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Neurotoxicity: Identifying and Controlling Poisons of the Nervous System
Chapter 5
Testing and Monitoring
US Congress Office of Technology Assessment April 1990
Note: The PDF file this document was copied from was not properly created and its quality is extremely low.
Therefore, the accuracy of this text is less than 100%. Some photos have not been included because of low quality.
"Over the last 10 to 15 years, cancer had dominated the discussion of occupational standards and it continues to remain terribly important. At the same time, information on neurotoxins has increased. The notion of chronic and subclinical neurotoxicity has developed. Although these things are progressive and don't occur overnight, you'll see more attention paid to neurotoxicity in the years ahead. "
Philip Landrigan
Occupational Hazards 49:36, 1987
"The reasons for inadequate neurobehavioral testing of chemicals. . relate to economic factors and political decisions, not to inadequacies of the test methods. "
Donald McMillan
Occupational Hazards 49:37, 1987
''We need to know a lot more about how toxicity is expressed in behavior. We need to be able to recommend tests for chemicals before they move into the marketplace. This is why we need more of what NIOSH is doing. As it is, we are still using workers as part of an early-warning system. "
Ronald Wood
Psychology Today, July 1982, p. 30
Back to Table of Contents
CONTENTS
INTRODUCTION
ANIMAL TOXICITY TESTS
Designing Useful Tests
Evaluating Chemicals for Neurotoxicity
Types of Animal Tests
Animal Testing Issues
ALTERNATIVES TO ANIMAL TESTS
In Vitro Neurotoxicity Test Development
Applications of In Vitro Techniques to Neurotoxicity Testing
Advantages and Limitations of In Vitro Testing
HUMAN TESTING
Overview of Human Tests
Human Exposure Studies
Legal and Ethical Considerations in Neurotoxicity Testing
and Monitoring
Prevention of Human Exposure to Neurotoxic Substances
MONITORING OF TOXIC SUBSTANCES
Specimen Banking
Biological Monitoring
Other Monitoring Programs
SUMMARY AND CONCLUSIONS
CHAPTER PREFERENCES
Boxes
5-A Tiered Animal Testing To Identify Adverse
Neurobehavioral Effects of Substances
5-B Conducting the EPA Functional Observational Battery
5-C Ethical Issues Associated With Chronic Exposure to a
Neurotoxic Agent
5-D Neurotoxicants Released Into the Environment by
Industry: The Toxics Release Inventory Supplies
New Evidence
Figures
5-1 The Effects of Toxic Substances on Motor Activity
5-2 Pattern Reversal Evoked Potential (PREP) and Flash
Evoked Potential (FEP) After Treatment With
Chlordimeform
5-3 Neurotoxic Substances Are Prominent Among the
Toxics Release Inventory's Top 25 Chemicals
Emitted Into the Air in 1967
Tables
5-l Subject Factors Influencing Neurobehavioral Test Results
5-2 Behavioral Test Battery for Toxicopsychological Studies
Used at the Institute of Occupational Health in Helsinki
5-3 WHO Neurobehavioral Core Test Battery
Chapter 5
Testing and Monitoring
INTRODUCTION
People are exposed to chemicals every day in the course of eating, working, and recreation. Some of these chemicals are synthetic; others, whose properties may be unknown, occur naturally in the environment and in food. Modern society could not exist without them. However, the same chemicals that contribute to our high standard of living may also produce unanticipated and undesired effects. Regulatory officials are concerned with weighing the benefits of use against the risks of adverse health effects.
All substances, even water, can be toxic at a high enough level of ingestion. Determining the risk posed to human health by toxic substances requires information about the potential hazard and about the expected level of exposure, resulting in an estimate of the probability that a substance will produce harm under certain conditions (see ch. 6) (105).
There are many approaches to testing for neurotoxicity, and each has both advantages and limitations. Toxic substances can be evaluated through whole animal (in vivo) tests, tissue and cell culture (in vitro) tests, and tests on human subjects. The latter is the best means of predicting the effects of potentially toxic substances on human health. This approach, however, is generally difficult, expensive, and in some circumstances unethical. Consequently, it is usually necessary to rely on animal or in vitro tests.
Most toxicity testing is performed on animals, usually mice and rats. Animals are used for several reasons, one of which is that, biologically, they resemble humans in many ways and can often serve as adequate models for toxicity studies. On the other hand, it can be difficult to extrapolate the results of animal studies to humans. It is also important to keep in mind that the biochemical and physiological processes underlying human neurological and psychiatric problems are highly complex and often cannot be modeled in any single system.
In vitro tests can be used to complement animal tests and reduce the number of animals used in routine toxicity testing. In vitro testing may also be less expensive and less time-consuming. By understanding the structure or function affected by a toxic substance in vitro, it is sometimes possible to predict adverse effects in the whole animal. Like all testing strategies, in vitro tests have limitations, including the inability to analyze behavioral effects such as loss of memory or irritability.
Some human toxicological data are derived from accidental exposures to industrial chemicals and some from epidemiological studies. Prescription drugs are tested on humans to determine safety and efficacy.
This chapter briefly describes some methods of neurotoxicity testing and the advantages and limitations of each. The first section addresses animal toxicity tests, including the types of neurotoxicity tests currently proposed for regulatory use by the U.S. Environmental Protection Agency (EPA). The second section describes alternatives to animal tests, including in vitro approaches, and the third section describes human testing. Finally, approaches to monitoring of toxic substances are briefly discussed.
ANIMAL TOXICITY TESTS
In designing animal tests and evaluating data, appropriate weight is given to the following factors on a case-by-case basis, taking into account the seriousness of the hazard and the assumptions needed to estimate human health risks (105):
the relationship between dose and response;
the effects at the molecular, cellular, organ, organ system, and whole organism levels;
the reproducibility of the study results and possible explanations for lack of reproducibility;
the effects of structurally similar substances on humans or animals;
any known metabolic differences between humans and the test species that could affect response;
statistical uncertainties and difficulties in extrapolating to a low dose; and
other factors, such as sex, species differences, and route of administration.
An Office of Technology Assessment (OTA) report, Alternatives to Animal Use in Research, Testing, and Education, contains a detailed discussion of the use of animals in research and associated ethical (105). The issues raised there will not be readdressed in this report.
Toxicity testing should aim to obtain all the data needed for accurate risk assessment at the lowest possible cost. Factors that influence cost include the number of appropriate test species, the nature of the parameters studied, the choice of test subjects, the controls required, and the skilled staff necessary to perform the studies. In addition, toxicity testing requires a substantial investment in labor. Aside from the maintenance needs of the animals used, many observations are necessary. Acute studies often involve observations of behavior and appearance as well as histopathological observations. Subchronic and chronic studies require more detailed pathological studies as well as weekly clinical examinations of all the animals used in the studies (92). Testing costs will be discussed in more detail in chapter 8.
Designing Useful Tests
Animal tests are used to determine the functional, structural, and biochemical effects of toxic substances. Experimental animal models have limitations, however, and the accuracy and reliability of a quantitative prediction of human toxicity depend on a number of conditions, such as choice of species, choice of tests, similarity of human and animal metabolism, design of the experiment, and method of extrapolation of animal data.
When designing animal toxicity tests, therefore, it is essential that the examiners clearly define the objective of their study and understand how the resulting data will be used. Several questions should be answered in advance: Will the data obtained from the animal tests be meaningful? Will the data be useful in the risk assessment process? Can the data be extrapolated from animals to humans?
The World Health Organization (WHO) recently suggested several general objectives of neurotoxicology testing (123):
identify whether the nervous system is altered by the toxic substance,
characterize the nervous system alterations associated with exposure,
ascertain whether the nervous system is the primary target for the chemical, and
determine dose and time-effect relationships to establish no observed adverse effect levels (NOAELs).
The initial goal is to determine whether or not the nervous system is affected by a substance for which no toxicological data exist. This often involves screening for neurotoxicity using tests that predict the potential of a substance to produce adverse effects. To be most effective, the tests should be simple, rapid, and economical to administer. Once a chemical is known to produce a neurotoxic effect, further studies can be performed in order to characterize the nature and mechanism of the alterations. Screens are generally designed to explore the consequences of exposure and to indicate whether or not the nervous system is adversely affected.
Chemicals are unlikely to affect all major components of the nervous system at the doses tested; therefore, it is important to use a variety of tests that measure different functional, morphological, or chemical alterations in order to maximize the probability of detecting neurotoxicity. The methods used may differ with the objective of the study, the age of the animal, and the species examined (123).
Potential neurotoxic risks are difficult to assess because of the complexity of the nervous system. Some of the problems in assessment are associated with the wide variations in response that can occur. Other problems are related to the examiner's incomplete understanding of what is being measured by a given test. Therefore, no single test can be used to examine the total functional capacity of the nervous system (123).
Animal Choice
In preliminary screening of known or suspected toxic substances, numerous economic factors influence the design of the evaluation. It is useful if there exist adequate anatomical, physiological, and toxicological databases on the species chosen for study to allow meaningful interpretations of effects and appropriate hypotheses about mechanisms and sites of action (123).
Most routine toxicity testing is carried out with only one or two species. For example, cancer bioassays frequently involve the use of rats and mice, and the monkey may be used for identifying the effects of MPTP, a byproduct in the illicit synthesis of a meperidine analog. Hens have been used to evaluate the neurotoxic potential of organophosphorous pesticides. Most other neurotoxicity screening studies use laboratory rats. Ideally, more than one animal species should be tested-if only a single species is tested, it is possible to conclude that human exposure is acceptable when in fact it is not. However, routine multi-species testing is a costly and demanding enterprise. The facilities and services needed for animal husbandry and the equipment and technical expertise needed to carry out the research make multi-species testing economically impractical in many instances (59).
There are other variables besides species that should be considered. For example, the sex of the test animal may influence results of the study. Some toxic substances may have a greater adverse effect on females than males or vice versa. Consequently, EPA testing guidelines require both male and female rats for neurotoxicity testing.
Another important factor is the age of the animal. The effects of a toxic substance may vary dramatically, depending on the stage of maturation of the animal. For example, cell loss in the nervous system due to natural aging processes may predispose an animal to the adverse effects of toxic substances. Most preliminary assessments are designed to provide information on the population with the greatest potential for exposure, namely, adults. However, aged populations or those undergoing rapid maturation are often especially vulnerable to environmental exposures; thus, tests to assess the neurobehavioral functioning of these populations are necessary for a complete evaluation.
The ideal tests are those that permit longitudinal assessment of animals of both sexes at any stage of development (i.e., at young childhood, prepuberty, and adulthood) (67). Whenever possible, the choice of animal model should take into account such factors as the differences in metabolism of substances between species, genetic composition of the species, and the sensitivity of the test animals to the toxic effects of the substances (50 FR 39458).
Dosing Regimen
Some compounds produce one kind of toxic effect following a single exposure and other effects following prolonged or repeated exposure. In environmental toxicology, a major objective is the detection of cumulative toxicity following continued (or intermittent) exposure. Thus, a multiple-dosing regimen is most commonly used. This is particularly important in neurobehavioral testing, since both quantitative and qualitative changes in the response to environmental factors can occur with repeated exposure, or at some later time following a single exposure (67,123). Normally, assessments are made for a period of time following termination of the dosing regimen, both to determine the reversibility of any observed effects and to see if any new effects appear (123).
Substances are administered in varying doses, the dose being a function of the concentration of the substance and the duration and frequency of exposure. Significant differences in response may occur when the same quantity of toxic material is administered over different exposure periods. Acute exposure to substances may produce both immediate and delayed toxic effects (such is the case for some organophosphorous pesticides). These effects may differ from the effects following long-term exposure. Repeated exposure to certain solvents may produce immediate effects after each dosing as well as delayed adverse effects from long-term exposure (47).
Acute toxic responses result when an animal is subjected to high concentrations of a substance over a short period of time. The acute response may be sudden and severe, and usually lasts for a brief period of time; in some cases, however, it is permanent. If the dose is sufficiently high, death may result. Lower doses (lower concentrations over longer periods of time) may not immediately cause death. As the dose decreases, the response is generally less severe and may take longer to develop. In chronic exposures, clinically adverse effects may take years to develop (47).
Route of Exposure
The most common routes by which toxic substances enter the body are, in descending order, inhalation (through the lungs), oral (through ingestion), and dermal (through the skin). Although substances generally produce t
Chapter 5
Testing and Monitoring
US Congress Office of Technology Assessment April 1990
Note: The PDF file this document was copied from was not properly created and its quality is extremely low.
Therefore, the accuracy of this text is less than 100%. Some photos have not been included because of low quality.
"Over the last 10 to 15 years, cancer had dominated the discussion of occupational standards and it continues to remain terribly important. At the same time, information on neurotoxins has increased. The notion of chronic and subclinical neurotoxicity has developed. Although these things are progressive and don't occur overnight, you'll see more attention paid to neurotoxicity in the years ahead. "
Philip Landrigan
Occupational Hazards 49:36, 1987
"The reasons for inadequate neurobehavioral testing of chemicals. . relate to economic factors and political decisions, not to inadequacies of the test methods. "
Donald McMillan
Occupational Hazards 49:37, 1987
''We need to know a lot more about how toxicity is expressed in behavior. We need to be able to recommend tests for chemicals before they move into the marketplace. This is why we need more of what NIOSH is doing. As it is, we are still using workers as part of an early-warning system. "
Ronald Wood
Psychology Today, July 1982, p. 30
Back to Table of Contents
CONTENTS
INTRODUCTION
ANIMAL TOXICITY TESTS
Designing Useful Tests
Evaluating Chemicals for Neurotoxicity
Types of Animal Tests
Animal Testing Issues
ALTERNATIVES TO ANIMAL TESTS
In Vitro Neurotoxicity Test Development
Applications of In Vitro Techniques to Neurotoxicity Testing
Advantages and Limitations of In Vitro Testing
HUMAN TESTING
Overview of Human Tests
Human Exposure Studies
Legal and Ethical Considerations in Neurotoxicity Testing
and Monitoring
Prevention of Human Exposure to Neurotoxic Substances
MONITORING OF TOXIC SUBSTANCES
Specimen Banking
Biological Monitoring
Other Monitoring Programs
SUMMARY AND CONCLUSIONS
CHAPTER PREFERENCES
Boxes
5-A Tiered Animal Testing To Identify Adverse
Neurobehavioral Effects of Substances
5-B Conducting the EPA Functional Observational Battery
5-C Ethical Issues Associated With Chronic Exposure to a
Neurotoxic Agent
5-D Neurotoxicants Released Into the Environment by
Industry: The Toxics Release Inventory Supplies
New Evidence
Figures
5-1 The Effects of Toxic Substances on Motor Activity
5-2 Pattern Reversal Evoked Potential (PREP) and Flash
Evoked Potential (FEP) After Treatment With
Chlordimeform
5-3 Neurotoxic Substances Are Prominent Among the
Toxics Release Inventory's Top 25 Chemicals
Emitted Into the Air in 1967
Tables
5-l Subject Factors Influencing Neurobehavioral Test Results
5-2 Behavioral Test Battery for Toxicopsychological Studies
Used at the Institute of Occupational Health in Helsinki
5-3 WHO Neurobehavioral Core Test Battery
Chapter 5
Testing and Monitoring
INTRODUCTION
People are exposed to chemicals every day in the course of eating, working, and recreation. Some of these chemicals are synthetic; others, whose properties may be unknown, occur naturally in the environment and in food. Modern society could not exist without them. However, the same chemicals that contribute to our high standard of living may also produce unanticipated and undesired effects. Regulatory officials are concerned with weighing the benefits of use against the risks of adverse health effects.
All substances, even water, can be toxic at a high enough level of ingestion. Determining the risk posed to human health by toxic substances requires information about the potential hazard and about the expected level of exposure, resulting in an estimate of the probability that a substance will produce harm under certain conditions (see ch. 6) (105).
There are many approaches to testing for neurotoxicity, and each has both advantages and limitations. Toxic substances can be evaluated through whole animal (in vivo) tests, tissue and cell culture (in vitro) tests, and tests on human subjects. The latter is the best means of predicting the effects of potentially toxic substances on human health. This approach, however, is generally difficult, expensive, and in some circumstances unethical. Consequently, it is usually necessary to rely on animal or in vitro tests.
Most toxicity testing is performed on animals, usually mice and rats. Animals are used for several reasons, one of which is that, biologically, they resemble humans in many ways and can often serve as adequate models for toxicity studies. On the other hand, it can be difficult to extrapolate the results of animal studies to humans. It is also important to keep in mind that the biochemical and physiological processes underlying human neurological and psychiatric problems are highly complex and often cannot be modeled in any single system.
In vitro tests can be used to complement animal tests and reduce the number of animals used in routine toxicity testing. In vitro testing may also be less expensive and less time-consuming. By understanding the structure or function affected by a toxic substance in vitro, it is sometimes possible to predict adverse effects in the whole animal. Like all testing strategies, in vitro tests have limitations, including the inability to analyze behavioral effects such as loss of memory or irritability.
Some human toxicological data are derived from accidental exposures to industrial chemicals and some from epidemiological studies. Prescription drugs are tested on humans to determine safety and efficacy.
This chapter briefly describes some methods of neurotoxicity testing and the advantages and limitations of each. The first section addresses animal toxicity tests, including the types of neurotoxicity tests currently proposed for regulatory use by the U.S. Environmental Protection Agency (EPA). The second section describes alternatives to animal tests, including in vitro approaches, and the third section describes human testing. Finally, approaches to monitoring of toxic substances are briefly discussed.
ANIMAL TOXICITY TESTS
In designing animal tests and evaluating data, appropriate weight is given to the following factors on a case-by-case basis, taking into account the seriousness of the hazard and the assumptions needed to estimate human health risks (105):
the relationship between dose and response;
the effects at the molecular, cellular, organ, organ system, and whole organism levels;
the reproducibility of the study results and possible explanations for lack of reproducibility;
the effects of structurally similar substances on humans or animals;
any known metabolic differences between humans and the test species that could affect response;
statistical uncertainties and difficulties in extrapolating to a low dose; and
other factors, such as sex, species differences, and route of administration.
An Office of Technology Assessment (OTA) report, Alternatives to Animal Use in Research, Testing, and Education, contains a detailed discussion of the use of animals in research and associated ethical (105). The issues raised there will not be readdressed in this report.
Toxicity testing should aim to obtain all the data needed for accurate risk assessment at the lowest possible cost. Factors that influence cost include the number of appropriate test species, the nature of the parameters studied, the choice of test subjects, the controls required, and the skilled staff necessary to perform the studies. In addition, toxicity testing requires a substantial investment in labor. Aside from the maintenance needs of the animals used, many observations are necessary. Acute studies often involve observations of behavior and appearance as well as histopathological observations. Subchronic and chronic studies require more detailed pathological studies as well as weekly clinical examinations of all the animals used in the studies (92). Testing costs will be discussed in more detail in chapter 8.
Designing Useful Tests
Animal tests are used to determine the functional, structural, and biochemical effects of toxic substances. Experimental animal models have limitations, however, and the accuracy and reliability of a quantitative prediction of human toxicity depend on a number of conditions, such as choice of species, choice of tests, similarity of human and animal metabolism, design of the experiment, and method of extrapolation of animal data.
When designing animal toxicity tests, therefore, it is essential that the examiners clearly define the objective of their study and understand how the resulting data will be used. Several questions should be answered in advance: Will the data obtained from the animal tests be meaningful? Will the data be useful in the risk assessment process? Can the data be extrapolated from animals to humans?
The World Health Organization (WHO) recently suggested several general objectives of neurotoxicology testing (123):
identify whether the nervous system is altered by the toxic substance,
characterize the nervous system alterations associated with exposure,
ascertain whether the nervous system is the primary target for the chemical, and
determine dose and time-effect relationships to establish no observed adverse effect levels (NOAELs).
The initial goal is to determine whether or not the nervous system is affected by a substance for which no toxicological data exist. This often involves screening for neurotoxicity using tests that predict the potential of a substance to produce adverse effects. To be most effective, the tests should be simple, rapid, and economical to administer. Once a chemical is known to produce a neurotoxic effect, further studies can be performed in order to characterize the nature and mechanism of the alterations. Screens are generally designed to explore the consequences of exposure and to indicate whether or not the nervous system is adversely affected.
Chemicals are unlikely to affect all major components of the nervous system at the doses tested; therefore, it is important to use a variety of tests that measure different functional, morphological, or chemical alterations in order to maximize the probability of detecting neurotoxicity. The methods used may differ with the objective of the study, the age of the animal, and the species examined (123).
Potential neurotoxic risks are difficult to assess because of the complexity of the nervous system. Some of the problems in assessment are associated with the wide variations in response that can occur. Other problems are related to the examiner's incomplete understanding of what is being measured by a given test. Therefore, no single test can be used to examine the total functional capacity of the nervous system (123).
Animal Choice
In preliminary screening of known or suspected toxic substances, numerous economic factors influence the design of the evaluation. It is useful if there exist adequate anatomical, physiological, and toxicological databases on the species chosen for study to allow meaningful interpretations of effects and appropriate hypotheses about mechanisms and sites of action (123).
Most routine toxicity testing is carried out with only one or two species. For example, cancer bioassays frequently involve the use of rats and mice, and the monkey may be used for identifying the effects of MPTP, a byproduct in the illicit synthesis of a meperidine analog. Hens have been used to evaluate the neurotoxic potential of organophosphorous pesticides. Most other neurotoxicity screening studies use laboratory rats. Ideally, more than one animal species should be tested-if only a single species is tested, it is possible to conclude that human exposure is acceptable when in fact it is not. However, routine multi-species testing is a costly and demanding enterprise. The facilities and services needed for animal husbandry and the equipment and technical expertise needed to carry out the research make multi-species testing economically impractical in many instances (59).
There are other variables besides species that should be considered. For example, the sex of the test animal may influence results of the study. Some toxic substances may have a greater adverse effect on females than males or vice versa. Consequently, EPA testing guidelines require both male and female rats for neurotoxicity testing.
Another important factor is the age of the animal. The effects of a toxic substance may vary dramatically, depending on the stage of maturation of the animal. For example, cell loss in the nervous system due to natural aging processes may predispose an animal to the adverse effects of toxic substances. Most preliminary assessments are designed to provide information on the population with the greatest potential for exposure, namely, adults. However, aged populations or those undergoing rapid maturation are often especially vulnerable to environmental exposures; thus, tests to assess the neurobehavioral functioning of these populations are necessary for a complete evaluation.
The ideal tests are those that permit longitudinal assessment of animals of both sexes at any stage of development (i.e., at young childhood, prepuberty, and adulthood) (67). Whenever possible, the choice of animal model should take into account such factors as the differences in metabolism of substances between species, genetic composition of the species, and the sensitivity of the test animals to the toxic effects of the substances (50 FR 39458).
Dosing Regimen
Some compounds produce one kind of toxic effect following a single exposure and other effects following prolonged or repeated exposure. In environmental toxicology, a major objective is the detection of cumulative toxicity following continued (or intermittent) exposure. Thus, a multiple-dosing regimen is most commonly used. This is particularly important in neurobehavioral testing, since both quantitative and qualitative changes in the response to environmental factors can occur with repeated exposure, or at some later time following a single exposure (67,123). Normally, assessments are made for a period of time following termination of the dosing regimen, both to determine the reversibility of any observed effects and to see if any new effects appear (123).
Substances are administered in varying doses, the dose being a function of the concentration of the substance and the duration and frequency of exposure. Significant differences in response may occur when the same quantity of toxic material is administered over different exposure periods. Acute exposure to substances may produce both immediate and delayed toxic effects (such is the case for some organophosphorous pesticides). These effects may differ from the effects following long-term exposure. Repeated exposure to certain solvents may produce immediate effects after each dosing as well as delayed adverse effects from long-term exposure (47).
Acute toxic responses result when an animal is subjected to high concentrations of a substance over a short period of time. The acute response may be sudden and severe, and usually lasts for a brief period of time; in some cases, however, it is permanent. If the dose is sufficiently high, death may result. Lower doses (lower concentrations over longer periods of time) may not immediately cause death. As the dose decreases, the response is generally less severe and may take longer to develop. In chronic exposures, clinically adverse effects may take years to develop (47).
Route of Exposure
The most common routes by which toxic substances enter the body are, in descending order, inhalation (through the lungs), oral (through ingestion), and dermal (through the skin). Although substances generally produce t
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