[Federal Register Volume 59, Number 158 (Wednesday, August 17, 1994)]
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[FR Doc No: 94-20033]


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[Federal Register: August 17, 1994]


_______________________________________________________________________

Part III





Environmental Protection Agency





_______________________________________________________________________




Final Report: Principles of Neurotoxicity Risk Assessment; Notice
ENVIRONMENTAL PROTECTION AGENCY

[FRL-5050-9]

 
Final Report: Principles of Neurotoxicity Risk Assessment

AGENCY: U.S. Environmental Protection Agency.

ACTION: Final Document.

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

SUMMARY: The U.S. Environmental Protection Agency is publishing a 
document entitled Final Report: Principles of Neurotoxicity Risk 
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 assessment.

SUPPLEMENTARY INFORMATION: This 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 Biologics 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

Contents
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
    2.1.3.1. Food and Drug Administration
    2.1.3.2. Occupational Safety and Health Administration
    2.1.3.3. National Institute for Occupational Safety and Health
    2.1.3.4. Environmental Protection Agency
    2.1.3.5. Consumer Product Safety Commission
    2.1.3.6. Agency for Toxic Substances and Disease Registry
    2.2. Basic Toxicological Considerations for Neurotoxicity
    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 System
    2.5. Special Considerations
    2.5.1. Susceptible Populations
    2.5.2. Blood-Brain and Blood-Nerve Barriers
    2.5.3. Metabolism
    2.5.4. Limited Regenerative Ability
3. Methods for Assessing Human Neurotoxicity
    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
    3.3.1.1. Test Batteries
    3.3.1.2. Investigator-Administered Test Batteries
    3.3.1.3. 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
    3.3.6.1. Mood Scales
    3.3.6.2. Personality Scales
    3.4. Approaches to Neurotoxicity Assessment
    3.4.1. Epidemiologic Studies
    3.4.1.1. Case Reports
    3.4.1.2. Cross-Sectional Studies
    3.4.1.3. Case-Control (Retrospective) Studies
    3.4.1.4. Prospective (Cohort, Followup) Studies
    3.4.2. Human Laboratory Exposure Studies
    3.4.2.1. Methodologic Aspects
    3.4.2.2. Human Subject Selection Factors
    3.4.2.3. Exposure Conditions and Chemical Classes
    3.4.2.4. Test Methods
    3.4.2.5. Controls
    3.4.2.6. Ethical Issues
    3.5. Assessment of Developmental Neurotoxicity
    3.5.1. Developmental Deficits
    3.5.2. Methodologic Considerations
    3.6. Issues in Human Neurotoxicology Test Methods
    3.6.1. Risk Assessment Criteria for Neurobehavioral Test Methods
    3.6.1.1. Sensitivity
    3.6.1.2. Specificity
    3.6.1.3. Reliability and Validity
    3.6.1.4. Dose Response
    3.6.1.5. Structure-Activity
    3.6.2. Other Considerations in Risk Assessment
    3.6.2.1. Mechanisms of Action
    3.6.2.2. Exposure Duration
    3.6.2.3. Time-Dependent Effects
    3.6.2.4. Multiple Exposures
    3.6.2.5. Generalizability and Individual Differences
    3.6.2.6. Veracity of Neurobehavioral Test Results
    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
    4.1.3.1. Susceptible Populations
    4.1.3.2. Dosing Scenario
    4.1.3.3. Other Factors
    4.1.3.4. Statistical Considerations
    4.2. Tiered Testing in Neurotoxicology
    4.2.1. Type of Test
    4.2.2. Dosing Regimen
    4.3. Endpoints of Neurotoxicity
    4.3.1. Introduction
    4.3.2. Behavioral Endpoints
    4.3.2.1. Functional Observational Batteries
    4.3.2.2. Motor Activity
    4.3.2.3. Neuromotor Function
    4.3.2.4. Sensory Function
    4.3.2.5. Learning and Memory
    4.3.2.6. Schedule-Controlled Behavior
    4.3.3. Neurophysiological Endpoints of Neurotoxicity
    4.3.3.1. Nerve Conduction Studies
    4.3.3.2. Sensory Evoked Potentials
    4.3.3.3. Convulsions
    4.3.3.4. Electroencephalography
    4.3.3.5. Electromyography
    4.3.3.6. Spinal Reflex Excitability
    4.3.4. Neurochemical Endpoints of Neurotoxicity
    4.3.5. Structural Endpoints of Neurotoxicity
    4.3.6. Developmental Neurotoxicity
    4.3.7. Physiological and Neuroendocrine Endpoints
    4.3.8. Other Considerations
    4.3.8.1. Structure-Activity Relationship
    4.3.8.2. In Vitro Methods
5. Neurotoxicology Risk Assessment
    5.1. Introduction
    5.2. The Risk Assessment Process
    5.2.1. Hazard Identification
    5.2.1.1. Human Studies
    5.2.1.2. Animal Studies
    5.2.1.3. Special Issues
    5.2.2. Dose-Response Assessment
    5.2.3. Exposure Assessment
    5.2.4. Risk Characterization
    5.3. Generic Assumptions and Uncertainty Reduction
6. General Summary
7. References
Tables
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 Neurotoxicity
4-2. Examples of Specialized Tests to Measure Neurotoxicity
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 labeling.

                 Table 1-1.--Major Regulatory Agencies                  
------------------------------------------------------------------------
              Agency                     Statute and sources covered    
------------------------------------------------------------------------
Food and Drug Administration (FDA).  Food, Drug, and Cosmetics Act for  
                                      food additives; color in          
                                      cosmetics; medical devices; animal
                                      drugs of medical and feed         
                                      additives.                        
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       Occupational Safety and Health Act 
 Administration (OSHA).               covers toxic chemicals in the     
                                      workplace.                        
A unit of the Department of Labor    ...................................
 that regulates workplace                                               
 conditions.                                                            
Environmental Protection Agency                                         
 (EPA).                                                                 
Independent agency (i.e., not part   Toxic Substances Control Act       
 of a Cabinet department);            requires premanufacture evaluation
 administers a number of diverse      of all new chemicals (other than  
 laws concerned with human health     foods, food additives, drugs,     
 and the environment.                 pesticides, 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      
                                      dumping.                          
Consumer Product Safety Commission                                      
 (CPSC).                                                                
Regulates a variety of consumer      Federal Hazardous Substances Act   
 products including household         covers ``toxic'' household        
 chemicals and fabric treatments.     products.                         
                                     Consumer Product Safety Act covers 
                                      dangerous consumer products.      
                                     Poison Prevention Packaging Act    
                                      covers packaging of dangerous     
                                      children's products.              
                                     Lead-Based Paint Poison Prevention 
                                      Act covers use of lead paint in   
                                      federally assisted housing.       
------------------------------------------------------------------------


                                                      Table 1-2.--Authorities for Toxicity Testing                                                      
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                                                  Authorities                           
                                                                                       -----------------------------------------------------------------
           Agency                         Law                       Coverage                Premarketing           Testing by                           
                                                                                              approval            manufacturer        Reporting of data 
--------------------------------------------------------------------------------------------------------------------------------------------------------
FDA.........................  Food, Drug, and Cosmetics    Drugs and foods............  x                     x                     x                   
                               Act.                                                                                                                     
                                                           Food additives and           x                     x                     ....................
                                                            cosmetics.                                                                                  
EPA.........................  Federal Insecticide,         Pesticides.................  x                     x                     x                   
                               Fungicide, and Rodenticide                                                                                               
                               Act.                                                                                                                     
                              Toxic Substances Control     Industrial chemicals.......  \1\x                  x                     x                   
                               Act.                                                                                                                     
                              Clean Air Act..............  Air pollutants.............  ....................  ....................  ....................
                              Resource Conservation and    Industrial waste...........                        x                     x                   
                               Recovery Act.                                                                                                            
OSHA........................  Occupational Safety and      Occupational exposure......  ....................  ....................  x                   
                               Health Act.                                                                                                              
CPSC........................  Federal Hazardous            Consumer products..........                        x                     ....................
                               Substances Act.                                                                                                          
                              Consumer Product Safety Act  Consumer products..........                                              x                   
--------------------------------------------------------------------------------------------------------------------------------------------------------
\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 assessment.
    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 Biologics 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 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 assessments.
    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 chapters.

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, 1984).
    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 g/dcl whole blood has 
been recommended (CDC, 1991). Lead exposure in the United States has 
decreased significantly during the last several years.

                 Table 2-1.--Human Neurotoxic Exposures                 
------------------------------------------------------------------------
    Year(s)          Location         Substance           Comments      
------------------------------------------------------------------------
370 B.C.........  Greece.........  Lead...........  Lead toxicity       
                                                     recognized in      
                                                     mining industry.   
1st century A.D.  Rome...........  Lead...........  Vapors recognized as
                                                     toxic.             
1837............  Scotland.......  Manganese......  Chronic manganese   
                                                     poisoning          
                                                     described.         
1924............  United States    Tetraethyl lead  Workers suffer      
                   (New Jersey).                     neurologic         
                                                     symptoms.          
1930............  United States    Tri-o-           Chemical contaminant
                   (Southeast).     cresylphosphat   added to Ginger    
                                    e (TOCP).        Jake, an alcoholic 
                                                     beverage           
                                                     substitute; more   
                                                     than 5,000         
                                                     paralyzed, 20,000  
                                                     to 100,000         
                                                     affected.          
1930's..........  Europe.........  Apiol..........  Drug containing TOCP
                                                     causes 60 cases of 
                                                     neuropathy.        
1932............  United States    Thallium.......  Contaminated barley 
                   (California).                     laced with thallium
                                                     sulfate poisons    
                                                     family, causing    
                                                     neurologic         
                                                     symptoms.          
1937............  South Africa...  TOCP...........  Paralysis develops  
                                                     after use of       
                                                     contaminated       
                                                     cooking oil.       
1946............  England........  Tetraethyl lead  Neurologic effects  
                                                     observed in people 
                                                     cleaning gasoline  
                                                     tanks.             
1950's..........  Japan (Mina-...  Methylmercury..  Fish and shellfish  
                  mata)..........                    contaminated with  
                                                     mercury are        
                                                     ingested, causing  
                                                     neurotoxicity.     
1950's..........  France.........  Organotin......  Medication          
                                                     (Stalinon)         
                                                     containing         
                                                     diethyltin diiodide
                                                     results in         
                                                     poisoning.         
1950's..........  Morocco........  Manganese......  Miners suffer       
                                                     chronic manganese  
                                                     intoxication.      
1950's..........  Guam...........  Cycad..........  Ingestion of plants 
                                                     associated with    
                                                     amyortrophic       
                                                     lateral sclerosis  
                                                     and Parkinson-like 
                                                     syndrome.          
1956............  Turkey.........  Hexachlorobenze  Hexachlorobenzene   
                                    ne.              causes poisoning.  
1956............  Japan..........  Clioquinol.....  Drug causes         
                                                     neuropathy.        
1959............  Morocco........  TOCP...........  Cooking oil         
                                                     contaminated with  
                                                     lubricating oil    
                                                     causes poisoning.  
1960............  Iraq...........  Methylmercury..  Mercury-treated seed
                                                     grain causes       
                                                     neurotoxicity.     
1964............  Japan..........  Methylmercury..  Methylmercury       
                                                     neurotoxicity.     
1968............  Japan..........  PCBs...........  Polychlorinated     
                                                     biphenyls are      
                                                     leaked into rice   
                                                     oil, causing       
                                                     neurotoxicity.     
1969............  Japan..........  n-Hexane.......  Neuropathy due to n-
                                                     hexane exposure.   
1969............  United States    Methylmercury..  Fungicide-treated   
                   (New Mexico).                     grain results in   
                                                     alkyl mercury      
                                                     poisoning.         
1971............  United States..  Hexachlorophene  Hexachlorophene-    
                                                     containing         
                                                     disinfectant is    
                                                     found to be toxic  
                                                     to nervous system. 
1971............  Iraq...........  Methylmercury..  Methylmercury used  
                                                     as fungicide to    
                                                     treat seed grain   
                                                     causes poisoning.  
1972............  France.........  Hexachlorophene  Hexachlorophene     
                                                     poisoning of       
                                                     children.          
1973............  United States    Methyl n-        Fabric production   
                   (Ohio).          butylketone.     plant employees    
                                                     exposed to MnBK    
                                                     solvent suffer     
                                                     polyneuropathy.    
1974-1975.......  United States    Chlordecone      Chemical plant      
                   (Virginia).      (Keptone).       employees exposed  
                                                     to insecticide     
                                                     suffer severe      
                                                     neurologic         
                                                     problems.          
1976............  United States    Leptophos        At least nine       
                   (Texas).         (Phosvel).       employees suffer   
                                                     serious neurologic 
                                                     problems after     
                                                     exposure to        
                                                     insecticide.       
1977............  United States    Dichloropropene  People hospitalized 
                   (California).    (Telone II).     after exposure to  
                                                     pesticide.         
1979-1980.......  United States    2-t-Butylazo-2-  Employees of        
                   (Texas).         hydroxy-5-       manufacturing plant
                                    methylhexane     experience serious 
                                    (BHMH) (Lucel-   neurologic         
                                    7).              problems.          
1980's..........  United States..  Methylphenyltet  Impurity in         
                                    rahydropyridin   synthesis of       
                                    e (MPTP).        illicit drug causes
                                                     Parkinson's disease-
                                                     like effects.      
1981............  Spain..........  Toxic oil......  People ingesting    
                                                     toxic substance in 
                                                     oil suffer severe  
                                                     neuropathy.        
1983-84.........  United States..  Vitamin B6.....  Excessive intake,   
                                                     causes sensory     
                                                     neuropathy,        
                                                     numbness,          
                                                     parathesia, and    
                                                     motor dysfunction. 
1985............  United States    Aldicarb.......  People experience   
                   and Canada.                       neuromuscular      
                                                     deficits after     
                                                     ingestion of       
                                                     contaminated       
                                                     melons.            
1987............  Canada.........  Domoic acid....  Ingestion of mussels
                                                     contaminated with  
                                                     domoic acid causes 
                                                     illnesses.         
1988............  India..........  TOCP...........  Ingestion of        
                                                     adulterated        
                                                     rapeseed oil cause 
                                                     polyneuritis.      
1989............  United States..  L-tryptophan-    Ingestion of a      
                                    containing       chemical           
                                    products.        contaminant        
                                                     associated with the
                                                     manufacture of L-  
                                                     tryptophan results 
                                                     in eosinophilia-   
                                                     myalgia syndrome.  
1991............  Nigeria........  Scopoletin.....  Natural component of
                                                     gari caused        
                                                     neuropathy         
                                                     associated with    
                                                     optic atrophy 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). 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 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 
(MPTP) (Langston et al., 1983). MPTP was a byproduct of a meperidine 
derivative sold illicitly as ``synthetic heroin.''
    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. paspali-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 Claviceps-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, 1990).
    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, NIOSH, and ATSDR, have been given the mandate to regulate or 
evaluate public exposure to toxic chemicals (Tilson, 1989).
2.1.3.1. 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 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 testing.
    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.
2.1.3.2. Occupational Safety and Health Administration.
    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 substance.
2.1.3.3. 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 all 
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).
2.1.3.4. Environmental Protection Agency.
    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.
2.1.3.5. Consumer Product Safety Commission.
    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. The FHSA also bans children's 
products that are hazardous and provides the CPSC with the authority to 
ban other hazardous products.
2.1.3.6. 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-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 Principles
    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 (Boettcher et al., 1987; Lim, 1986; 
Bhattacharyya and Dayal, 1984).
    The nervous system is a highly complex and integrated organ. It is 
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 possible.

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 m 
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 cells).
    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 transport.
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 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 axon.
    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 System

    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 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 convulsions.
    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 Cadividedivide 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 Cadividedivide 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, 1987).
    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; 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 substance.
2.5.2. Blood-Brain and Blood-Nerve Barriers
    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. 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 CNS.

3. Methods for Assessing Human Neurotoxicity

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 
neuroanatomic 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 
are 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 effects.
    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 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 assessment.
    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 neurotoxicology.

3.3. Current Neurotoxicity Testing Methods

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 Methods                   
------------------------------------------------------------------------
     Neurobehavioral function                       Test                
------------------------------------------------------------------------
Sensation..........................  Flicker Fusion.                    
                                     Lanthony (color                    
                                     vision).                           
Motor/Dexterity....................  Pursuit Aiming.                    
                                     Finger Tapping.                    
                                     Postural Stability.                
                                     Reaction Time.                     
                                     Santa Ana Peg Board.               
Cognition..........................  Benton Visual Retention.           
                                     Continuous Performance Task.       
                                     Digit-Symbol.                      
                                     Digit Span.                        
                                     Dual Tasks.                        
                                     Paired-Associate.                  
                                     Symbol-Digit Task.                 
                                     Wechsler Adult Intelligence Scale--
                                      Revised> (Components). 
                                     Wechsler Memory Scale.> 
Affect.............................  Profile of Mood States> 
                                      (POMS).                           
------------------------------------------------------------------------

    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.
3.3.1.1. 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.
3.3.1.2. Investigator-administered test batteries.
    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 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.
3.3.1.3. 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 NO2, ethanol, and toluene (Letz, 1990).
    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 PNS 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 toxicity.
    Noninvasive neurophysiologic test methods used in neurotoxicity 
evaluations include the electroencephalogram (EEG), visually evoked 
response (VER), somatosensory evoked potential (SEP), and the brainstem 
auditory evoked response (BAER). The EEG 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 EEG changes in 
chemically exposed (especially solvents and styrene) individuals have 
been noted (Seppalainen and Harkonen, 1976). EEG 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 dependent.
    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 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 studies.
    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 curve.
    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 conditions.
    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 
pharmacokinetic 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 EEG 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 observation.
    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; 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.
3.3.6.1. 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 insult.
    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 g/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 
assessment.
3.3.6.2. 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 Assessment

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.
3.4.1.1. 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.
3.4.1.2. 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.
3.4.1.3. Case-control (retrospective) studies.
    Last (1986) defines a case-control study as one that ``starts with 
the identification of persons with the disease (or other outcome 
variable) of interest, and a suitable control population (comparison, 
reference) group of persons without the disease.'' He states that the 
relationship of an ``attribute'' to the disease is measured by 
comparing the diseased with the nondiseased with regard to how 
frequently the attribute is present in each of the groups. The cases 
are assembled from a population of persons with and without exposure 
and the comparison group is selected from the same population; the 
relative distribution of the potential risk factor (exposure) in both 
groups is evaluated by computing an odds ratio that serves as an 
estimate of the strength of the association between the disease and the 
potential risk factor. The statistical significance of the ratio is 
determined by calculating a p-value and is used to approximate relative 
risk.
    The case-control approach to the study of potential 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.
3.4.1.4. Prospective (cohort, followup) studies.
    In a prospective study design, a healthy group of people is 
assembled and followed forward in time and observed for the development 
of disease. Such studies are invaluable for determining the time course 
for development of disease (e.g., followup studies performed in various 
cities on the effects of lead on child development). This approach 
allows the direct estimate of risks attributed to a particular exposure 
since disease incidence rates in the cohort 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 study.
    A special type of cohort study is the retrospective cohort study in 
which the investigator goes back in time to select the study groups and 
traces them over time, often to the present. The studies usually 
involve specially exposed groups and have provided much assistance in 
estimating risks due to occupational exposures. Occupational 
retrospective cohort studies rely on company records of past and 
current employees that include information on the dates of employment, 
age at employment, date of departure, and whether diseased (or dead in 
the case of mortality studies). Workers can then be classified by 
duration and degree of exposure. 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 Studies
    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/
sensitization, sleep deprivation, motivation, etc., can be studied.
3.4.2.1. 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.
3.4.2.2. 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.
3.4.2.3. 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 substances.
3.4.2.4. 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.
3.4.2.5. Controls.
    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.
3.4.2.6. 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 Neurotoxicity

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 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 environment.
    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).
3.6.1.1. 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 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).
3.6.1.2. 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.
    The 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.
3.6.1.3. 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.
3.6.1.4. 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 humans 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.
3.6.1.5. 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
3.6.2.1. 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.
3.6.2.2. 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.
3.6.2.3. 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.
3.6.2.4. 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 exposures.
    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 individual sensitivity to indoor air contaminants 
suggest that other neurobehavioral mechanisms also may be operating.
3.6.2.5. Generalizability and individual differences
    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.
3.6.2.6. Veracity of neurobehavioral test results
    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 needed.
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 across species is of 
paramount importance for neurotoxicity risk assessment.

4. Methods to Assess Animal Neurotoxicity

4.1. Introduction

4.1.1. Role of Animal Models
    Determining the risk posed to human health from chemicals requires 
information about the potential toxicological 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 obtainable from 
humans due to ethical considerations. Frequently, results from animal 
studies are used to guide the design of toxicological studies in 
humans.
    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 chemicals.
    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 positives.
4.1.3. Special Considerations in Animal Models
4.1.3.1. Susceptible populations.
    Like most other measures of toxicological 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 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 maturational 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.
4.1.3.2. 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.
4.1.3.3. 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.
4.1.3.4. 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 model.

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, 1989).
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 levels.

      Table 4-1.--Examples of Potential Endpoints of Neurotoxicity      
                                                                        
                                                                        
Behavioral Endpoints:                                                   
  Absence or altered occurrence, magnitude, or latency of sensorimotor  
   reflex                                                               
  Altered magnitude of neurological measurements, 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 posture, 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 associated signal transduction        
  Alterations in membrane-bound enzymes regulating neuronal activity    
  Decreases in brain AChE                                               
  Inhibition of NTE                                                     
  Altered developmental patterns of neurochemical systems               
  Altered proteins (c fos, substance P)                                 
Structural Endpoints:                                                   
  Accumulation, proliferation, or rearrangement 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 4-2.

                        Table 4-2. Examples of Specialized Tests to Measure Neurotoxicity                       
----------------------------------------------------------------------------------------------------------------
         Function                               Procedure                            Representative-agents      
----------------------------------------------------------------------------------------------------------------
Neuromuscular:                                                                                                  
Weakness..................  Grip strength; swimming endurance; suspension     n-hexane, methyl butylketone,     
                             from rod; discriminative motor function;          carbaryl.                        
                             hindlimb splay.                                                                    
Incoordination............  Rotorod, gait measurements......................  3-acetylpyridine, ethanol.        
Tremor....................  Rating scale, spectral analysis.................  Chlordecone, Type I pyrethroids,  
                                                                               DDT.                             
Myoclonia, spasms.........  Rating scale, spectral analysis.................  DDT, Type II pyrethroids.         
Sensory:                                                                                                        
Auditory..................  Discriminated conditioning Reflex modification..  Toluene, trimethyltin.            
Visual toxicity...........  Discriminated conditioning......................  Methyl mercury.                   
Somatosensory toxicity....  Discriminated conditioning......................  Acrylamide.                       
Pain sensitivity..........  Discriminated conditioning (titration);           Parathion.                        
                             functional observational battery.                                                  
Olfactory toxicity........  Discriminated conditioning......................  3-methylindole methylbromide.     
Learning/Memory:                                                                                                
Habituation...............  Startle reflex..................................  Diisopropyl-flurophosphate (DFP). 
Classical conditioning....  Nictitating membrane............................  Aluminum.                         
                            Conditioned flavor aversion.....................  Carbaryl.                         
                            Passive avoidance...............................  Trimethyltin, IDPN.               
                            Olfactory conditioning..........................  Neonatal trimethyltin.            
Operant or instrumental     One-way avoidance...............................  Chlordecone.                      
 conditioning.                                                                                                  
                            Two-way avoidance...............................  Neonatal lead.                    
                            Y-maze avoidance................................  Hypervitaminosis A.               
                            Biel water maze.................................  Styrene.                          
                            Morris water maze...............................  DFP.                              
                            Radial arm maze.................................  Trimethyltin.                     
                            Delayed matching to sample......................  DFP.                              
                            Repeated acquisition............................  Carbaryl.                         
                            Visual discrimination learning..................  Lead.                             
----------------------------------------------------------------------------------------------------------------

4.3.2.1. Functional observational batteries.
     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 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) function. 

 Table 4-3.--Summary of Measures in the Functional Observational Battery and the Type of Data Produced by Each  
                                                                                                                
                                                                                                                
Home cage and open field              Manipulative                          Physiologic                         
Posture (D)                           Ease of removal (R)                   Body temperature (I)                
Convulsions, tremors (D)              Handling reactivity (R)               Body weight (I)                     
Palpebral closure (R)                                                                                           
Lacrimation (R)                       Approach response (R)                                                     
Piloerection (Q)                      Click response (R)                                                        
Salivation (R)                        Touch response (R)                                                        
Vocalizations (Q)                     Tail pinch response (R)                                                   
Rearing (C)                           Righting reflex (R)                                                       
Urination (C)                         Landing foot splay (I)                                                    
Defecation (C)                        Forelimb grip strength (I)                                                
Gait (D, R)                           Hindlimb grip strength (I)                                                
Arousal (R)                           Pupil response (Q)                                                        
Mobility (R)                                                                                                    
Stereotypy (D)                                                                                                  
Bizarre behavior (D)                                                                                            
D = descriptive data; R = rank order data; Q = quantal data;                                                    
I = interval data; C = count data                                                                               

    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.
4.3.2.2. 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 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 ``habituation.''
    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.
4.3.2.3. 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 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.
4.3.2.4. 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 humans.
4.3.2.5. 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, 1989).
    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 the pairing 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).
4.3.2.6. 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 Neurotoxicity
    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 function.
    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.
4.3.3.1. 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.
4.3.3.2. 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 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.
4.3.3.3. 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.
4.3.3.4. Electroencephalography (EEG)
    EEG analysis is used widely in clinical settings for the diagnosis 
of neurological disorders and less often for the detection of subtle 
toxicant-induced dysfunction (WHO, 1986; Eccles, 1988). The basis for 
the use of EEG in either setting is the relationship between specific 
patterns of EEG 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 EEG. 
Dissociation of EEG activity and behavior can, however, occur after 
exposure to certain chemicals. Normal patterns of transition between 
sleep stages or between sleeping and waking states are known to remain 
disturbed for prolonged periods of time following exposure to certain 
chemical classes (e.g., organophosphates). Changes in the pattern of 
the EEG can be elicited by stimuli producing arousal (e.g., lights, 
sounds) and neuroactive drugs. In studies with toxicants, changes in 
EEG pattern can sometimes precede alterations in other objective signs 
of neurotoxicity. EEG experiments must be done under highly controlled 
conditions, and the neurotoxicological significance of chemical-induced 
changes in the EEG 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 EEG.
4.3.3.5. 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.
4.3.3.6. 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 Neurotoxicity
    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 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.

     Table 4-4.--Neurotoxicants With Known Neurochemical Mechanisms     
------------------------------------------------------------------------
          Site of attack                          Examples              
------------------------------------------------------------------------
1. Neurotoxicants acting on ionic                                       
 balance                                                                
    A. Inhibit sodium entry.......  Tetrodotoxin.                       
    B. Block closing of sodium      p,p\1\--DDT, pyrethroids (I).       
     channel.                                                           
    C. Increase permeability to     Batrachotoxin.                      
     sodium.                                                            
    D. Increase intracellular       Chlordecone.                        
     calcium.                                                           
2. Cytotoxicants--depend on uptake  MPTP.                               
 into nerve terminal.                                                   
3. Uptake blockers................  Hemicholinium.                      
4. Metabolic poisons..............  Cyanide.                            
5. Receptor hyperactivators.......  Domoic acid.                        
6. Transmitter release (ACh)        Botulinum toxin.                    
 blockers.                                                              
7. Transmitter degradation (ACh)    Organophosphates, carbamates.       
 inhibitors.                                                            
8. Microtubule disruptors.........  Vincristine.                        
------------------------------------------------------------------------

    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 Neurotoxicity
    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                                
----------------------------------------------------------------------------------------------------------------
                                                             Corresponding        Disease or neurodegenerative  
       Site of attack              Neuropathology            neurotoxicant                 condition            
----------------------------------------------------------------------------------------------------------------
Neuron cell body...........  Neuronopathy..............  Methylmercury.......  Minamata disease.                
                                                         A.E.T.T.............  Ceroid lipofuscinoses.           
                                                         Quinolinic acid.....  Huntington's disease.            
                                                         3-acetylpridine.....  Cerebellar ataxia.               
                                                         Aluminum............  Alzheimer's disease.             
Nerve terminal.............  Neurodegeneration.........  MPTP................  Parkinson's disease.             
Schwann cell myelin........  Myelinopathy..............  Lead Buckthorn toxin  Neuropathy of metachromatic      
                                                                                leukodystrophy.                 
Central-peripheral distal    Distal axonopathy.........  Acrylamide            Vitamin deficiency.              
 axon.                                                    Hexacarbons Carbon                                    
                                                          disulfide.                                            
Central axons..............  Central axonopathy........  Clioquinol..........  Subacute myelooptico-neuropathy. 
Proximal axon..............  Proximal axonopathy.......  B,B'-imminodi-        Motor neuron disease.            
                                                          proprionitrile.                                       
----------------------------------------------------------------------------------------------------------------

    In general, chemical effects lead to two types of primary cellular 
alteration: (l) 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   
                              Neurotoxicity                             
                                                                        
                                                                        
Alcohols                             Methanol, ethanol                  
Antimitotics                         X-radiation, azacytidine           
Insecticides                         DDT, kepone, organophosphates      
Metals                               Lead, methylmercury, cadmium       
Polyhalogenated hydrocarbons         PCB, PBB                           
Psychoactive drugs                   Cocaine, phenytoin                 
Solvents                             Carbon disulfide, toluene          
Vitamins                             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 exposure.
    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 testing.
    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 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 
tested.

4.3.7. Physiological and Neuroendocrine Endpoints

    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 neuropeptides 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
4.3.8.1. 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.
4.3.8.2. 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 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 way.
    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 
assessment.

5.2. The Risk Assessment Process

5.2.1. Hazard Identification
    Agents that adversely affect the neurophysiological, neurochemical, 
or 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.
5.2.1.1. 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).
5.2.1.2. 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.
5.2.1.3. Special issues
    5.2.1.3.1. 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.
    5.2.1.3.2. 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).
    5.2.1.3.3. 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.
5.2.1.3.4. Weight of evidence.
    A ``weight of evidence'' approach to identifying an agent as a 
neurotoxic 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 toxicologically 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 parameters).
    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 gender is generally used as the basis for estimating the 
RfD (OTA, 1990). 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 determined.
    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 less than 0.003, etc. Because 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. . . .''
    The RfD approach relies on single experimental observations (the 
NOAEL or LOAEL) 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 
determined.
    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, 1992).
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 process.
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 RfD approach to all 
neurotoxicants is unlikely to be biologically defensible in light of 
mechanistic data (NRC, 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). 

     Table 5-1.--General Assumptions That Underlie Traditional Risk     
                             Assessmentsa,b                             
                                                                        
                                                                        
1. A threshold dose exists for noncancer endpoints.                     
2. NOAEL/LOAEL uncertainty- or safety-factor approaches are reasonable. 
3. Variability in the toxic response to the chemical exposure is not due
 to a heterogeneous population response.                                
4. Average dose or total dose is a reasonable measure of exposure when  
 doses are not equivalent in time, rate, or route of administration 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.    
aThis is not intended to be an exhaustive list.                         
bModified 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 species.
    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 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., 1989).
    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 be 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.

6. 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 workplace.
    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. Reiter, USEPA, Chair
Hugh A. Tilson, USEPA, Executive Secretary
John Dougherty, NIOSH
G. Jean Harry, NIEHS
Carol J. Jones, OSHA
Suzanne McMaster, USEPA
William Slikker, NCTR/FDA
Thomas J. Sobotka, FDA

Ad Hoc Interagency Committee on Neurotoxicology

William Boyes, U.S. Environmental Protection Agency
Joy Cavagnaro, Food and Drug Administration
Selene Chou, Agency for Toxic Substances and Disease Registry
Murray Cohn, U.S. Consumer Product Safety Commission
Joseph F. Contrera, Food and Drug Administration
Miriam Davis, National Institute of Environmental Health Sciences, 
National Institutes of Health
Joseph DeGeorge, Food and Drug Administration
Robert Dick, National Institute for Occupational Safety and Health
John Dougherty, National Institute for Occupational Safety and 
Health
Lynda Erinoff, National Institute on Drug Abuse
Joseph P. Hanig, Food and Drug Administration
G. Jean Harry, National Institute of Environmental Health Sciences
David G. Hattan, Food and Drug Administration
Norman A. Krasnegor, National Institutes of Health
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. Reiter, 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 
Engineering Center
Bernard A. Schwetz, National Institute of Environmental Health 
Sciences
William F. Sette, U.S. Environmental Protection Agency
William Slikker, Jr., National Center for Toxicological Research
D. Stephen Snyder, National Institute on Aging
Thomas J. Sobotka, Food and Drug Administration
Hugh A. Tilson, U.S. Environmental Protection Agency
Mildred Williams-Johnson, Agency for Toxic Substances and Disease 
Registry

[FR Doc. 94-20033 Filed 8-16-94; 8:45 am]
BILLING CODE 6560-50-P