Redbook 2000: IV.C.10. Neurotoxicity Studies July 2007
Toxicological Principles for the Safety Assessment of Food Ingredients
Chapter IV.C.10. Neurotoxicity Studies
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- Evaluating Neurotoxicity
This chapter defines neurotoxicity and the broad spectrum of adverse effects to the nervous system that may occur in the adult and developing organism. Emphasis is placed on the need to effectively minimize the risk of human neurotoxicity by assessing the neurotoxic potential of food ingredients. The chapter proceeds with explicating the nature and extent of information needed for an assessment of neurotoxic potential and suggests a strategy for obtaining this information as a routine part of the toxicological testing to evaluate the safety of chemicals proposed for use as food ingredients. Consistent with the basic strategy advocated by FDA for toxicological testing, the assessment of neurotoxic potential would be most effectively carried out through a structured process of tiered testing in which chemicals are initially screened for signs of neurotoxicity as part of those toxicity studies recommended for entrance-level testing of proposed food ingredients . Chemicals identified as possible neurotoxicants become candidates for subsequent special neurotoxicity testing designed to confirm and characterize the scope of nervous system involvement and to determine dose-response characteristics, including a quantitative determination of the no-observed-adverse-effect level. The basic elements of a neurotoxicity screen and of special neurotoxicity testing are presented and the principle points to consider in protocol design are discussed.
In 1982, the FDA issued guidelines for toxicological testing of food ingredients(2). Although neurotoxicity was neither explicitly discussed nor defined in these guidelines, there were certain elements included in the conventional toxicity studies which have traditionally been used to assess nervous system toxicity. In general, these included a routine pathological evaluation of neuronal tissue and cage-side observations for clinical signs of toxicity. In 1985, FDA commissioned the Federation of American Societies for Experimental Biology (FASEB) to assess the utility of these current FDA guidelines for detecting neurotoxic hazards.(3) One conclusion of the FASEB report was that the current guidelines are too broad and nonspecific with respect to the nature and extent of information which needs to be provided to the FDA for an evaluation of a chemical's neurotoxic potential. The limited information derived from conventional toxicity screening studies, as currently conducted and reported, enables little more than the detection of clearly evident nervous system toxicity associated with general neuropathology and overt neurological dysfunction. Little consistent or systematically documented information is typically available about other possibly less severe, but equally important, types of neurotoxic effects including, for example, behavioral and physiological dysfunction and developmental neurotoxicity. Incomplete documentation about the range of adverse effects to the structural and functional integrity of the nervous system limits the effective evaluation of the full spectrum of neurotoxic hazards.(4) The present FDA guidelines are intended to explicate more clearly the nature and extent of information deemed necessary for the assessment of neurotoxic potential and to suggest a strategy for obtaining this information as part of the safety evaluation process.
Until recently, neurotoxicity was equated with neuropathy involving frank neuropathological lesions or overt neurological dysfunctions, such as seizure, paralysis or tremor. Examples of chemically induced neuropathy in humans (for example, from exposure to domoic acid, lead, organic mercury, hexane, carbon disulfide, and tri-ortho-cresylphosphate) emphasize the need for assessing the neurotoxic potential of chemicals to which humans may be exposed.(5) Although neuropathy is appropriately recognized as a manifestation of neurotoxicity, it is now clear that there are numerous other endpoints which may signal nervous system toxicity.(6) Ongoing research on nervous system toxicity continues to reveal the diversity of biochemical, structural, and functional abnormalities that toxicants can elicit, both directly and indirectly.(7) Neurotoxic chemicals invariably initiate their effects at the molecular level, altering cellular neurochemical processes. The qualitative nature of these alterations or their magnitude may be such as to result in cytoarchitectural changes and neuropathological effects accompanied by nervous system dysfunction expressed as physiological or behavioral abnormalities.(8) Motor incoordination, sensory deficits, learning and memory impairment, changes in emotion, and altered states of arousal in the adult and the developing organism are examples of deficits recognized as functional indices of possible neurotoxicity. Notably, physiological or behavioral dysfunctions may occur prior to, or even in the absence of, evident neuropathology or other signs of toxicity.(9) This is exemplified by the behavioral dysfunctions associated with exposure to such neuroactive chemicals as barbiturates, amphetamines, ethanol, lead, and carbon monoxide at exposure levels that elicit little or no apparent signs of neuropathy.(10) This dissociation of neuropathology and functional changes may involve a number of factors, including the intrinsic toxicity of a chemical and, particularly, the dose and regimen of exposure. Continued reliance on neuropathy as the primary criterion of neurotoxicity is overly simplistic and does not adequately reflect contemporary concerns about the broader spectrum of potential neurotoxic effects on the adult and developing organism.
Among the various approaches that can be used for assessing neurotoxicity, behavioral testing in conjunction with neuropathological evaluation represent a practical means of obtaining a relatively comprehensive assessment of the functional development and integrity of the nervous system within the context of a standard toxicity study.(11) Behavior is an adaptive response of an organism, orchestrated by the nervous system, to some set of internal and external stimuli. A behavioral response represents the integrated end product of multiple neuronal subsystems including sensory, motor, cognitive, attentional, and integrative components, as well as an array of physiological functions.(12) As such, behavior can serve as a measurable index of the status of multiple functional components of the nervous system. Since behavioral testing is non-invasive, it can be applied repeatedly for longitudinal assessment of the neurotoxicity of a test compound, including persistent or delayed treatment-related effects.(13) Furthermore, since neuronal function can be influenced by the status of other organ systems in the body (e.g., cardiovascular, endocrine, and immunologic systems), certain types of behavioral changes may indirectly reflect significant primary toxicity in other organ systems. For this reason it is important to emphasize that the assessment of neurotoxicity necessitates an integrated interpretation of all toxicologic data.
Behavioral testing has been established as a reliable toxicological index in safety assessment. Considerable progress has been made in the standardization and validation of neurobehavioral and neurodevelopmental testing procedures.(14) As a result, a variety of behavioral methodologies is available for use in determining the potential of chemical substances to affect adversely the functional integrity of the nervous system in adult and developing organisms.(15) Behavioral testing can be readily incorporated into toxicity testing protocols and, together with neuropathological evaluation, can enhance the ability to assess neurotoxic hazard(16).
Because of the impact that nervous system toxicity can have on human health, assessing the neurotoxic potential of a chemical proposed for use as a food ingredient should be an essential element in that chemical's toxicological profile.(17) Current scientific technology provides ample means of effectively assessing neurotoxic potential of chemical substances(18). To effectively minimize the risk of potential neurotoxicity in humans, it is important that the best available science be used to develop the necessary information. It should be clear that neurotoxic effects identified in experimental animal models may not always compare exactly with what may occur in humans. Nonetheless, these effects are still interpreted as being indicative of treatment related effects on the nervous system and predictive of possible adverse health effects in humans. As advances in the neurosciences continue to evolve, our understanding of the processes underlying neurotoxicity will become increasingly clear. This will enhance our ability to assess neurotoxicity in a manner that is more predictive of potential human risk and to apply the available neurotoxicological information more reliably in support of regulatory decisions.(19)
III. Evaluating Neurotoxicity
The reliability of assessing the full spectrum of neurotoxic potential for a test substance is directly related to the extent to which the detection and evaluation of neurotoxicity is explicitly included as a specific, defined objective of routine toxicity testing.(20) Consistent with the basic strategy advocated by the FDA for toxicological testing and with the recommendations by expert committees, scientific panels and health-related organizations, the assessment of neurotoxic potential is most efficiently carried out through a structured process of tiered testing.(21) Each stage of testing would focus on different aspects of assessment. In the first stage of testing chemicals would be initially screened across a range of dose levels for any clinical or pathological signs of toxicity, including those involving the nervous system. Those chemicals showing evidence of adversely affecting the nervous system may be presumptively identified as candidates for subsequent specific neurotoxicity testing to confirm and further characterize the scope of nervous system involvement (i.e., characterization of effects) and to determine dose-response kinetics (i.e., dose-response determination), including a quantitative determination of the no-observed-adverse-effect level (NOAEL).
A tiered approach to neurotoxicity testing and evaluation allows for multiple decision points at which scientifically based decisions can be made about the adequacy of available information and the need for additional testing. To facilitate such decisions, specific summary statements regarding the neurotoxic potential of the test compound should be included in the evaluation of the results of each level of testing. Since the nervous system interacts dynamically with certain other organ systems in the body, adverse effects to the nervous system should be evaluated within the context of a comprehensive assessment of all significant toxic effects for a test compound. In this regard, the neurotoxicity summary statements should reflect an integrated assessment of all relevant toxicology data which are available. This would include information derived not only from tests specifically focused on the detection of nervous system toxicity (e.g., neuropathology, behavioral dysfunctions, neurochemical alterations or physiological changes), but also from more conventional toxicological testing that focuses on other measures of toxicity, for example, general organ pathology and adverse changes in growth, development, food or water intake, or endocrine status.
The neurobiological implications of some conventional endpoints of toxicity are certainly more evident than others. For example, a compound that induces specific teratogenicity of the nervous system, even at high dose levels, would be suspect for adversely affecting the development of nervous system function at lower doses. The neurotoxicological significance of other types of toxicity, however, may be less obvious. For instance, chemicals found to alter hormonal balance might also be suspected of affecting the structural or functional integrity of the nervous system, since endocrine status and the nervous system are interrelated. Altered growth, which is considered an index of general toxicity, may also signal the presence of neurotoxicity. In the developing organism, abnormal growth may reflect a treatment related neurotoxicity of the mother involving poor care of the nursing offspring. In the adult, altered growth stemming from changes in food or water intake may reflect underlying nervous system dysfunction, since both eating and drinking are consummatory behaviors with neuromuscular and physiological components under neuronal control. It should be clear, however, that such generic toxicological endpoints, by themselves, are not to be taken as evidence of neurotoxicity. Rather, when viewed in conjunction with other available data, such effects may serve to indicate the possibility of treatment related effects on the nervous system. Again, it is important to emphasize the need for integrated interpretation of all available toxicological data in the process of assessing neurotoxic potential.
The first stage in assessing neurotoxicity involves a process of screening to identify those chemicals that exhibit any potential for adversely affecting the nervous system. It should be clear that the primary objective of screening is detection. Chemicals identified as exhibiting a significant potential for neurotoxicity would typically be considered as a possible candidate for additional more specific neurotoxicity testing. Under such conditions, the nature and extent of information which is typically developed by screening methods would not provide a sufficient basis for determining the NOAEL for neurotoxicity. Rather, additional more specific neurotoxicity information developed in subsequent stages of testing would be needed to accurately determine the NOAEL. If significant neurotoxic potential is not identified in screening, then there would typically be neither a basis nor a need to define a NOAEL for neurotoxicity.
There are basically three sources of neurotoxicity screening information. One involves the use of structure activity relationships (SAR), the second relies on published literature and other sources of documentation, and the third involves empirical testing. The usefulness and reliability of SAR for identifying potential neurotoxicants is, at the present time, rather limited due to the fact that SAR databases for neurotoxicity are still being developed. The use of published literature or other types of documented information, to the extent that this type of information is available and appropriate for regulatory application, can be of significant value in identifying chemicals that may affect the nervous system. However, this type of information is usually scattered and typically not available for many food ingredients. At the present time, the primary means of obtaining neurotoxicity screening data is through empirical testing. The experimental data needed to screen chemicals for potential neurotoxicity should be routinely obtained as part of those toxicity studies recommended for "entrance-level" testing of proposed food ingredients. Neurotoxicity screening information could be developed most appropriately in short-term (e.g., 14 to 28-day rodent and non-rodent) studies to screen adult animals exposed to the test chemical across a range of relatively high doses for brief periods of time, in subchronic (e.g., 90-day rodent and non-rodent) and long-term (e.g., one-year non-rodent) studies to screen adult animals following more prolonged exposure across a range of relatively lower doses, and reproduction/developmental studies to screen for potential developmental neurotoxicity in perinatally exposed offspring. The development of neurotoxicity screening information in other types of toxicity studies (e.g., chronic studies) would certainly be acceptable and encouraged.
Screening for neurotoxicity involves the use of valid, cost-effective procedures which can be carried out rapidly and routinely on large numbers of chemicals to detect the presence or absence of immediate or delayed adverse effects on the nervous system.(22) Neurotoxicity can appear as a wide range of morphological and functional abnormalities involving the nervous system at very specific or multiple levels of its organization.(23) Under the previous guidelines for toxicity testing of proposed food ingredients the identification of neurotoxic effects was based on information derived from a general pathological evaluation of a few sections of neuronal tissue and an unstructured casual observation of test animals in their cages for overt signs of toxicity.(24) This approach focused detection on the more severe forms of neurotoxicity. To maximize the scope of detection, screening should be sufficiently comprehensive to enable the detection of a representative variety of pathological changes and functional disorders of the peripheral, central and autonomic segments of the nervous system.(25) In reproduction/developmental studies, age-appropriate neurotoxicity screening should enable the detection of treatment-related effects on the physical and functional development of the offspring.
1. Elements of a Neurotoxicity Screen
The elements of a basic neurotoxicity screen should include a specific histopathological examination, in conjunction with a systematic clinical evaluation.
Specific histopathological examination
A specific histopathological examination should be made of tissue samples representative of all major areas and elements of the brain, spinal cord and peripheral nervous system. Emphasis should be placed more on the carefulness of the histopathological examination of the neuronal tissue and the documentation of findings rather than on the numbers of sections used, provided that all major areas and elements of the nervous system are included. For purposes of screening, either immersion fixation or in situ perfusion of tissues is acceptable. Typically, the initial examination may be carried out on tissues from the control and the highest dose group. Positive findings would then be followed by examination of tissues from the other dose groups. The concept of age-appropriateness should also be considered in the morphological evaluation of the immature nervous system.(26)
Systematic clinical evaluation
A systematic clinical evaluation of experimental animals should be conducted inside and outside of their cages using a clearly defined battery of clinical tests and observations selected to detect signs of significant neurological disorders, behavioral abnormalities, physiological dysfunctions, and any other signs of nervous system toxicity. Typically, in addition to the animal's physical appearance, body posture and weight, the clinical screen should provide sufficient information to assess the incidence and severity of such endpoints as seizure, tremor, paralysis or other signs of neurological disorder; the level of motor activity and alertness; the animals' reactivity to handling or other stimuli; motor coordination and strength; gait; sensorimotor response to primary sensory stimuli; excessive lacrimation or salivation; piloerection; diarrhea; polyuria; ptosis; abnormal consummatory behavior; and any other signs of abnormal behavior or nervous system toxicity. To accommodate age-appropriate testing, screening for potential developmental neurotoxicity could include measures of postnatal development of representative physical landmarks (for example, body weight and development of external genitalia) and functional milestones (for example, righting reflex, startle response, and motor development) in the experimental offspring. In carrying out the functional evaluation screen, animals should be initially observed in their home cages and then removed to an open arena for the completion of the observations and manipulative testing. As appropriate, more sensitive and objective indices of neurotoxicity, such as tests of learning and memory, and quantitative measures of sensory function and motor behavior, could be included as part of the screen.(27) Further, it is important that the neurotoxicity screening information be supplemented with any other relevant toxicological findings.
2. Considerations in Protocol Design for Neurotoxicity Screening
There are a number of available publications to guide in the design and conduct of neurotoxicity screens appropriate for the adult organism(28) and for the developing and adult offspring.(29) The process of protocol design for deriving neurotoxicity screening information should include consideration of the following:
- Each testing laboratory should develop and maintain an historical database demonstrating its continuing competence in the assessment of neurotoxicity. The neurotoxicity screen should consist of valid test methodology administered by personnel who, in compliance with GLP requirements, are adequately trained to conduct the procedures appropriately. The reliability and sensitivity of the proposed screening to be used for detecting neurotoxic effects should be documented by the availability of historical or concurrent positive control data.
To help ensure the complete and consistent application of the neurotoxicity screen throughout a particular study, each study protocol should include a detailed description of the particular screen to be used in that study, including its composition, the test procedures to be followed, the time periods at which the screen is to be carried out, the neuronal structures to be examined, the endpoints to be used, and the methods for recording and analyzing the data. During the conduct of the studies, the detailed clinical evaluation should be carried out systematically, using a prepared checklist of tests and observations when appropriate. All experimental procedures should be documented.
- Since neurotoxicity screening is intended to be a routine part of both general and reproductive toxicity studies, the specific composition of the screen and the endpoints to be recorded should be consistent with the particular focus of the study and, specifically, be appropriate for the age (and species) of the animals to be tested. For example, to screen for potential developmental neurotoxicity, it would be appropriate for a systematic evaluation to be carried out on representative male and female offspring from each experimental litter in reproduction studies and to include measures of the ontogenetic development and maturation of representative physical landmarks (for example, body weight and development of external genitalia) and functional milestones (for example, righting reflex, startle response, and motor development) in those offspring. The evaluation of offspring during the preweaning period should be planned so as to maintain the integrity of the primary reproductive study, for example by minimizing the period of pup separation from the dams. The optional inclusion of other, more sensitive, or more objective indices of neurotoxicity, such as tests of learning/memory and quantitative measures of sensory and motor function, to supplement the basic screening of the developing and/or mature offspring would be encouraged in separate or satellite litters. The concept of age-appropriateness should also be considered in the morphological evaluation of the immature nervous system.(30) There are a number of available publications to guide the design and conduct of clinical testing appropriate for neurotoxicity screening of the adult organism(31) and for developing and adult offspring.(32)
- Testing should be carried out at representative intervals throughout the duration of the study (including, when feasible, a pretreatment baseline) to provide information about the consistency of the neurotoxic effect(s), and, as possible, about their onset, duration and reversibility.
- At the discretion of the sponsor or testing laboratory, satellite groups of animals could be used to carry out the neurotoxicity screen testing.
- A sufficient number of male and female animals from each experimental and control group should be used (as recommended in the guidelines for the primary toxicity protocols) to ensure valid statistical analyses giving consideration to the variability of the endpoints being measured. As possible, the selection of tests should afford the best level of detectability with use of the smallest number of animals. In adult studies the individual animal is routinely used as the statistical unit, whereas in developmental studies the litter is typically considered to be the appropriate statistical unit. For screening purposes, the initial histochemical examination could involve tissues from control and high dose animals. If treatment-related effects are found, the subsequent examination of tissues from the lower dose groups would be warranted.
- The experimental design should include measures to minimize inadvertent bias, for example by using random assignment to treatment groups and, as feasible, carrying out testing with the experimenters blind to treatment conditions. Appropriate procedures should be followed to control for potentially confounding variables, such as housing conditions, diet and nutritional status, circadian cycles, test to test interactions, environmental conditions, and handling. For example, in the process of screening for potential developmental neurotoxicity the direct clinical evaluation of the pregnant or lactating dams should be limited to minimize influence of such handling on maternal behavior.
- To take full advantage of the neurotoxicity screening information routinely developed in toxicological testing, experimental data should be accurately recorded, documented and reported to the FDA. Summary tables of all positive effects should be presented. In addition, all data collected (positive and negative) should be submitted to the FDA to enable review personnel the opportunity of examining the actual study results. As appropriate, data should be analyzed using suitable and acceptable statistical procedures. This information, together with any other pertinent toxicity data, should be incorporated into an integrated assessment of the potential for the test chemical to adversely affect the structural or functional integrity of the nervous system. Based on this assessment, an explicit statement should be made as to whether or not the test chemical represents a potential neurotoxic hazard which may require special neurotoxicity testing. Study protocols for additional neurotoxicity testing should be developed using valid state-of-the-art methodology.
- Throughout the process of protocol design and testing in the assessment of neurotoxic potential, the opportunity for consultation with FDA is available and encouraged.
Increasing attention is being devoted to the development of in vitro systems for assessing the neurotoxicological impact of chemical agents.(33) In vitro methods would have practical advantages, such as minimizing the use of live animals, but validation studies remain to be done to correlate in vitro results with neurotoxicological responses in whole animals. Such systems, once appropriately validated, may have particularly useful application in screening for potential neurotoxicity and in helping to elucidate mode of action or mechanistic information.
The information collected during screening is used to determine whether or not the test chemical represents a potential neurotoxic hazard and whether additional tests to confirm and characterize the neurotoxicity, to define NOAELs, and to develop other necessary information should be recommended. A number of considerations enter into the scientific interpretation of the neurotoxicity screening information when making this evaluation. These include the adequacy and completeness of the screening assessment; the nature and severity of the effects detected; consistency of effects across dose; consistency of effects across testing intervals within a study; replicability of effects across different types of toxicity studies; presence of other toxic effects; and the margin of difference between the doses producing neurotoxicity and those producing other toxic effects. The extent to which screening provides the information to address these issues adds to the level of confidence in identifying a potential neurotoxic hazard and aids in determining the need to proceed from screening to the development of more comprehensive neurotoxicity information. The decision to proceed with such specialized neurotoxicity testing should be made in consultation with the FDA.
B. Special Neurotoxicity Testing
When a chemical is presumptively identified by SAR, empirical screening, or other sources of information as producing neurotoxicity, that chemical becomes a candidate for additional neurotoxicity testing. Chemicals not identified as having neurotoxic effects during screening will generally not be recommended for subsequent neurotoxicity testing, although exceptions may occur. Special neurotoxicity testing focuses on the characterization of the neurotoxic effects and the determination of dose-response relationships:
1. Characterization of Effects
Following the presumptive identification of chemicals that adversely affect the nervous system, the next level of testing focuses attention on determining the nature and extent to which the nervous system is affected by that chemical (characterization). At this level the neurotoxic effects found during screening are further characterized and studies are conducted to determine whether the test chemical has any other, possibly more subtle, effects on the structural and functional integrity of the nervous system in mature and developing organisms. The in-depth assessment of neurotoxicity at this stage of testing should include information about the nature and severity of effects, the temporal pattern of onset of effects (particularly when delayed neurotoxicity occurs), and the duration of effects. To enhance detection of subtle neuropathological findings, tissues should be perfusion-fixed in situ and a detailed histopathological examination (more thorough than the histopathology examination performed during screening) should be carried out involving the use of special stains to highlight relevant neural structures.(34)
The neurofunctional assessment at this level should routinely include a core battery of behavioral and physiological tests designed to detect adverse changes to the primary subfunctions (e.g., cognitive, sensory, motor, and autonomic) of the nervous system in the mature and developing nervous system.(35) The need for additional special tests may logically follow from information obtained during screening; for example, if a chemical is observed to induce convulsions during screening, the seizure potential and pro-convulsant properties of that chemical should be more specifically characterized during the second level of testing.
2. Dose-Response Relationships
A critical element used in defining a chemical's neurotoxic hazard is the no-observed-adverse-effect level (NOAEL), typically using the most relevant and sensitive endpoint identified in previous testing. To enable a more quantitative determination of the NOAEL, ample data should be obtained to thoroughly characterize the dose-response and dose-time relationships in repeated exposure studies, e.g., intermittent and continuous exposure regimes, typically using the most relevant and sensitive endpoint.
The protocols for special neurotoxicity testing, which should be designed in consultation with FDA, should take into consideration elements similar to those involved in the development of protocols for neurotoxicity screening, including the appropriateness and reliability of the test procedures, the suitability of the control measures, and the adequacy of the experimental design and schedule of testing (frequency and duration). Consistent with the guidelines for the primary toxicity testing protocols, special neurotoxicity testing would initially be carried out using rodents as the principal species of choice. However, as appropriate and in consultation with FDA, neurotoxicity studies using non-rodent species may be recommended, on a case-by-case basis, to develop information needed for more reliable cross-species extrapolation of data.(36)
At the stage of special neurotoxicity testing, efforts to develop additional relevant information for a more comprehensive assessment of neurotoxic hazard are certainly encouraged. For example, information regarding the occurrence of treatment-related neurochemical changes, the pharmacokinetic properties of the test compound, or the factors that may modulate the sensitivity of the organism to the test compound could contribute to a better understanding of the neurobiological processes underlying the chemically induced neurotoxicity. This mechanistic type of information would enable a more reliable interpretation of the available animal data for predicting neurotoxic risk in humans.
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