal lead.
          Hypervitaminosis A.
          Lead. Functional observational

  Functional observational batteries are
first-tier tests designed to detect and
quantify major overt behavioral,
physiological, and other neurotoxic
effects (Moser, 1989). A number of
                       batteries have been used (Tilson and
                       Moser, 1992), each consisting of tests
                       generally intended to evaluate various
                       aspects of sensorimotor function. Most
                       FOB are similar to clinical neurological
                       examinations that rate presence or
                       absence and, in some cases, the relative
                       degree of neurological signs. A  typical
                                                  PRODUCED BY EACH
FOB, as summarized in Table 4-3,
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)

Home cage and open field
Posture (D)
Convulsions, tremors (D)
Palpebral closure (R)
Lacrimation (R)
Piloerection (Q)
Salivation (R)
Vocalizations (Q)
Rearing (C)
                       Ease of removal (R)
                       Handling reactivity (R)

                       Approach response (R)
                       Click response (R)
                       Touch response (R)
                       Tail pinch response (R)
                       Righting reflex (R)
Body temperature (I)
Body weight (I)

                Federal Register / Vol.  59,  No. 158 / Wednesday, August  17,  1994  /  Notices
                                          PRODUCED BY EACH—Continued
Urination (C)
Defecation (C)
Gait (D, R)
Arousal (R)
Mobility (R)
Stereotypy (D)
Bizarre behavior (D)
  Landing foot splay (I)
  Forelimb grip strength (I)
  Hindlimb grip strength (I)
  Pupil response (Q)
  D = descriptive data; R = rank order data; Q
  I = interval data; C = count data
  The major advantages of FOB tests are
that they can be administered within the
context of other ongoing toxicological
tests and provide some indication of the
possible neurological alterations
produced by exposure. Potential
problems include insufficient
interobserver reliability, difficulty in
defining certain endpoints, and the
tendency toward observer bias. The
latter can be controlled by using
observers unaware of the actual
treatment of the subjects. Some FOB
tests may not be very sensitive to agent-
induced sensory loss (i.e., vision,
audition) or alterations in cognitive or
integrative processes such as learning
and memory. FOB data may be used to
trigger experiments performed at the
next tier of testing.
   FOB data may be interval, ordinal, or
continuous (Creason, 1989). The
relevance of statistically significant test
results from an FOB is judged according
to the number of signs affected, the
dose(s) at which neurotoxic signs are
observed, and the nature, severity, and
 persistence of the effects. Data from the
 FOB may provide presumptive evidence
 of adverse effects and neurotoxicity. If
 only a  few unrelated measures in the
 FOB are affected or the effects are
 unrelated to dose, there is less concern
 about neurotoxic potentials of a
 chemical. If dose is associated with
 other overt signs of toxicity, including
 systemic toxicity, large decreases in
 body weight, or debilitation, the data
 must be interpreted carefully. In cases
 where several related measures  in a
 battery of tests are affected and the
 effects appear to be dose dependent, the
 level of concern about the potential of
 a chemical is higher. Motor activity.
    Movement within  a defined
 environment is a naturally occurring
 response and can be affected by
 environmental agents. Motor activity
 represents a broad class of behaviors
 involving coordinated participation of
 sensory, motor, and integrative
: quanta! data;
 processes. Motor activity measurements
 are noninvasive and can be used to
 evaluate the effects of acute and
 repeated exposure to chemicals
 (MacPhail et al., 1989). Motor activity
 measurements have also been used in
 humans to evaluate disease states,
 including disorders of the nervous
 system (Goldstein and Stein, 1985). The
 assessment of motor activity is often
 included in first-tier evaluations, either
 as part of the FOB or as a separate
 quantitated measurement.
   There are many different types of
 activity measurement devices, differing
 in size, shape, and method of movement
 detection (MacPhail et al., 1989).
 Because of the accuracy and ease of
 calibration, devices with photocells are
 widely used. In general, situating the
 apparatus to minimize extraneous noise,
 movements, or lights usually requires
 that the recording devices be placed  in
 light- and sound-attenuating chambers
 during the testing period. A number of
 different factors,  including age, gender,
 and time of day,  can affect motor
  activity, and should be controlled or
  counterbalanced. Different strains of
  animals may have significantly different
  basal levels of activity, making
  comparisons across studies difficult. A
  major factor in activity studies is the
  duration of the testing session. Motor
  activity levels are generally highest at
  the beginning of the session and
  decrease to a low level throughout the
  session. The rate of decline during the
  test session is frequently termed
    Motor activity measurements are
  typically included as part of a battery of
  tests to detect or characterize
  neurotoxicity. Agent-induced alterations
  in motor activity associated with overt
  signs of toxicity (e.g.,  loss of body
  weight, systemic toxicity) or occurring
  in non-dose-related fashion are of less
  concern than changes that are dose
  dependent, related to structural or other
  functional changes in the nervous
  system, or occur in the absence of life-
threatening toxicity and are generally
convincing evidence of neurotoxicity. Neuromotor function.
  Motor dysfunction is a common
neurotoxic effect, and many different
types of tests have been devised to
measure time- and dose-dependent
effects. Anger (1984) reported 14 motor
effects of 89 substances, which could be
classified into four categories: weakness,
incoordination, tremor, and myoclonia
or spasms. Chemical-induced changes
in motor function can be determined
with relatively simple techniques such
as the FOB. More specialized tests to
assess weakness include measures of
grip strength, swimming endurance,
suspension from a hanging rod,
discriminitive motor function, and
hindlimb splay. Rotarod and gait
assessments measure incoordination,
while rating scales and spectral analysis
techniques quantify tremor and other
abnormal movements (Tilson and
Mitchell, 1984).
   An example of a second-tier
procedure to assess motor function has
been described by Newland (1988), who
trained squirrel monkeys to hold a bar
within specified limits (i.e.,
 displacement) to receive positive
 reinforcement. The bar was also
 attached to a rotary device, which
 allowed measurement of chemical-
 induced tremor. Spectral analysis was
 used to characterize the tremor, which
 was found to be similar to that seen in
 humans exposed to neurotoxicants or
 with such neurologic diseases as
 Parkinson's disease.
   Incoordination and  performance
 changes can be assessed with
 procedures that measure chemical-
 induced alterations in force (Fowler,
 1987). The accuracy of performance may
 reflect neuromotor function and is
 sensitive to the debilitating effects of
 many psychoactive drugs (Walker et al.,
 1981; Newland, 1988). Gait, an index of
 coordination, has been measured in rats
 under standardized conditions and can
 be a sensitive indication of specific

Federal  Register / Vol. 59. No.  158 / Wednesday. August 17, 1994 / Notices
 damage to the basal ganglia and motor
 cortex (Hruska et al., 1979) as well as
 damage to the spinal cord and
 peripheral nervous system.
   Procedures to characterize chemical-
 induced motor dysfunction have been
 used extensively in neurotoxicology.
 Most require preexposure training
 (including alterations of motivational
 state) of experimental animals, but such
 tests might be useful, in as much as
 similar procedures are often used in
 assessing humans. Sensory function.

   Alterations in sensory processes (e.g.,
 paresthesias and visual or auditory
 impairments) are frequently reported
 signs or symptoms in humans exposed
 to toxicants (Anger, 1984). Several
 approaches have been devised to
 measure sensory deficits. Data from tests
 of sensory function must be interpreted
 within the context of changes in body
 weight, body temperature, and other
 physiological endpoints. Furthermore,
 many tests assess the behavioral
 response of an animal to a specific
 sensory stimulus; such responses are
 usually motor movements that could be
 directly affected by chemical exposure.
 Thus, care must be taken to determine
 whether proper controls were included
 to eliminate the possibility that changes
 in response to a sensory stimulus may
 have been related to agent-induced
 motor dysfunction.
  Several first-tier testing procedures
 have been devised to screen for overt
 sensory deficits. Many rely on
 orientation or the response of an animal
 to a stimulus. Such tests are usually
 included in the FOB used in screening
 (e.g., tail-pinch or click responses).
 Responses are usually recorded as being
 either present, absent, or changed in
 magnitude (Moser, 1989; O'Donoghue,
 1989). Screening tests for sensory
 deficits are typically not suitable to
 characterize chemical-induced changes
 in acuity or fields of perception. The
 characterization of sensory deficits
 usually necessitates psychophysical
 methods that study the relationship
 between the physical dimensions of a
 stimulus and the behavioral response it
 generates (Maurissen, 1988).
  One second-tier approach to the
characterization of sensory function
 involves  the use of reflex-modification
techniques (Crofton, 1990). Chemical-
induced changes in the stimulus
frequency or threshold required to
inhibit a reflex are taken as possible
changes in sensory  function. Prepulse
inhibition has been used only recently
in neurotoxicology  (Fechter and  Young,
1983) and can be used to assess sensory
                      function in humans as well as in
                      experimental animals.
                        Various behavioral procedures require
                      that a learned response occur only in
                      the presence of a specific stimulus (i.e.,
                      discriminated or conditioned
                      responding). Chemical-induced changes
                      in sensory function are determined by
                      altering the physical characteristics of
                      the stimulus (e.g., magnitude or
                      frequency) and measuring the alteration
                      in response rate  or accuracy. In an
                      example of the use of a discriminated
                      conditional response to assess chemical-
                      induced sensory dysfunction, Maurissen
                      et al. (1983) trained monkeys to respond
                      to the presence of a vibratory or electric
                      stimulus applied to the fingertip.
                      Repeated dosing with acrylamide
                      produced a persistent decrease in
                      vibration sensitivity; sensitivity to
                      electric stimulation was unimpaired.
                      That pattern of sensory dysfunction
                      corresponded well to known sensory
                      deficits in humans. Discriminated
                      conditional response procedures have
                      been used to assess the ototoxicity
                      produced by toluene (Pryor et al., 1983)
                      and the visual toxicity produced by
                      methylmercury (Merigan, 1979).
                       Procedures to characterize chemical-
                      induced sensory dysfunction have been
                      used often in neurotoxicology. As in the
                      case of most procedures designed to
                      characterize nervous system
                      dysfunction, training and motivational
                      factors can be confounding factors.
                      Many tests designed to assess sensory
                      function for laboratory animals can also
                     be applied with some adaptation to

            Learning and memory.
                       Learning and memory disorders are
                     neurotoxic effects of particular
                     importance. Impairment of memory is
                     reported fairly often by adult humans as
                     a consequence of toxic exposure.
                     Behavioral deficits in children have
                     been caused by lead exposure (Smith et
                     al., 1989), and it is hypothesized (Calne
                     et al., 1986) that chronic low-level
                     exposure to toxic agents may have a role
                     in the pathogenesis of senile dementia.
                       Learning can be defined as an
                     enduring change  in the mechanisms of
                     behavior that results from experience
                     with environmental events (Domjan and
                     Burkhard, 1986).  Memory is a change
                     that can be either short-lasting or long-
                     lasting (Eckerman and Bushnell, 1992).
                     Alterations in learning and memory
                     must be inferred  from changes in
                     behavior. However, changes in learning
                     and memory must be separated from
                     other changes in behavior that do not
                     involve cognitive or associative
                     processes (e.g., motor function, sensory
                     capabilities, and motivational factors),
 and an apparent toxicant-induced
 change in learning or memory should be
 demonstrated over a range of stimuli
 and conditions. Before it is concluded
 that a toxicant alters learning and
 memory, effects should be confirmed in
 a second learning procedure. It is well
 known that lesions in the brain can
 inhibit learning. It is also known that
 some brain lesions can facilitate some
 types of learning by removing
 behavioral tendencies (e.g., inhibitory
 responses due to stress) that moderate
 the rate of learning under normal
 circumstances. A discussion of learning
 procedures and examples of chemicals
 that can affect learning and memory
 have appeared in recent reviews (Heise,
 1984; WHO, 1986; Peele and Vincent,
  One simple index of learning and
 memory, which can be measured as a
 first-tier endpoint, is habituation.
 Habituation is defined as a gradual
 decrease in the magnitude or frequency
 of a response after repeated
 presentations of a stimulus. A toxicant
 can affect habituation by increasing or
 decreasing the number of stimulus
 presentations needed to produce
 response decrements (Overstreet, 1977).
 Although habituation is a  very simple
 form of learning, it can also be
 perturbed by a number of chemical
 effects not related to learning.
  A more complicated approach to
 studying the effects of a chemical on
 learning and memory involves the
 pairing of a novel stimulus with a
 second stimulus that produces a known,
 observable, and quantifiable response
 (i.e., classical "Pavlovian"
 conditioning). The novel stimulus is
 known as the conditioned stimulus, and
 the second, eliciting stimulus is the
 unconditioned stimulus. With repeated
 pairings of the two stimuli, the
 conditioned stimulus comes to elicit a
 response similar to the response elicited
 by the unconditioned stimulus. The
 procedure has been used in behavioral
 pharmacology and, to a lesser extent, in
 neurotoxicology. Neurotoxicants that
 interfere with learning and memory
 would alter the number of presentations
 of the pair of stimuli required to
 produce conditioning or learning.
Memory would be  tested by determining
how long after the last presentation of
 the two stimuli the conditioned
stimulus would still elicit a response
 (Yokel, 1983). Other classically
conditioned responses known to be
affected by psychoactive or neurotoxic
agents are conditioned taste aversion
(Riley and Tuck, 1985) and conditioned
suppression (Chiba and Ando, 1976).
  Second-tier procedures to assess
learning or memory typically involve

                Federal Register / Vol.  59,  No. 158 / Wednesday, August 17, 1994 / Notices
	r	j of a response with a
stimulus that increases the probability
of future response through
reinforcement. Response rate can be
increased by using positive
reinforcement or removing negative
reinforcement. Learning is usually
assessed by determining the number of
presentations or trials needed to
produce a defined frequency of
response. Memory can be defined
specifically as the maintenance of a
stated frequency of response after initial
training. Neurotoxicants may adversely
affect learning by increasing or
decreasing the number of presentations
required to achieve the designated
criterion. Decrements in memory may
be indicated by a decrease in the
probability or frequency of a response at
some time after initial training.
Toxicant-induced changes in learning
and memory should be interpreted
within the context of possible toxicant-
induced changes in sensory, motor, and
motivational factors. Examples of
instrumental learning procedures used
in neurotoxicology are repeated
acquisition (Schrot et al., 1984). passive
and active avoidance, Y-maze
avoidance, spatial mazes (radial-arm
maze), and delayed matching to sample
 (Heise, 1984; WHO, 1986; Tilson and
Mitchell, 1984).  Schedule-controlled behavior.
   Another type of second-tier procedure
 is schedule-controlled operant behavior
 (SCOB), which involves the
 maintenance of behavior (performance)
 by response-dependent reinforcement
 (Rice, 1988). Different patterns of
 behavior and response rates are
 controlled by the relationship between
 response and later reinforcement. SCOB
 affords  a measure of learned behavior
 and with appropriate experimental
 design may be useful for studying
 chemical-induced effects on motor,
 sensory, and cognitive function.
   The  primary endpoints for evaluation
 are agent-induced changes in response
 rate  or frequency and the temporal
 pattern of responding. Response rate is
 usually related to an objective response,
 such as lever press or key peck, and
 differs according to the schedule of
 reinforcement. Response rates are
 expressed per unit of time. For some
 classes of chemicals, the direction of an
 effect on response rate can differ
 between low and high doses. Agent-
  induced changes in temporal pattern of
  responding can occur independently of
  changes in the rate and require analysis
  of the distribution of responses relative
  to reinforcement schedule.
    SCOB has been used to study the
  effects of psychoactive drugs on
behavior and is sensitive to many
neurotoxicants, including
methylmercury, solvents, pesticides,
acrylamides, carbon monoxide, and
organic and inorganic lead (Paule and
McMillan, 1984; MacPhail 1985; Cory-
Slechta, 1989; Rice, 1988). The
experimental animal often serves as its
own control, and the procedure
provides an opportunity to study a few
animals extensively over a relatively
long period. SCOB typically requires
motivational procedures, such as food
deprivation, and training sessions are
usually required to establish a stable
baseline of responding. Because of its
sensitivity to neuroactive chemicals,
SCOB has great potential for use in
second-tier assessments.
4.3.3. Neurophysiological  Endpoints of
   Neurophysiological studies are those
that assess function either directly
through measurements of the electrical
activity of the nervous system
 (electrophysiology) or indirectly
through measurements of peripheral
 organ functions controlled or modulated
by the nervous system (general
 physiology) (Dyer, 1987). When
 performed properly, neurophysiological
 techniques provide information on the
 integrity of defined portions of the
 nervous system. Many of the endpoints
 used in animals have also been used in
 humans to determine chemical-induced
 alterations in neurophysiological
   The term "electrophysiology" refers
 to the set of neurophysiological
 procedures that study neural function
 through the direct measurement of the
 electrical activity generated by the
 nervous system (Dyer, 1987). A variety
 of electrophysiological procedures are
 available for application to
 neurotoxicological problems, which
 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. The
 latter types of procedures have
 historically been used in studies to
 detect or characterize the potential
 neurotoxicity of agents of regulatory
 interest. Several macroelectrode
  procedures are discussed below. Nerve conduction studies.
    Nerve conduction studies are
  generally performed on peripheral
  nerves and can be useful in
  investigations of possible peripheral
  neuropathy. Most peripheral nerves
  contain mixtures of both individual
sensory and motor nerve fibers, which
may or may not be differentially
sensitive to neurotoxicants. It is possible
to distinguish sensory from motor
effects in peripheral nerve studies by
measuring activity in purely sensory
nerves such as the sural to study
sensory effects or by measuring the
muscle response evoked by nerve
stimulation to measure motor effects.
While a number of endpoints can be
recorded, the most commonly used
variables are (1) Nerve conduction
velocity, and (2) response amplitude. In
well-controlled studies, decreases in
nerve conduction velocity typically are
evidence of neurotoxicity (Dyer,  1987).
While a decrease in nerve conduction
velocity is a reliable measure of
demyelination, it frequently occurs
rather late 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 necessarily rule out peripheral
axonal degeneration if other signs of
 peripheral nerve dysfunction are
 present. Increases in conduction
 velocity of adult organisms following
 treatment with neurotoxic compounds,
 in the absence of hypothermia, are
 atypical responses and may, in fact,
 reflect experimental or statistical errors.
 Decreases in response amplitude reflect
 a loss of active nerve fibers, and may
 occur prior to decreases in conduction
 velocity in the course of peripheral
 neuropathy. Hence changes in response
 amplitude may be more sensitive
 measurements of axonal degeneration
 than conduction velocity.
 Measurements of response amplitude,
 however, are more variable and  require
 careful experimental techniques, a
 larger sample size, and greater statistical
 power than measurements of velocity to
 detect changes. Alterations in peripheral
 nerve function are associated with
 abnormal peripheral sensations such as
 numbness, tingling, or burning or with
 motor impairments such as weakness.
  Examples of compounds that alter
  peripheral nerve function in humans or
  experimental animals at some level of
  exposure include acrylamide, carbon
  disulfide, hexacarbons, lead, and some
  organophosphates. Sensory evoked potentials.
    Sensory evoked potentials are
  electrophysiological procedures that
  involve measuring the response elicited
  by the presentation of a defined sensory
  stimulus such as a tone, a light, or a
  brief electrical pulse to the skin.
  Sensory evoked potentials reflect
  sensory function, and can be used to

Federal Register / Vol. 59, No. 158  /  Wednesday, August 17, 1994 / Notices
 investigate visual, auditory, or
 somatosensory (body sensation) systems
 (Rebert, 1983; Mattsson and Albee,
 1988). The data are in the form of a
 voltage record over time, which can be
 quantified in several ways. Commonly,
 the positive and negative voltage peaks
 are identified and measured as to their
 latency (time from stimulus onset) and
 amplitude (voltage).
   Changes in peak amplitudes or
 equivalent measures reflect changes in
 the magnitude of the neural population
 that is responsive to stimulation. Both
 increases and decreases in amplitude
 are possible following exposure to
 neurotoxicants because (1) The brain
 normally operates in a careful balance
 between excitatory and inhibitory
 systems, and disruption of this balance
 can produce either positive or negative
 shifts in the voltages recorded in evoked
 potential experiments, and (2) excitatory
 or inhibitory neural activity is translated
 into a positive or negative deflection in
 the sensory evoked potential depending
 on the physical orientation of the
 electrode with respect to the tissue
 generating the response, which is
 frequently unknown. Within any given
 sensory system, the neural circuits that
 generate the different evoked potential
 peaks differ as a function of peak
 latency. In general, early latency peaks
 reflect the transmission of afferent
 sensory information, and changes  in
 either the latency or amplitude of these
 peaks generally indicate a neurotoxic
 change 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. Thus, the
 neurotoxicological significance of
 changes in later latency evoked
 potential peaks must be interpreted in
 light of the behavioral status of the
 subject. Convulsions.
  Observable behavioral convulsions in
 animals may be indicative of central
 nervous system seizure activity.
 However, behavioral convulsions that
 occur only at lethal or near lethal dose
 levels may reflect an indirect effect
 secondary to systemic toxicity and not
 directly on the nervous system.
Convulsions occurring at dose levels
that are clearly sublethal, and in the
absence of apparent systemic toxicity,
are more likely due to a direct effect on
the nervous system. In such cases,
neurophysiological recordings of
electrical activity in the brain that are
indicative of seizures may provide
                      additional evidence of direct
                      neurotoxicity. In addition to producing
                      seizures, chemicals may also affect
                      seizure susceptibility, altering the
                      frequency, severity, duration, or
                      threshold for eliciting seizures produced
                      through other means. Such changes can
                      occur after acute exposure or after
                      repeated exposure to dose levels below
                      the acute threshold, and are considered
                      neurotoxic. Agents that produce
                      convulsions include lindane, DDT,
                      pyrethroids, and trimethyltin (WHO,
                      1986). Some agents, including many
                      solvents, act to raise the threshold for
                      eliciting seizures through other means
                      or otherwise act to reduce the severity
                      or duration of the elicited convulsions.
                      These agents are difficult to classify as
                      neurotoxic based on such data, but
                      frequently have other effects on which
                      a determination of neurotoxic potential
                      can be based.

             Electroencephalography (EEC)
                        EEC analysis is used widely in
                      clinical settings for the diagnosis of
                      neurological disorders and less often for
                      the detection of subtle toxicant-induced
                      dysfunction (WHO, 1986; Eccles, 1988).
                      The basis for the use of EEC in either
                      setting is the relationship between
                      specific patterns of EEC waveforms and
                      specific behavioral states. Because states
                      of alertness and the 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 EEC.
                      Dissociation of EEC 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 following
                      exposure to certain chemical classes
                      (e.g.,  organophosphates). Changes in the
                      pattern of the EEC can be elicited by
                      stimuli producing arousal (e.g., lights,
                      sounds) and neuroactive drugs. In
                      studies with toxicants, changes in EEC
                      pattern can sometimes precede
                      alterations in other objective signs of
                      neurotoxicity. EEC experiments must be
                      done  under highly controlled
                      conditions, and the neurotoxicological
                      significance of chemical-induced
                      changes in the EEC in the absence of
                      other signs of neurotoxicity must be
                      considered on a case-by-case basis.
                      Many chemicals, including metals,
                      solvents, and pesticides, would be
                      expected to affect the EEC.

             Electromyography (EMG).
                        EMG involves making electrical
                      recordings from muscle and has been
                      used  extensively in human clinical
 studies in the diagnosis of certain
 diseases of the muscle (WHO, 1986).
 Changes in the EMG include amplitude
 and firing frequency of spontaneous
 firing; evoked muscle responses to nerve
 stimulation can be used to study
 alterations in the neuromuscular
 junction. EMG has been used to study
 toxicant-induced changes in
 neuromuscular function, including
 organophosphate insecticides, methyl n-
 butyl ketone, and botulinum and
 tetanus toxin. Spinal reflex excitability.
   Segmental spinal monosynaptic and
 polysynaptic reflexes are relatively
 simple functions in the central nervous
 system that can be evaluated by
 quantitative techniques (WHO, 1986).
 Many of the procedures used in animals
 are similar to procedures used clinically
 to perform neurological tests in humans.
 One approach infers the functional state
 of a reflex arc from either the latency
 and magnitude of the reflex response
 evoked by stimuli  of predetermined
 intensity or from the stimulus intensity
 required to elicit a detectable response
 (i.e., the threshold). This approach is
 used best in a screening context and the
 significance of effects in this test should
 be considered on a case-by-case basis.
  A second more involved approach
 records electrophysiologically the time
 required for a stimulus applied to a
 peripheral nerve to reach the spinal
 cord and return to the site of the original
 stimulation. Data from  this procedure
 can indicate the excitability of the
 motoneuron pool, an effect seen with
 many volatile solvents. Although this
 approach is more invasive and time-
 consuming than the noninvasive
 procedure, it provides better data
 concerning the possible site of action. In
 addition, the manner in which the
 invasive procedure is carried out (i.e., in
 decerebrated animals) precludes
 repeated testing on the same animal.
The significance of effects in this
 procedure should also be considered on
 a case-by-case basis.

 4.3.4. Neurochemical Endpoints of
  Neuronal function within the nervous
system is dependent on synthesis and
release of specific neurotransmitters and
activation of their receptors in specific
neuronal pathways. With few
exceptions, neurochemical
measurements are invasive and
therefore used infrequently in human
risk assessment. There are many
different neurochemical endpoints that
could be measured in
neurotoxicological studies (Bondy,
 1986; Mailman, 1987; Morell and

                Federal Register / Vol. 59, No. 158 / Wednesday, August 17, 1994  / Notices
Mailman, 1987). Neurotoxicants can
interfere with the ionic balance of a
neuron, act as a cytotoxicant after being
transported into a nerve terminal, block
uptake of neurotransmitter precursors,
act as a metabolic poison, overstimulate
receptors, block transmitter release, and
inhibit transmitter degradation. Table 4-
4 lists several chemicals with known
neurochemical effects. Many
neuroactive agents can increase or
decrease neurotransmitter levels in the
                                     brain. Dose-related changes on these
                                     endpoints may indicate a chemical
                                     effect on the nervous system, but the
                                     neurotoxicological significance of such
                                     changes must be interpreted in the
                                     context of other signs of neurotoxicity.
Site of attack
1 . Neurotoxicants acting on ionic balance

7. Transmitter degradation (ACh) inhibitors 	
8. Microtubule disrupters 	
p,p1 — DDT, pyrethroids (I).
Domoic acid.
Botulinum toxin.
   Some chemicals, such as the
 organophosphate and carbamate
 insecticides, are known to interfere with
 a specific enzyme, acetylcholinesterase
 (AChE) (Costa, 1988). Inhibition of this
 enzyme in brain may be considered
 evidence of neurotoxicity, whereas
 decreases in AChE in the blood, which
 can be easily determined in humans, are
 only suggestive of a neurotoxic effect. A
 subset of organophosphate agents
 produces organophosphate-induced
 delayed neuropathy (OPIDN) after acute
 or repeated exposure. Neurotoxic
 esterase (or neuropathy target enzyme,
 NTE) has been associated with agents
 that produce OPIDN (Johnson, 1990).
   The ultimate functional significance
 of many biochemical changes is not
 known; therefore it may be difficult to
 determine if a specific biochemical
change can be considered adverse or
convincing evidence of neurotoxicity.
Any such change, however, is
potentially adverse and each
determination of adversity requires a
judgment to be made. Likewise, the
absence of specific biochemical testing
protocols does not mean biochemical
changes are of no concern, but instead
reflects a lack of understanding of the
significance of changes at the
biochemical level.
4.3.5. Structural Endpoints of
  The central nervous system (brain and
spinal cord) comprises nerve cells or
neurons, which consist of a neuronal
body, axon, and dendritic processes.
Various types of neuropathological
lesions may be classified according to
                                       their nature or the site where they are
                                       found (WHO, 1986; Krinke. 1989;
                                       Griffin, 1990). Lesions may be classified
                                       as neuropathy (changes in the neuronal
                                       body), axonopathy (changes in the
                                       axons), myelinopathy (changes in the
                                       myelin sheaths), neurodegeneration
                                       (changes in the nerve terminals), and
                                       peripheral neuropathy (changes in the
                                       peripheral nerves). For axonopathies, a
                                       more precise location of the changes
                                       should be described (i.e., proximal,
                                       central, or distal axonopathy).  In some
                                       cases, agents produce neuropathic
                                       conditions that resemble naturally
                                       occurring neurodegenerative disorders
                                       in humans (WHO, 1986). Table 4-5 lists
                                       examples of such chemicals, their
                                       known site of action, the type of
                                       neuropathology produced, and the
                                       disease or condition that each  typifies.
                              TABLE 4-5.—EXAMPLES OF KNOWN NEUROPATHIC AGENTS
Site of attack

Neurodegeneration ....
Distal axonopathy 	
Central axonopathy ...
Proximal axonopathy .
Methylmercury ..
Quinolinic acid ..
3-acetylpridine ..
Lead Buckthorn
Carbon disul-
Disease or
neurodegenerative condi-
Minamata disease.
Ceroid lipofuscinoses.
Huntington's disease.
Cerebellar ataxia.
Alzheimer's disease.
Parkinson's disease.
Neuropathy of metachro-
matic leukodystrophy.
Vitamin deficiency.
Subacute myelooptico-neu-
Motor neuron disease.

Federal Register / Vol. 59, No.  158 / Wednesday. August  17,  1994  /  Notices
   In general, chemical effects lead to
 two types of primary cellular alteration:
 (1) the accumulation, proliferation, or
 rearrangement of structural elements
 (e.g., intermediate filaments,
 microtubules) or organelles
 (mitochondria) and (2) the breakdown of
 cells, in whole or in part. The latter can
 be associated with regenerative
 processes that may occur during
 chemical exposure. Such changes are
 considered to be neurotoxic.
   While most neurotoxic damage is
 evident at the microscopic level, gross
 changes in morphology can be reflected
 by a significant change in the weight of
 the brain. Weight changes (absolute or
 relative to body weight), discoloration,
 discrete or massive cerebral
 hemorrhage, or obvious lesions in nerve
 tissue are generally considered
 neurotoxic effects.
   Chemical-induced injury to the
 central  nervous system is associated
 with astrocytic hypertrophy at the site
 of damage. Assays of glial fibrillary
 acidic protein (GFAP), the major
 intermediate filament protein of
 astrocytes, has been proposed as a
 biomarker of this response (O'Callaghan,
 1988). A number of chemicals known to
 injure the central nervous system,
 including trimethyltin, methylmercury,
 cadmium, 3-acetylpyridine, and MPTP,
 have been shown to increase GFAP. In
 addition, increases in GFAP may be
 seen at dosages below those necessary to
 produce cytopathology as determined
 by Nissl-based stains used in standard
 neuropathological examinations.
 Because increases in GFAP may be an
 early indicator of neuronal injury in the
 adult, exposure level-dependent
 increases in GFAP should be viewed
 with concern.
  Chemical-induced alterations in the
 structure of the nervous system are
 generally considered neurotoxic effects.
 To ensure reliable data, it is important
 that neuropathological studies minimize
 fixation artifacts and potential
 differences in the section(s) of the
 nervous system sampled and control for
 variability due to the age, sex, and body
 weight of the subject (WHO, 1986).

 4.3.6. Developmental Neurotoxicity
  Exposure to chemicals during
 development can result in effects other
 than death, gross structural abnormality,
 or altered growth. There are several
 instances in which functional
alterations have resulted from exposure
during the period between conception
and sexual maturity (Riley and Vorhees,
 1986; Vorhees, 1987). Table 4-6 lists
several examples of chemicals known to
produce developmental neurotoxicity in
experimental animals. Animal models
                      of developmental neurotoxicity have
                      been shown to be sensitive to several
                      environmental chemicals known to
                      produce developmental toxicity in
                      humans, including lead, ethanol,
                      methylmercury, and PCBs (Kimmel et
                      al., 1990).

                      TABLE 4-6.—PARTIAL LIST OF AGENTS
                        BELIEVED TO HAVE DEVELOPMENTAL



                      Polyhalogenated hy-
                      Psychoactive drugs

Methanol, ethanol
DDT, kepone,
Lead, methylmercury,

Cocaine, phenytoin
Carbon disulfide, tolu-
Vitamin A
                        Sometimes functional defects are
                      observed at dose levels below those at
                      which other indicators of
                      developmental toxicity are evident
                      (Rodier, 1986). Such effects may be
                      transient or reversible in nature, but
                      generally are considered adverse effects.
                      Data from postnatal studies, when
                      available, are considered useful for
                      further assessment of the relative
                      importance and severity of findings in
                      the fetus and neonate. Often, the long-
                      term consequences of adverse
                      developmental outcomes noted at birth
                      are unknown and further data on
                      postnatal development and function are
                      necessary to determine the full
                      spectrum of potential developmental
                      effects. Useful data also can be derived
                      from well-conducted multigeneration
                      studies, although the dose  levels used in
                      these studies may be much lower than
                      those in studies with shorter-term
                       Much of the early work in
                      developmental neurotoxicology was
                      related to behavioral evaluations. Recent
                      advances in this area have  been
                      reviewed in several publications (Riley
                      and Vorhees, 1986; Kimmel et al., 1990).
                      Several expert groups have focused on
                      the functions that should be included in
                      a behavioral testing battery, including
                      sensory systems, neuromotor
                      development, locomotor activity,
                      learning and memory, reactivity and
                      habituation, and reproductive behavior.
                      No testing battery  has fully addressed
                      all of these functions, but it is important
                      to include as many as possible, and
                      several testing batteries have been
                      developed and evaluated for use in
   Direct extrapolation of functional
 developmental effects to humans is
 limited in the same way as for other
 endpoints of developmental toxicity,
 i.e., by the lack of knowledge about
 underlying toxicological mechanisms
 and their significance. It can be assumed
 that functional effects in animal studies
 indicate the potential for altered
 development in humans, although the
 types of developmental effects seen in
 experimental animal studies will  not
 necessarily be the same as those that
 may be produced in humans. Thus,
 when data from functional
 developmental toxicity studies are
 encountered for particular agents, they
 should be considered in the risk
 assessment process.
   Agents that produce developmental
 neurotoxicity at a dose that is not toxic
 to the maternal animal are of special
 concern because the developing
 organism is affected but toxicity is not
 apparent in the adult. More commonly,
 however, adverse developmental effects
 are produced only at doses that cause
 minimal maternal toxicity; in these
 cases, the developmental effects are still
 considered to represent developmental
 toxicity and should not be discounted as
 secondary to maternal toxicity. At doses
 causing excessive maternal toxicity (that
 is, significantly greater than the minimal
 toxic dose), information on
 developmental effects may be difficult
 to interpret and of limited value.
 Current information is inadequate to
 assume that developmental effects at
 maternally toxic doses result only from
 maternal toxicity; it may be that the
 mother and developing organism are
 sensitive to that dose level. Moreover,
 whether developmental effects are
 secondary to maternal toxicity or not,
 the maternal effects may be reversible
 while effects on the offspring may be
 permanent. These are  important
 considerations for agents to which
 humans may be exposed at minimally
 toxic levels either voluntarily  or
 involuntarily, because several agents are
 known to  produce adverse
 developmental effects at minimally
 toxic doses in adult humans (e.g.,
 smoking, alcohol).
  Although interpretation of functional
 developmental neurotoxicity data  may
be limited at present, it is clear that
 functional effects must be evaluated in
 light of other toxicity data, including
other forms of developmental  toxicity
 (e.g., structural abnormalities, perinatal
death, and growth retardation). The
level of confidence in an adverse effect
may be as important as the type of
change seen, and confidence may be
increased by such factors as replicability
of the effect either in another study of

                 Federal Register  /  Vol. 59. No.  158 / Wednesday,  August 17. 1994  / Notices          42393
the same function or by convergence of
data from tests that purport to measure
similar functions. A dose-response
relationship is considered an important
measure of chemical effect; in the case
of functional effects, both monotonic
and biphasic dose-response curves are
likely, depending on the function being
4.3.7. Physiological and Neuroendocrine
   One of the key roles played by the
nervous system is to orchestrate the
general physiological functions of the
body to help maintain homeostasis. To
this end, the nervous system and many
of the peripheral organ systems are
integrated and functionally
interdependent. For example, specific
neuronal processes are intimately
involved in maintaining or modulating
respiration, cardiovascular function,
body temperature, and gastrointestinal
function. Because many peripheral
organ functions involve neuronal
components, changes in such
physiological endpoints as blood
pressure, heart rate, EKG, body
temperature, respiration, lacrimation, or
salivation may indirectly reflect
possible treatment-related effects on the
functional integrity of the nervous
system. However, since physiological
endpoints also depend on the integrity
 of the related peripheral organ itself,
 changes in physiological function also
 may reflect a systemic toxicity involving
 that organ. Consequently, the
 neurotoxicological significance of a
 physiological change must be
 interpreted within the context of other
 signs of toxicity. A variety of general
 physiological procedures can be applied
 to neurotoxicological problems. These
 procedures range in scale from simple
 measurements, for example, of body
 temperature, respiration, lacrimation,
 salivation, urination, and defecation,
 which may be included in routine
 functional observational batteries used
 for chemical screening, to more
 involved procedures involving
 measurements of blood pressure,
 endocrine responses, cardiac function,
 gastrointestinal function, etc. The latter
 would be more appropriate for second-
 level tests to characterize the scope of
 chemically related toxicity.
   The central nervous system also
 regulates the outflow of the endocrine
 system, which together with the
 influence of the autonomic nervous
 system, can affect immunologic function
 (WHO, 1986). Hormonal balance results
 from the integrated action of the
 hypothalamus, located in the central
 nervous system, and the pituitary,
  which regulates activities of endocrine
target organs. Each site is susceptible to
disruption by neurotoxic agents.
Neuroendocrine dysfunction may occur
because of a disturbance in the
regulation and modulation of the
neuroendocrine feedback systems. One
major indicator of neuroendocrine
function is secretions of hormones from
the pituitary. Hormones from the
anterior pituitary are important for
reproduction (follicle-stimulating
hormone, luteinizing hormone), growth
(thyroid-stimulating hormone), and
response to stress (adrenocorticotropic
hormone). Hypothalamic control of
anterior pituitary secretions occurs
through the release of hypothalamic-
hypophysiotropic hormones. Hormones
from the posterior hypothalamus
(prolactin, melanocyte-stimulating
hormone, and growth hormone) are  also
involved in a number of important
bodily functions.
   Many types of behaviors (e.g.,
reproductive behaviors, sexually
dimorphic behaviors) are  dependent on
the integrity of the hypothalamic-
pituitary system, which could represent
an important site for neurotoxic action.
Pituitary secretions arise from a number
of different cell types in this gland and
neurotoxicants could affect these cells
either directly or indirectly.
Morphological changes in follicular
cells, chromophobe cells, somatotropic
 cells, prolactin cells, gonadotropic cells,
 follicle-stimulating hormone secreting
 cells, luteinizing hormone-containing
 cells, thyrotropic cells, and cortico  cells
 might be associated with  adverse effects
 on the pituitary,  which could  ultimately
 affect behavior and the functioning of
 the nervous system.
   Biochemical changes in the
 hypothalamus also may be used as
 indices of potential changes in
 neuroendocrine  function. However, the
 neuroendocrine  significance of changes
 in hypothalamic neurotransmitters and
 neuropcptides is usually only
 inferential  and data must be considered
 on a case-by-case basis.
    Most anterior  pituitary hormones are
 subject to negative feedback control by
 peripheral endocrine glands and, if
 neurotoxicants modify peripheral
 secretions, neuroendocrine changes can
 result from this  altered feedback.
 Modifications in the functioning of
 these endocrine secretions could occur
 after toxic  exposure; a number of agents
 have been  shown to alter blood levels of
 glucocorticoids, thyroxine, estrogen,
 corticosterone, and testosterone.
 Although such changes are not
 necessarily due to direct
 neuroendocrine effects, target organ
 changes often can be a first indication
  of neuroendocrine changes.
4.3.8. Other Considerations Structure-activity relationship.
  Because of a general lack of
epidemiologic or toxicologic data on
most chemical substances, attempts
have been made in toxicology to predict
activities based on chemical structure.
The basis for inference from structure-
activity relationships (SARs) can be
either comparison with structures
known to have biologic activity or
knowledge of structural requirements of
a receptor or macromolecular site of
action. However, given the complexity
of the nervous system and the lack of
information on biologic mechanisms of
neurotoxic action, there are relatively
few well-characterized SARs in
neurotoxicology. Since SARs cannot be
used to rule out all neurotoxic activity,
it is not acceptable to use them as a
basis for excluding potential
neurotoxicity. Caution is warranted in
interpreting SARs in anything other
than the most preliminary analyses. Use
of SARs requires detailed knowledge
not only of structure, but also of each
critical step in the pathogenetic
mechanism of neurotoxic injury. Such
knowledge is still generally unavailable.
   SAR approaches are more successful
when the range of possible sites of
action or mechanisms of action is
 narrow. Thus, SARs have had more use
 in relation to carcinogenicity and
 mutagenicity than in other kinds of
 toxicity. The SAR approaches used  in
 the development of novel
 neuropharmacologic structures deserve
 consideration in neurotoxicology, but
 their utility depends on  a better
 understanding of neurotoxic
 mechanisms. In vitro methods.
    In vitro procedures  for testing have
 practical advantages, but studies must
 be done to correlate the  results with
 responses in whole animals. One
 advantage of validated in vitro tests is
 that they minimize the use of live
 animals. Some of the more developed in
 vitro tests might be simple and might
 not have to be conducted by highly
 trained personnel, but, as with many in
 vivo tests, the analysis and
 interpretation of results are likely to
 require expertise. Experience with  the
 Ames test for mutagenesis confirms the
 advantages of in vitro procedures, but
 also illustrates the problems that arise
 when an assay is used to predict an
  endpoint that is not exactly what it
  measures (e.g., carcinogenicity rather
  than specific aspects of genotoxicity). In
  vitro changes can be markers for
  toxicity, even when the structural or
  functional consequences are not known

Federal Register / Vol. 59.  No. 158 / Wednesday. August  17, 1994  / Notices
 or predicted. In addition, in vitro
 methods can examine the more
 evolutionarily conserved elements of
 the nervous system and improve
 neurotoxicity detection and could also
 provide suitable systems for studying
 developmental neurotoxicity.
   A broad range of tissue-culture
 systems are available for assessing the
 neurologic impact of environmental
 agents, including cell lines, dissociated
 cell cultures, reaggregate cultures,
 explant cultures, and organ cultures
 (Veronesi, 1991).
   Neuronal and glial cell lines are used
 extensively in neurobiology and have
 potential for neurotoxicological studies.
 They consist of populations of
 continuously dividing cells that, when
 treated appropriately, stop dividing and
 exhibit differentiated neuronal or glial
 properties. Neuronal lines can develop
 electric excitability,  chemosensitivity,
 axon formation, neurotransmitter
 synthesis and secretion, and synapse
 formation. Large quantities of cells can
 be generated routinely to develop
 extensive dose-response or other
 quantitative data.
   When neural tissue, typically from
 fetal animals, is  dissociated into a
 suspension of single cells, and the
 suspension is inoculated into tissue-
 culture dishes, the neurons and glia
 survive, grow, and establish functional
 neuronal networks. Such preparations
 have been made from most regions of
 the CNS and exhibit  highly
 differentiated, site-specific properties
 that constitute an in  vitro model of
 different portions of  the CNS. Most of
 the neuronal transmitter and receptor
 phenotypes can be demonstrated, and a
 variety of synaptic interactions can be
 studied. Glial cells are also present,  and
 neuroglial interactions are a prominent
 feature of the cultures. A substantial
battery of assays (neurochemical and
 neurophysiologic) is now available to
assess the development of the cultures
 and to indicate toxic effects of test
 agents added to the culture medium.
 Relatively pure populations of different
 cell types can be isolated and cultured,
 so that effects on specific cell types can
be assessed independently. Pure glial
 cells or neurons, or even specific neural
 categories,  can be prepared in this way
 and studied separately, or interaction
 between neurons and glial cells can  be
 studied at high resolution. The
 neurobiologic measures used to assess
 the effect of any agent can be very
 specific (for example, activity of
neurotransmitter-related enzyme or
binding of a receptor ligand) or global
 (for example, neuron survival or
concentration of glial fibrillary acidic
protein). The two-dimensional character
                      of the preparations makes them
                      particularly suited for morphologic
                      evaluation, and detailed
                      electrophysiologic studies are readily
                      performed. The toxic effects and
                      mechanisms of anticonvulsants,
                      excitatory amino acids, and various
                      metals and divalent cations have been
                      assessed with these preparations. The
                      cerebellar granular cell culture system,
                      for example, has been exploited recently
                      in studies of the mechanism of alkyllead
                      toxicity (Verity et al., 1990).
                        A related preparation made from
                      single-cell suspensions of neural tissue
                      is the reaggregate culture. Instead of
                      being placed in culture dishes and
                      allowed to settle onto the surface of the
                      dishes, the cells are kept in suspension
                      by agitation; under appropriate
                      conditions, they  stick to one another
                      and form aggregates of controllable size
                      and composition. Typically, the cells in
                      an aggregate organize and exhibit
                      intercellular relations that are a function
                      of, and bear some resemblance to, the
                      brain region that was the source of the
                      cells. The cells establish a three-
                      dimensional, often laminated structure.
                      Reaggregate cultures lend themselves to
                      large-scale, quantitative experiments in
                      which neurobiologic variables can be
                      examined, although morphologic and
                      ligand-binding studies are performed
                      less readily than  with surface cultures.
                        Organotypic explant cultures also are
                      closely related to the intact nervous
                      system. Small pieces or slices of neural
                      tissue are placed in culture and can be
                      maintained for long periods with
                      substantial maintenance of structural
                      and cell-cell relations of intact tissue.
                      Specific synaptic relations develop  and
                      can be maintained and evaluated, both
                      morphologically and
                      electrophysiologically. Because all
                      regions of the nervous system are
                      amenable to this sort of preparation, it
                      is possible to analyze toxic agents that
                      are active only in specific regions of the
                      central or peripheral nervous system.
                      Explants can be made from  relatively
                      thin slices of neural tissue, so detailed
                      morphologic and intracellular
                      electrophysiologic studies are possible.
                      Their anatomic integrity is such that
                      they capture many of the cell-cell
                      interactions characteristic of the intact
                      nervous system while allowing a direct,
                      continuing evaluation of the effects of a
                      potentially neurotoxic compound added
                      to the  culture medium. The process of
                      myelination has been studied
                      extensively in explant cultures, and
                      considerable neurotoxicologic
                      information has been gained. A
                      preparation similar to an explant culture
                      is the organ culture, in which an entire
                      organ, such as the inner ear or a
 ganglion, rather than slices or fragments,
 is grown in vitro. Obviously, only
 structures so small that their viability is
 not compromised can be treated in this
   In general, the technical ease of
 maintaining a culture varies inversely
 with the degree to which it captures a
 spectrum of in vivo characteristics of
 nervous system behavior. The problem
 of biotransformation of potentially
 neurotoxic compounds is shared by all,
 although the more complete systems
 (explant or organ cultures) might
 alleviate this problem in specific
 instances. In many culture systems,
 complex and ill-defined additives—
 such as fetal calf serum, horse serum,
 and human placental serum—are used
 to promote cell survival. A number of
 thoroughly described synthetic media
 are now available, however, and such
 fully defined culture systems can be
 used where necessary.
 5. Neurotoxicology Risk Assessment
 5.1. Introduction
  Risk assessment is an empirically
 based process used to estimate the risk
 that exposure of an individual or
 population to a chemical, physical, or
 biological agent will be associated with
 an adverse effect. Generally, such effects
 can be quantified and the relative
 probability of their occurrence can be
 calculated. The risk assessment process
 usually involves four  steps: hazard
 identification, dose-response
 assessment, exposure assessment, and
 risk characterization (NRC, 1983). Risk
 management is the process that applies
 information obtained through the risk
 assessment process to determine
 whether the assessed risk should be
 reduced and, if so, to what  extent (NRC,
 1983). In some cases, risk is the only
 factor considered in a decision to
 regulate exposure to a substance.
 Alternatively, the risk posed by a
 substance is weighed against social,
 ethical, and  medical benefits and
 economic and technological factors in
 formulating a risk management
 decision. The risk-balancing approach is
 used by some agencies to consider the
 benefits as well as the risks associated
 with unrestricted or partially restricted
use of a substance. The purpose of this
chapter is to describe the risk
assessment process as it has currently
evolved in neurotoxicology and present
available options for quantitative risk

 5.2. The Risk Assessment Process

 5.2.1. Hazard Identification
  Agents that adversely affect the
neurophysiological, neurochemical, or

                Federal Register  /  Vol.  59. No. 158 / Wednesday, August  17.  1994  /  Notices
structural integrity of the nervous
system or the integration of nervous
system function expressed as modified
behavior may be classified as
neurotoxicants (Tilson, 1990b). For
hazard identification, the best or most
generalizable studies would measure
these changes in humans. With the
exclusion of therapeutic agents,
information on effects in humans is
usually derived from case reports of
accidental exposures and
epidemiological studies. This type of
data affords less certainty regarding
generalizability as well as less specific
exposure information. As discussed  in
chapter 4, a common alternative method
of data generation for hazard
identification is the use of animal
models. Animal models that measure
behavioral, neurophysiological,
neurochemical, and structural effects
have been developed and validated.
Studies that employ these models to
evaluate specific  potential hazards are
used to predict the outcome of exposure
to the same hazard in humans. Human studies
   Information obtained through the
evaluation of human exposure data
provides direct identification of
neurotoxic hazards. This type of
 information is generally available from
 clinical trials required for the approval
 of therapeutic products for human use.
 For the purposes of risk assessment of
 nontherapeutic substances, data on
 effects of exposure to humans come
 primarily from two types of studies,
 case reports and  epidemiological
 (Friedlander and Hearn, 1980) (see
 chapter 3). Case studies can supply
 evidence of an agent's toxicity, but  are
 often limited by both the qualitative
 nature of the signs and symptoms
 reported and the nature of the exposure
 data.  Epidemiological studies can
 provide data on the types of neurotoxic
 effects and the possible susceptibilities
 of certain populations. Under
 appropriate considerations, they can
 generally  provide convincing and
 reliable evidence of potential human
 neurotoxicity. As with case studies,
 however,  often only qualitative
 estimates of exposure can be obtained.
 Controlled laboratory studies have  the
 potential to provide adequate exposure
 and effects data  for accurate hazard
 identification, but ethical considerations
 place moral and practical restrictions on
 such studies except in those instances
 where direct benefit to the subjects, as
 in the case of therapeutic agents, may be
  expected. Excluding instances of
  therapeutic product development,  most
  studies are limited to measuring the
  effects of acute,  rather than long-term,
exposure. This limits their utility in risk
assessment because the effect of long-
term, low-level exposure to a potentially
toxic agent is often the issue of concern.
  Methods available to evaluate
neurotoxicity in humans include
examination of neurophysiological and
behavioral parameters.  Specific tests to
measure neuromuscular strength and
coordination, alterations in sensation,
deficits in learning and memory,
changes in mood and personality, and
disruptions of autonomic function are
frequently employed (see chapter 3). Animal studies
  As discussed in chapter 4, animal
models for many endpoints of
neurotoxicity are available and widely
used for hazard identification. Data from
animal studies are frequently
extrapolated to humans. For example,  if
exposure to an agent produces
neuropathology in an animal model,
damage to a comparable structure in
humans is predicted. Similarly,
biochemical and physiological effects
observed in animals are commonly
extrapolated to humans. Agents that
produce alterations in  the levels of
specific enzymes in one animal species
generally have the same effect in other
 species, including humans.
 Neurophysiological endpoints also tend
 to be affected by the same
 manipulations across species. Thus, an
 agent interfering with nerve conduction
 in an  animal study is often assumed to
 have the same effect in humans.
 Behavioral studies in animals are also
 applied to human hazard identification,
 although the correspondence between
 methods employed in animals and
 humans is sometimes not as obvious.
 For this reason, behavioral methods
 developed for neurotoxic hazard
 identification need to  be considered on
 a case-by-case basis. Special issues Animal-to-human
 extrapolation. The use of animal data to
 identify hazard to humans is not
 without controversy. Relative sensitivity
 across species as well as between sexes
 is a constant concern. Overly
 conservative risk assessments, based on
 the assumption that humans are always
 more sensitive than a tested animal
 species, can result in poor risk
 management decisions. Conversely, an
 assumption of equivalent sensitivity in
 a case where humans actually are more
 sensitive to a given agent can result in
 underregulation that might have a
 negative impact on human health. Susceptible populations. A
  related controversy concerns the use of
  data collected from adult organisms.
animal or human, to predict hazards in
potentially more sensitive populations,
such as the very young and the elderly,
or in other groups, such as the
chronically ill. In some cases,
identification of neurotoxicity hazard
does not generally include subjects from
either end of the human life span or
from other than healthy subjects.
Uncertainty factors are used to adjust for
more sensitive populations. In addition,
single or multigeneration reproductive
studies in animals may provide a source
of information on neurological
disorders, behavioral changes,
autonomical dysfunction,
neuroanatomical anomalies, and other
signs of neurotoxicity in the developing
animal (chapter 4). Reversibility. For the most
part, the basic principles of hazard
identification are the same for
neurotoxicity as for any adverse effect
on health. One notable exception,
however, concerns the issue of
reversibility and the special
consideration that must be given to the
inherent redundancy and plasticity of
the nervous system.
   For many health effects, temporary, as
opposed to permanent, effects are
repaired during a true recovery. Damage
to many organ  systems, if not severe,
can be spontaneously repaired. For
 example, damaged liver cells that may
 result in impaired liver function often
 can be replaced with new cells that
 function normally. The resulting
 restoration of liver function can be
 viewed as recovery. In the central
 nervous system, cells generally do not
 recover from severe damage and new
 cells do not replace them. When
 nervous system recovery is observed, it
 may represent compensation requiring
 activation of cells that were previously
 performing some other function,
 reactive synaptogenesis, or recovery of
 moderately injured cells. While a
 damaged liver may recover due to the
 addition of new cells, severe damage to
 nervous system cells results in a net loss
 of cells. This loss of compensatory
 capacity may not be noticed for many
 years and, when it does appear, it may
 be manifest in a way seemingly
 unrelated to the original neurotoxic
 event. Lack of ability to recover from a
 neurotoxic event later in life or
 premature onset of signs of normal aging
 may result. It is therefore important to
 consider the possibility that significant
 damage to the nervous system may have
 occurred in experiments where effects
 appear to be reversible. Weight of evidence.
    A "weight of evidence" approach to
  identifying an agent as a neurotoxic

Federal  Register / Vol. 59, No.  158 / Wednesday,  August 17, 1994  / Notices
 hazard is almost always necessary. With
 the exception of therapeutic products, a
 single, complete, controlled study of an
 agent's effects on the nervous system,
 conducted in an appropriate
 representative sample of humans, is
 rarely, if ever, possible. Rather, those
 individuals charged with identifying
 hazard are usually confronted with a
 collection of imperfect studies, often
 providing conflicting data (Barnes and
 Dourson, 1988).
   There are several possible approaches,
 depending on the quality of the
 evidence. Two examples are the use of
 data from only the most sensitive
 species tested and the use of data from
 only species responding most like the
 human for any given endpoint. In
 assessing neurotoxicity of therapeutic
 products, when  human data are
 available and neurotoxic endpoints
 detected in animals can be clinically
 measured, the human findings
 supersede those of the nonclinical data
 base. Assuming  that all available
 evidence is to be included,
 considerations necessary for formulating
 a conclusion include the relative
 weights that should be given to positive
 and negative studies. Sometimes
 positive studies  are given more weight
 than negative ones, even when the
 quality of the studies is comparable.
 Experimental design factors such as the
 species tested, the number and gender
 of subjects evaluated, and the duration
 of the test are given different weights
 when data from different studies are
 combined. The route of exposure in a
 given study and  its relevance  to
 expected routes of human exposure are
 often a weighted factor. The issue of
 statistical significance is frequently
 debated.  Some argue that an effect
 occurring at a statistically insignificant
 level may nevertheless represent a
 biologically or toxicologicnlly
 significant event, and should be
 afforded the same weight as if the
 finding were statistically significant. In
 general, however, only statistically
 significant measures should be
 considered in hazard identification. The
 power of various statistical measures is
 also considered.

 5.2.2. Dose-Response Assessment

  In the second step of the risk
 assessment process, the dose-response
 assessment, the relationship between
 the extent of damage or toxicity and
 dose of a toxic substance for various
conditions of exposure is determined.
 Because several different kinds of
responses may be elicited by a single
agent, more than one dose-response
relationship may need to be developed
                      (e.g., neurochemical and morphological
                        When quantitative human dose-effect
                      data are not available for a sufficient
                      range of exposures, other methods must
                      be used to estimate exposure levels
                      likely to produce adverse effects in
                      humans. In the absence of human data,
                      the dose-response assessment may be
                      based on tests performed in laboratory
                      animals. Evidence for a dose-response
                      relationship is an important criterion in
                      assessing neurotoxicity, although this
                      may be based on limited data from
                      standard studies that often use only
                      three dose groups and a control group
                      (Barnes and Dourson, 1988).
                        The most frequently used approach
                      for risk assessment of neurotoxicants
                      and other noncancer endpoints is the
                      uncertainty- or safety-factor approach
                      (Barnes and Dourson, 1988; Kimmel,
                      1990). For  example, within the EPA,
                      this approach involves the
                      determination of reference doses (RfDs)
                      by dividing a no observed adverse effect
                      level (NOAEL) by uncertainty factors
                      that presumably account for interspecies
                      differences in sensitivity (Barnes and
                      Dourson, 1988). Generally, an
                      uncertainty factor of 10 is used to allow
                      for the potentially higher sensitivity in
                      humans than in animals and another
                      uncertainty factor of 10 is used to allow
                      for variability in sensitivity among
                      humans. Hence,  the RfD is equal to the
                      NOAEL divided  by 100. If the NOAEL
                      cannot be established, it is replaced by
                      the lowest observed adverse effect level
                      (LOAEL) in the RfD calculation and an
                      additional uncertainty factor of 10 is
                      introduced (i.e., the RfD equals the
                      LOAEL divided by 1000).
                       If more than one effect is observed in
                      the animal  bioassays, the effect
                      occurring at the lowest dose in the most
                      sensitive animal  species and gonder is
                      generally used as this basis for
                      estimating the RID (OTA, 19<)0).
                      Sometimes, different RfDs can be
                      calculated,  depending on endpoint or
                      species selected. Selection of safety
                      factors may be influenced by several
                     considerations, including data available
                      from humans, weight of evidence, type
                     of toxic insult, and probability of
                     variations in responses among
                     susceptible populations (e.g., very
                     young or very old). Established
                     guidelines have been accepted by
                     several agencies that use the safety-
                     factor approach to account for
                     intraspecies variability, cross-species
                     extrapolation, and exposure duration. In
                     some instances, comparisons between
                     these predicted values and experimental
                     data have been conducted and the
                     results appear comparable for some
 selected examples (Dourson and Stara,
 1983; McMillan, 1987).
   The uncertainty-factor approach is
 based on the assumption that a
 threshold does exist, that there is a dose
 below which an effect does not change
 in incidence or severity. The threshold
 concept is complicated and
 controversial. As described by Sette and
 MacPhail (1992), there are several
 different  ways in which the term
 threshold is used. Thresholds are
 defined, in part, by the limit of
 detection of an assay. As the sensitivity
 of the analytical method or bioassay is
 improved, the threshold might be
 adjusted downward, indicating that the
 true threshold had not been previously
   Another problem inherent with an
 observation of no discernible effects at
 low doses is that it is impossible to
 determine whether the risk is actually
 zero (i.e., the dose is below a threshold
 dose) or whether the statistical resolving
 power of  a study is inadequate to detect
 small risks (Gaylor and Slikker, 1992).
 Every study has a statistical limit of
 detection that depends on the number of
 individuals or animals involved. For
 example,  it would be relatively unusual
 to conduct an experiment on a
 neurotoxicant with as many as 100
 animals per dose. If no deleterious
 effects were observed in 100 animals at
 a particular dose, it might be concluded
 that this dose level is below the
 threshold dose. However, we can only
 be 95 percent confident that the true
 risk is less than 0.03. That is, if 3
 percent of the animals in a  population
 actually develop a toxic effect at this
 dose, there is a 5 percent chance that a
 group of 100 animals would not show
 any effect. The observation of no toxic
 effects in an extremely large sample of
 1,000 animals only indicates with 95
 percent confidence that the true risk is
 loss than 0.003, ok:. Docauso thresholds
 cannot be realistically demonstrated,
 they are therefore assumed.
  The notion of threshold may be useful
 in explaining mechanisms associated
 with specific types of toxicity. What
 little is known about mechanisms of
 neurotoxicity suggests that  both
 threshold  and nonthreshold scenarios
 are possible (Silbergeld, 1990).
 However,  for one of the most studied
 neurotoxicants, lead, there has been a
 steady decline in exposure  levels shown
 to have effects, suggesting to some that
 no threshold dose is apparent (Bondy,
 1985). Sette and MacPhail (1992) also
consider the threshold as a
mathematical assumption and as a
population sensitivity and conclude that
"the idea of no threshold seems
experimentally untestable.  . . ."

                Federal Register / Vol. 59, No. 158  /  Wednesday,  August  17.  1994  /  Notices	42397
  The RfD approach relies on single
experimental observations (the NOAEL
or LO AEL) instead of complete dose-
response curve data to calculate risk
estimations. Chemical interactions with
biological systems are often specific,
stereoselective, and saturable. Examples
include enzyme-substrate binding
leading to substrate metabolism,
transport, and receptor-binding, any or
all of which may be a requirement of an
agent's effect or toxicity. Therefore, a
chemical's dose-response curve may not
be linear. The certainty of low-dose
extrapolation has been determined to be
markedly affected by the shape of the
dose-response curve (Food and Drug
Administration Advisory Committee on
Protocols for Safety Evaluation, 1971).
Therefore, the appropriate use of dose-
response curve data should enhance the
certainty of risk estimations when
thresholds are not assumed or
  Dose-response models have generated
considerable interest as more
appropriate and quantitative
alternatives to the safety-factor approach
in risk assessment. Rather than
routinely applying a "fixed" safety
factor to the NOAEL (based on a  single
dose) to obtain a "safe" dose, another
approach uses data from the entire dose-
response curve.
   Two fundamentally different
 approaches in the use of dose-response
 data to estimate risk have been
 developed. Dews and coworkers (Dews,
 1986; Glowa and Dews, 1987; Glowa et
 al., 1983) and Crump (1984)
 demonstrated an approach in which
 they used information on the shape of
 the dose-response curve to estimate
 levels of exposure associated with
 relatively small effects (i.e., a 1,  5, or 10
 percent change in a biological
 endpoint). Both Dews and Crump fit a
 mathematical function to the data and
 provided an estimate of the variability
 in exposure levels associated with a
 relatively small effect.
    An alternative approach developed by
 Gaylor and Slikker (1990) first
 establishes a mathematical relationship
 between a biological effect and the dose
 of a given chemical. The second step
 determines the distribution (variability)
 of individual measurements of
 biological effects about the dose-
 response curve. The third step
 statistically defines an adverse or
 "abnormal" level of a biological effect in
 an untreated population. The fourth
 step estimates the probability of an
  adverse or abnormal level as a function
  of dose utilizing the information from
  the first three steps. The advantages of
  these dose-response models are that
  they encourage the generation and use
of data needed to define a complete
dose-response curve.
  Although more quantitative dose-
response assessment models have
emerged in recent years, uncertainty
remains as to what biological endpoints
from which species with what dosing
regimen should be analyzed. Within a
species, a given agent may produce a
variety of effects, including
neurochemical, neuropathological, and
behavioral effects. In other instances, a
chemical may produce alterations of one
endpoint but not others (Slikker et al.,
1989). Species selection may also
dramatically affect the outcome of risk
assessments. The Parkinson-like
syndrome produced by single doses of
MPTP in the human or nonhuman
primate is not observed in rats given
comparable MPTP doses (Kopin and
Markey, 1988). Although endpoint and
species selection appear to have a
tremendous effect on the outcome of an
assessment, only a few studies have
systematically investigated the effect on
assessment outcome of varying either
the species or the endpoint within a
species (McMillan,  1987; Hattis and
Shapiro, 1990; Gaylor and Slikker,
 5.2.3. Exposure Assessment
   This step of the risk assessment
 process determines the source, route,
 dose, and duration  of human exposure
 to an agent. The results of the dose-
 response assessment are combined with
 an estimate of human exposure to obtain
 a quantitative estimate of risk. As either
 the effect of or the exposure to an agent
 approaches zero, the risk of
 neurotoxicity approaches zero. It should
 be recognized that exposures to multiple
 agents may produce synergistic or
 additive effects.
   Exposure can occur via many routes,
 including ingestion, inhalation, or
 contact with skin. Sources of exposure
 may include soil, food, air, water, or
 intended vehicle (e.g., drug
 formulation). The degree of exposure
 may be strongly influenced by a number
 of factors, for example, the occupation
 of the individual involved.
   The duration of exposure (i.e., acute
 or chronic) and interval of exposure
 (i.e., episodic or continuous) are
 variables of exposure that are  common
 to all types of risk  assessments,
 including carcinogenicity (OSTP, 1985).
    Although not routinely used,
 biological markers or biomarkers of
  exposure could theoretically improve
  the exposure assessment process and,
  thereby, improve the overall risk
  assessment of neurotoxicants. Exposure
  biomarkers may include either the
  quantitation of exogenous agents or the
complex of endogenous substances and
exogenous agents within the system
(Committee on Biological Markers,
1987). A limited number of examples of
biomarkers of exposure have been
reviewed by Slikker (1991) and include
blood or dentine lead concentrations
(Needleman, 1987), cerebrospinal fluid
concentrations of dopamine metabolites
following MPTP administration (Kopin
and Markey, 1988), cerebrospinal fluid
concentrations of a serotonin metabolite
following MDMA exposure (Ricaurte et
al., 1986), and serum esterase
concentrations following
organophosphate exposure (Levine et
al., 1986). The use  of muscarinic
receptor binding in peripheral plasma
lymphocytes has also been described as
a potential biomarker of exposure for
the organophosphates (Costa et al.,
1990). These examples suggest that
biomarkers of exposure are available for
some agents, but more effort will be
required to demonstrate that these
biomarkers  can routinely be used to
improve the exposure assessment
5.2.4. Risk Characterization
   The final step of the risk assessment
process combines  the hazard
identification, the dose-response
 assessment, and the exposure
 assessment to produce the
 characterization of risk. As previously
 stated, the current practice is to divide
 the NOAEL by the appropriate safety
 factor to obtain the RfD. The magnitudes
 of the safety factors used to determine
 RfDs [interspecies extrapolation  (10),
 intraspecies extrapolation (10), and
 acute vs. chronic exposure (10) = 1000]
 are based more on conservative
 estimates than on actual data (Sheehan
 et al., 1989; McMillan, 1987) and have
 been questioned for empirical reasons
 (Gaylor and Slikker, 1990). Uncertainty
 factors may be decreased as more data
 become available. Modifying factors are
 also employed under certain
 circumstances to  account for the
 completeness of data sets. Along with
 this RfD numerical value, any
 uncertainties and assumptions inherent
 in the risk  assessment should also be
 stated (OTA, 1990). Although the RfD
 provides a single  numerical  value, it
 does not provide  information
 concerning the uncertainty of this
 number nor does the RfD approach
 attempt to estimate the potential risk as
 a function of dose or consider the
 potential risk at the NOAEL. The risk at
 the NOAEL generally is greater  than
 zero and has been estimated to be as
 high as about 5 percent (Crump, 1984;
 Gaylor, 1989). Concern has been
  expressed that the application of the

Federal Register / Vol. 59, No. 158  / Wednesday, August 17, 1994 / Notices
 RED approach to all neurotoxicants is
 unlikely to be biologically defensible in
 light of mechanistic data (NRG, 1992).
 Several other quantitative risk
 assessment procedures have recently
 emerged as alternatives to the RfD
 approach (Kimmel and Gaylor, 1988).
   Quantitative risk assessment may be
 defined as a data-based process that
 uses dose-response information and
 measurements of human exposure to
 arrive at estimates of risk. Assumptions
 are required to extrapolate results from
 high to low doses, to extrapolate from
 animal results to humans, and to
 extrapolate across  different routes and
 durations of exposure.
   In a step toward quantitative risk
 assessment. Crump (1984) suggested the
 use of a benchmark dose defined as "a
 statistical lower confidence limit
 corresponding to a small increase in
 effect over the background level." The
 benchmark dose is determined with a
 mathematical model and is less affected
 by the particular shape of the dose-
 response curve. Although the
 benchmark approach avoids several
 problems inherent in the RfD approach
 (e.g., lack of precision in  defining the
 LOAEL; Kimmel. 1990), the same final
 step of dividing by arbitrary safety
 factors is obligatory.
  Another approach to quantitative risk
 assessment is the statistical or curve-
 fitting approach. If quantal information
 concerning the proportion of response at
 a given dose is available but
 mechanistic information is lacking,
 statistical models can be used to fit
 population data (Wyzga, 1990). This
 approach has been used to fit various
 models to data of lead toxicity. The data
 were sufficient to allow discrimination
 of several models in terms of goodness
 of fit; the nerve-conduction velocity
 data from children exposed to
 environmental lead as a function of
blood lead concentration fit a "hockey-
 stick" type dose-response curve rather
 than a logistic or quadratic model
 (Schwartz et al., 1988). These statistical
 approaches not only provide a method
 to extrapolate data to lower exposure
 conditions but also can provide
 circumstantial evidence to support a
 proposed mechanism of action.
  The development of quantitative risk
 assessment approaches depends, in part,
 on the availability  of information on the
mechanism of action and
pharmacokinetics of the agent in
question. In the development  of a
biologically based, dose-response model
for MDMA neurotoxicity, Slikker and
Gaylor (1990) considered several factors,
including the pharmacokinetics of the
parent chemical, the target tissue
concentrations of the parent chemical or
                      its bioactivated proximate toxicant, the
                      uptake kinetics of the parent chemical
                      or metabolite into the target cell and
                      membrane interactions, and the
                      interaction of the chemical or metabolite
                      with presumed receptor site(s). Because
                      these theoretical factors contain a
                      saturable step due to limited amounts of
                      required enzyme, reuptake, or receptor
                      site(s), a nonlinear, saturable dose-
                      response curve was predicted. In this
                      case of neurochemical effects of MDMA
                      in the rodent, saturation mechanisms
                      were hypothesized and indeed
                      saturation curves provided relatively
                      good fits to the experimental results.
                      The conclusion was that use of dose-
                      response models based on plausible
                      biological mechanisms provide more
                      validity to prediction than purely
                      empirical models. Concomitant with
                      attempts to develop quantitative risk
                      assessment procedures, it is imperative
                      that regulatory policy or risk
                      management procedures also be
                      developed to use appropriately the type
                      of data generated by quantitative risk
                      assessment. However, until alternative
                      risk assessment procedures have been
                      validated, the available RfD approach
                      with its  limitations will most likely
                      continue to be used.

                      5.3. Generic Assumptions and
                      Uncertainty Reduction
                        The purpose of risk assessment is to
                      determine the risk associated with
                      human exposure to a hazard. The
                      quality of the  data from toxicological
                      studies differs. In the case of therapeutic
                      products where human effects
                      information is available, risk
                      assessments rely primarily on the result
                      of controlled clinical trials. Even when
                      clinical trial data are available, however,
                      conducting a risk assessment is
                      complicated by many  uncertainties. In
                      the  face  of these uncertainties,
                      conservative assumptions are usually
                      made at  several steps in the risk
                      assessment process. For example, unless
                      adequate clinical data are available, the
                      most sensitive experimental species is
                      frequently used. While conservative
                      assumptions may  lead to a risk
                      assessment that adequately protects the
                      human population, this may result in an
                      increased financial burden on the public
                      (e.g., manufacturing costs or loss of
                      jobs); even then it is impossible to be
                      certain that the total population will be
                      protected. Conversely, errors leading to
                      allowable exposure levels that are too
                      high reduce the safety margin for human
                      health and increase health care costs.
                      Thus, there are compelling public
                      health and economic reasons to obtain
                      more precise risk assessments; all
                      assumptions cannot be completely
 eliminated, but the degree of
 uncertainty associated with certain
 specific assumptions can at least be
 reduced (Sheehan et al., 1989).
   Risk assessment for neurotoxicity
 shares many common features with
 other noncancer toxicities such as
 developmental toxicity and
 immunotoxicity. As such, there are
 several generic assumptions that apply
 to all traditional, noncancer endpoint
 risk assessment procedures (Table 5-1).

 1. A  threshold dose exists (or  noncancer
 2. NOAEL/LOAEL uncertainty- or  safety-fac-
  tor approaches are reasonable.
 3. Variability in  the toxic response  to the
  chemical exposure is not due to a hetero-
  geneous population response.
 4. Average dose or total dose is a reason-
  able measure of exposure when doses are
  not  equivalent in time, rate, or route of ad-
  ministration and the average (or  total) dose
  is proportional to adverse effect.
 5. Structure-activity correlations can be used
  to predict human toxicity.
 6. The mechanism of action  is the same at
  all doses for all species.

  a This is not intended to be an exhaustive
  b Modified from Sheehan et al., 1989.
  One approach to reducing some of the
uncertainties is to critically define and
examine the assumptions made in the
risk assessment process. Several of the
more  generic of these assumptions are
listed in Table 5-1. Despite their
diversity, these assumptions share the
attribute of being partially replaceable
by factual information. If, for example,
the assumption of 100 percent
absorption of a toxicant from a
contaminated food source is replaced by
data demonstrating that 90 percent of
the toxicant is not biologically available
under human exposure conditions, then
a revised risk assessment could allow a
10-fold greater exposure from that
source; i.e., the former risk assessment
was too conservative by a factor of 10.
As another example, many risk
assessments employ data from two
  If experimental animals and humans
absorb or metabolize the same  fraction
of a dose, the potency estimate would
not change when extrapolating from
animals to humans. Therefore, it is
necessary to have information on both
human and animal rates before changes
in potency estimates are made. If a
toxicant acts via a reactive intermediate
and humans produce 10-fold more of

                 Federal Register / Vol. 59, No. 158  / Wednesday. August  17.  1994  /  Notices
the intermediate than either of the test
species under similar conditions, then
allowable human exposure should be
decreased 10-fold (i.e., the allowable
exposure levels are 10-fold too high) or
an increased danger to human health
exists. These  findings could then
replace the "most sensitive species"
principle with facts concerning relevant
human exposure and susceptibility. In
these examples, the identification of the
assumption helps define research needs
or knowledge gaps (Sheehan et al.,
  In general,  the knowledge gaps are
many and complex, but some can be
filled with practical solutions. The
combination  of ample dose-response
data and a quantitative risk assessment
process can eliminate assumptions 1
(existence of a threshold) and 2
(reasonableness of safety factors) of the
six generic assumptions (Table 5-1).
The uncertainty of assumption 4
(exposure comparisons) could be at least
reduced with the proper application of
appropriate pharmacokinetic data.
Likewise, the uncertainty  of generic
assumption 3 (variability of
heterogeneous populations) can
theoretically be reduced with the use of
biomarkers of exposure and biomarkers
of effect, to more accurately define the
relationship  between exposure and
biological effect in a large population.
   Many assumptions remain, however,
and uncertainty reduction by filling
knowledge gaps will ultimately require
greater understanding of biological
mechanisms underlying neurotoxicity.
A single risk assessment model may not
he adequate  for all conditions of
 exposure,  for all endpoints, or for all
 agents. Risk  assessment models of the
 future may well include biomarkers of
 both effect and exposure as well as
 biologically  based mechanistic
 considerations  derived from both
 epidemiologic and experimental test
 system data.
 G. General Summary
   It is now generally accepted that some
 chemicals, including industrial agents,
 pesticides, therapeutic agents, drugs of
 abuse, food-related chemicals, and
 cosmetic ingredients, can have adverse
 effects on the structure and function of
 the nervous system. It has recently been
 proposed that exposure to
 neurotoxicants might also be associated
 with Parkinsonism and Alzheimer's
 disease. Several Federal agencies have
 initiated research programs in
 neurotoxicology, developed
 neurotoxicology testing guidelines, and
 used neurotoxic endpoints to regulate
 chemicals in the environment and
  The scientific basis for identifying
and characterizing chemical-induced
neurotoxicity has advanced rapidly
during the last several years. The
manifestation of neurotoxicity depends
on the relationship between exposure
(applied dose) and the dose at the site
of toxic action (delivered or target dose)
and response. Chemical-induced
changes in the structure or function of
the nervous system at the cellular or
molecular level can be observed as
alterations in sensory, motor, or
cognitive function at the level of the
whole organism. Several important
features about the nervous system make
it particularly vulnerable to chemical
insult, including differential
susceptibilities at different stages of
maturation, the presence of blood brain
and nerve barriers that may be the target
of toxic action, high metabolic rate, and
limited regenerative capability
following damage.
  Methods devised to detect and
quantify agent-induced changes in
nervous system function in humans
include clinical evaluations and
neurotoxicity testing methods such as
neurobehavioral, neurophysiological,
neurochemical, imaging, and self-
reporting procedures. Experimental
approaches used in human
neurotoxicology include
epidemiological studies and, to a
limited extent, human laboratory
exposure studies. There are several
important unresolved issues in human
neurotoxicology, including the
 development of commonly accepted risk
 assessment criteria and animal-to-
 human extrapolation.
   It is generally assumed that if physical
 or chemical-induced neurotoxicity is
 observed in animal models, then
 neurotoxicity will be produced in
 humans. Considerable research has been
 performed to demonstrate the validity of
 many animal models in an experimental
 context and to show predictive validity.
 Methods in animal neurotoxicology are
 frequently used in a tier-testing
 framework with simpler, more cost-
 effective tests to screen or identify
 neurotoxic potential. In hazard
 identification, the presence of
 neurotoxicity at the first tier is used to
 make decisions about subsequent
 development of a chemical or  about the
 need to conduct additional experiments
  to define the level at which
 neurotoxicity will be observed. A
  number of methods have been devised
  for studies in animal neurotoxicology,
  including neurobehavioral,
  neurophysiological, neurochemical, and
  neuroanatomical techniques. It is
  known that the neuroendocrine system
  may be affected adversely by
neurotoxicants and that there are
populations that are differentially
vulnerable to neurotoxic agents.
Considerable research is in progress to
employ structure-activity relationships
to predict neurotoxicity and newly
developed in vitro procedures are being
used to augment or complement
currently existing in vivo approaches.
  Principles of risk assessment for
neurotoxicity are evolving rapidly. At
the present time, neurotoxicity risk
assessment is generally limited to
qualitative hazard identification.
Neurotoxicological risk assessments
have been generally based on a no
observed adverse effect level and
uncertainty factors. As with other
noncancer endpoints, there is a need to
consider more information about the
shape of the dose-response curve and
mechanisms of effect in quantitative
neurotoxicology risk assessment.
Research is needed to develop dose-
response models that incorporate
biologic information and mechanistic
hypotheses into quantitative
extrapolation of dose-response
relationships across species and from
high to low dose exposures.
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                        Prepared by
                        Working Party on Neurotoxicology
                        Subcommittee on Risk Assessment
                        Federal Coordinating Council on Science,
                          Engineering, and Technology
                        Lawrence W. Ruiter, USEPA, Chair
                        Hugh A. Tilson, USEPA, Executive Secretary
                        John Dougherty, NIOSH
                        G. Jean Harry, NIEHS
                        Carol J. Jones, OSHA
                        Suzanne McMaster, USEPA
                        William Slikkur, NGTR/FDA
                        Thomas J. Sobotka, FDA

                        Ad Hoc Inturaguncy Committee on
                        William Boyes, U.S. Environmental
                          Protection Agency
                        Joy Cavagnaro, Food and Drug
                        Selene Chou, Aguncy for Toxic Substances
                          and Disease Registry
                        Murray Cohn, U.S. Consumer Product Safety
                        Joseph F. Contrera, Food and Drug
                        Miriam Davis, National Institute of
                          Environmental Health Sciences, National
                          Institutes of Health
 Joseph DeGeorge, Food and Drug
 Robert Dick, National Institute for
   Occupational Safety and Health
 John Dougherty, National Institute for
   Occupational Safety and Health
 Lynda Erinoff, National Institute on Drug
 Joseph P. Hanig, Food and Drug
 G. Jean Harry, National Institute of
   Environmental Health Sciences
 David G. Hattan, Food and Drug
 Norman A. Krasnegor, National Institutes of
 Robert C. MacPhail, U.S. Environmental
   Protection Agency
 Suzanne McMaster, U.S. Environmental
   Protection Agency
 Lakshmi C. Mishra, U.S. Consumer Product
   Safety Commission
 Andres Negro-Vilar, National Institute of
   Environmental Health Sciences
 James K.  Porter, U.S. Department of
   Agriculture/Agricultural Research Service
 Lawrence W. Reiler, U.S. Environmental
   Protection Agency
 Jane Robens, U.S. Department of Agriculture/
   Agricultural Research Service
 Barry Rosloff, Food and Drug Administration
 Harry Salem, U.S. Army Chemical Research,
   Development, and Enginooring Center
 Bernard A. Schvvetz, National Institute of
   Environmental Health Sciences
 William F. Sotte, U.S. Environmental
   Protection Agency
 William Slikker,  Jr., National Center for
  Toxicological Research
 D. Stephen Snyder, National Institute on
Thomas J. Sobotka, Food and Drug
Hugh A. Tilson, U.S. Environmental
  Protection Agency
Mildred Williams-Johnson, Agency for Toxic
  Substances and Disease Registry

|FR Doc. 94-20033 Filed U-1G-94; 8:45 am|