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Methods and Issues in Evaluating the Neuroloxic Effects of Organic SoTvents Beverly M. Kulig The term "organic solvents" refers to a chemically heterogeneous class of compounds and mixtures which are typically liquid between 0 and 250 C and are used industrially to extract, dissolve, or suspend materials not soluble in water. Solvents are derived from many chemical classes and are present in products such as paints, glues, adhesives, coatings, and degreasing agents. Solvents are also used to manufac- ture a wide variety of products including polymers, dyes, plastics, textiles, printing inks, agricultural products, and pharmaceuticals (World Health Organization, 1985~. Because of the widespread application of these compounds in in- dustrial products and processes, the volume of organic solvents pro- duced and the number of workers exposed are considerable. In the United States, for example, some 49 million tons is produced on an annual basis, and the National Institute for Occupational Safety and Health (NIOSH) estimates that approximately 9.8 million American workers are potentially exposed (NIOSH, 1987~. Understandably, much of the concern regarding the effects of organic solvents focuses on the occupational setting where exposures can be relatively high. As a result, recommended occupational exposure limits for many of these compounds have been proposed (American Conference of Gov- ernmental Industrial Hygienists, 1986; NIOSH, 1987~. Despite the emphasis on the occupational setting, however, solvent exposure is not limited to the workplace. Organic solvents are ubiquitous in the 159

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160 BEVERLY M. KULIG environment and are detectable in air, drinking water, and foodstuffs, including human milk (Packard, 1985~. Considerable controversy exists regarding the extent to which or- ganic solvents are toxic to the nervous system and at what exposure levels (Cranmer and Goldberg, 1986; Grasso et al., 1984~. For several compounds (e.g., n-hexane, methyl n-butyl ketone, and carbon disul- fide), a sufficiently large data base has been established whereby the consequences of long-term overexposure can be evaluated (Spencer, 1985~. For the vast majority of organic solvents, however, very few data regarding nervous system effects are available. Until recently, the majority of animal studies examining the neuro- toxicity of organic solvents concentrated either on the involvement of the nervous system in the acute lethality of these compounds or on the morphological changes that may result from chronic overexposure. Of growing concern, however, is the possibility that long-term solvent exposure is accompanied by subclinical changes which reflect a reduction in the adaptive capabilities of the nervous system. Occupational studies, for example, have suggested that long-term exposure may be associ- ated with a number of neurobehavioral changes including psychomotor slowing, attention and memory impairments, and changes in affective behavior (Baker and Fine, 1986~. Although occupational studies are an important source of information regarding the possible long-term effects of overexposure, results from such studies are often difficult to interpret. By applying animal neurobehavioral methods to the study of solvents, the possibility of addressing many of the questions regarding solvent neurotoxicity is becoming increasingly feasible. The present chapter considers some of the interpretative issues that have developed regarding the risks to human health associated with solvent exposure and examines the possible use of animal behavioral methods in this area. ACUTE SOLVENT EFFECTS The central nervous system (CNS) is a primary target organ for the acute toxic effects of organic solvents. The acute toxicity of inhaled solvent vapors is characterized, both in animals and in humans, by reversible signs of CNS depression. At moderate levels of overexposure, human subjects often complain about nausea, incoordination, and feelings of intoxication. At sufficiently high exposure levels, uncon- sciousness and death by respiratory arrest can occur (NIOSH, 1987~. A number of factors contribute to the susceptibility of the CNS to the acute effects of organic solvents. First, during the initial stages of absor Cation of an inhaled dose of solvent vapors, distribution to different

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 161 body organs is related to regional blood flow, and as a result, entry into the brain proceeds rapidly (Baker and Rickert, 1981~. Further, because of their lipophilic nature, organic solvents accumulate in tissues with a high lipid content, thus making brain and nerve potential depots for these compounds and, in some cases, their toxic metabo- lites (Bus et al., 1981~. One experimental approach to determining behaviorally effective levels is to expose human volunteers to controlled atmospheres of solvent vapors and to examine changes in psychometric measures designed to assess different aspects of motor, sensory, and cognitive performance (Dick and Johnson, 1986~. In general, the results of experimental exposure studies have demonstrated that one of the most consistent effects of inhaled organic solvents in humans is a slowing in behavioral performance evidenced by increased response latencies in simple and complex reaction time tasks (Gamberale, 1985~. However, the results of such studies also seem to indicate that the level at which behavioral effects occur tends to be on the higher end of the scale for human occupational exposures. As pointed out by Dick and Johnson (1986), there are a number of difficulties in conducting human experimental exposure studies. In addition to the narrow dose-response range that can ethically be examined, the duration of exposure which is acceptable to human volunteer subjects is often less than that encountered in the occupational situation. Thus, human exposure studies rarely employ an 8-hour exposure regimen even at Threshold Limit Values (TLVs) and, in most instances, are restricted to 2-4 hours of actual exposure. This may explain, in part, the mod- est effects seen in human exposure studies and the limited dose- response data obtained. Application of Animal Behavioral Methods Acute Effects of Single Exposures Animal studies using operant techniques are being employed increasingly to examine the effects of inhaled organic solvent vapors on behavioral performance, and guidelines for the use of schedule- controlled operant techniques for neurotoxicity evaluation have recently been proposed by the U.S. Environmental Protection Agency (USEPA, 1985~. Because the aim of many of the studies employing operant techniques has been to demonstrate the usefulness of these methods in neurotoxicity screening, experimental protocols involving relatively high-level, short-duration exposure schedules have typically been employed (e.g., Glowa and Dews, 1983; Moser et al., 1985~. Although

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162 BEVERLY M. KULIG such studies can provide highly reliable, quantitative information on which to judge the relative potency of different compounds to affect behavior in that animal test system, they also give the impression that very high concentrations of inhaled solvents are necessary to affect learned behavior in rodents. Perhaps as a result, very few animal studies have examined occupationally relevant concentrations and exposure durations for the purposes of risk assessment. Although it is unclear at present just how sensitive measures of learned behavior are to the acute effects of inhaled solvents, there is some evidence that animal studies using low-level exposure may be a worthwhile approach for estimating concentrations at which behavioral effects can be expected to begin to occur in humans. Kishi and his coworkers (1988), for example, have recently demonstrated that inhalational exposure to toluene at 125 ppm for 4 hours produced significant effects on signaled avoidance both during the initial stages of exposure and for several hours following the end of exposure. Results from acute exposure studies conducted in our laboratory using positive reinforcement also indicate measurable effects of solvents at occupationally relevant levels. In a recent study, for example, the effects of low aromatic white spirits were examined in rats working on a two-choice visual discrimination task for water reward. Rats were first trained in operant chambers equipped with two levers, two light panels located above each lever, and a pump for delivering water reward in daily sessions consisting of 100 trials. The rat's task was to depress the lever under the illuminated panel in order to obtain a drop of water. Following stabilization of performance, rats were randomly assigned to one of four groups and exposed by inha- lation to low aromatic white spirits at O (controls), 1,200 mg/m (~200 ppm), 2,400 mg/m3 (~400 ppm), and 4,800 mg/m3 (~800 ppm) for 8 hours and tested immediately following the termination of exposure. As the left panel in Figure 1 demonstrates, exposure to white spir- its at these concentrations produced no observable effects on dis- crimination accuracy. Speed of responding (right panel), however, was affected at all concentrations tested, with exposure to 1,200 ma/ m3, 2,400 mg/m3, and 4,800 mg/m3 producing a mean increase in trial response latency of 47, 96, and 156 percent, respectively. The effects on two-choice response speed did not appear to be the result of changes in motivation for water reward: All groups consumed the same number of reinforcements and the latency to obtain reinforce- ment following a correct trial response was similar for all groups. Although only several human experimental exposure studies have been conducted with white spirits and the length of exposure in the human studies was considerably less than 8 hours, the data that are

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 100 90 80 Q - in A o CL in 40 G o 70 60 30 20 10 o _ 6 ~ 41~ GOD All ~~ Amp mg/m3 163 GOD ` mg/m3 FIGURE 1 Effects of a single 8-hour exposure to white spirits on accuracy (left panel) and response speed (right panel) of rats performing on a two-choice visual discrimina- tion task. available indicate relatively good agreement between the levels of exposure producing changes in learned performance in both types of studies. For example, in studies conducted with young healthy male volunteers, Gamberale and his coworkers (1975) reported that exposure to white spirits at 4,000 mg/m3 for 50 minutes produced a small (i.e., 10 ms) but significant increase in response latencies measured in a simple reaction time task. Further, in a study employing a total exposure duration of 7 hours and concentration levels of 34, 100, 200, and 400 ppm, significant effects on response speed measures were found at 100 ppm and higher, with effects on other cognitive and motor tasks appearing at higher concentrations (Cohr et al., 1980~. Thus, although the number of studies aimed at examining the acute behavioral effects of low-level solvent exposure is limited, the data available suggest that the rat may prove to be a more useful model for estimating human observable effect levels than might be expected on the basis of the results obtained in high-level, short-duration ex- posure studies.

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164 BEVERLY M. KULIG Acute Effects in the Context of Chronic Exposures Except for instances of accidental poisoning in which lethal sol- vent concentrations are reached, all signs of CNS depression, even those resulting from a single high-level intoxication, appear to be readily reversible and no evidence exists to indicate that acute sol- vent exposure is accompanied by neuropathological or persistent neurofunctional sequelae. As a result, acute solvent effects are usually mentioned only in passing in discussions of solvent neurotoxicity (Baker and Fine, 1986; Grasso et al., 1984; Spencer, 1985~. Different lines of evidence from the human literature, however, indicate that a need exists for more careful consideration of acute solvent effects particularly in the context of chronic exposure. First, there is a considerable amount of evidence from the occupa- tional literature demonstrating deficits in behavioral functioning in exposed workers. Discussions of these effects often imply that because neurobehavioral effects were measured in persons exposed on a chronic basis, the effects themselves are chronic in nature. Behavioral testing in occupational studies, however, is often conducted in workers who have just left an acute exposure situation (i.e., the worksite) or in workers tested within a day or two following an exposure period (e.g., the work week). Thus, it is not unlikely that acute effects could contribute to the changes in behavior often reported in the human literature. Further, there are indications from the human literature that sensitivity to the acute effects of solvents can change in a repeated exposure situation and that such changes may be important in monitoring the potential hazards of these compounds. On one hand, there are both anecdotal and experimental reports indicating that tolerance develops to the effects of organic solvents, i.e., that acute effects are attenuated with repeated exposures (Gotell et al., 1972~. On the other hand, patients with suspected solvent-induced toxic encephalopathy often complain of an increased sensitivity to the acute effects of solvents. In such patients, signs of acquired intolerance are usually characterized by dizziness and nausea when they are exposed to even very low concentrations, despite the fact that they had been occupationally exposed for years to higher concentrations without subjective symp- toms (Gyntelberg et al., 1986~. From the vast literature on the development of tolerance to ethyl alcohol and other CNS depressants, a number of behavioral paradigms are available for examining tolerance development to industrial solvents as well. Himnan (1984), for example, demonstrated rapid development of tolerance to the effects of repeated short-duration, high-level toluene

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 90 80 v 70 o CO CO z g In fir o a: z 60 50 40 30 20 10 _ O PreWk Day 1 Day 2 \\ ~ - ~, . r Exposure ~ Controls - sty 100 ppm O sty 340 ppm ~ - sty 1,22S ppm 165 Day 3 Post Post Wk FIGURE 2 Effects of repeated inhalational exposure to styrene on the number of short- latency two-choice responses of rats performing on a visual discrimination task. exposures on measures of ataxia and rearing. However, the develop- ment of tolerance appeared to depend on the behavioral functions examined, with increased activity and head shakes showing a slowly developing reverse tolerance. In studies investigating the effects of styrene on learned discrimination performance, we have also found a rapidly developing tolerance to the effects of styrene on speed and accuracy measures of discrimina- tion performance during the first week of exposure. As Figure 2 shows, rats exposed to 100, 350, and 1,225 ppm of styrene for 18 hours a day and tested on a visual discrimination task (described above) all showed a reduction on Day 1 in the number of two-choice responses made within 2 s of trial onset. However, as exposure continued, the effects of styrene on short-latency responding, particularly in the highest concentration group, showed a marked attenuation. Further, results from a chronic exposure study (Kulig, 1988) indicated that the rapidly developing tolerance to styrene seen in the first week of exposure persisted throughout chronic exposure. In contrast, results from studies examining the effects of trichloro- ethylene (TCE) on discrimination indicated a quite different profile

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66 100 80 o In Z 2 V) m IL 40 o G m 20 at o BEVERLY M. KULIG A:, _~ Ok 1 1 1 ~ ~ iL~:,~? Exposure ~ I i Controls TCE 500 TCE 1 ,000 TCE 1,500 ppm 1 1 ~ I I 1 1 1 0 3 6 9 12 15 18 21 24 WEEK OF EXPERIMENT FIGURE 3 Changes In the acute effects of trichloroethylene (TCE) on the number of short-latency two-choice responses of rats during and following 18 weeks of exposure. Redrawn with permission from Kulig, 1987, Pergamon Press PLC. in the time course of effects during chronic exposure. Groups of rats were exposed to TCE by inhalation at 0, 500, 1,000 and 1,500 ppm for 16 hours a day, 5 days per week, for 18 weeks (Kulig, 1987~. Similar to styrene, exposure to TCE led to a within-week development of tolerance to the effects of TCE on response speed of discrimination performance but only during initial stages of exposure. As exposure became chronic, within-week tolerance was lost, with the result that the acute effects of TCE on response speed became more pronounced as exposure continued (Figure 3~. Despite the very marked disturbances seen in the behavior of rats chronically exposed to TCE, the effects were apparently acute in nature because no evidence for a carryover of effects into the postexposure period could be demonstrated. Taken together, the results of our studies as well as those of other investigators indicate that different profiles of tolerance and reverse tolerance develop for different behavioral effects and for different organic solvents. Given the indications from the human literature that similar phenomena also occur in exposed workers, and the problems associated with the interpretation of the nature of the deficits seen in occupational behavioral studies, it appears that animal studies examining

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 167 acute effects in the context of the chronic exposure situation might provide a worthwhile approach to resolving some of these issues. Reinforcing Properties of Organic Solvents Unlike heavy metals and pesticides, organic solvents possess a be- havioral property that is not seen with other industrially used chemicals, namely, the potential for abuse. Although it may be argued that possible self-exposure to organic solvents for their euphoric effects has little to do with evaluating the neurotoxicity of these compounds, the reinforcing effects of these compounds and the possible cross- tolerance with recreational drugs such as ethyl alcohol are important considerations in evaluating the potential risk to human health posed by these chemicals both in exposed workers and in the general population. Similar to the effects of ethyl alcohol, acute high-level exposure to solvents can produce feelings of euphoria, and intentional inhalational solvent abuse has become a serious health problem, particularly among children and adolescents Johnston et al., 1984~. The most commonly abused products include glues, paints and paint thinners, gasoline, lighter refills, and cleaning fluids (King, 1982~. Although toluene-containing products appear to be particularly popular, the chemical diversity of the different compounds which are abused suggests that the potential for abuse is not limited to a single compound. In addition to the social, psychological, and economic consequences of any addiction, the long-term abuse of solvent-containing products has been associated with a number of neurotoxic effects, including psychosis, hallucinations, sensory and motor disturbances, and convulsions. Although the acute encephalopathy produced by solvent inhalant abuse appears reversible in most cases, reports indicate that in some patients severe CNS effects may persist indefinitely (Boor and Hurtig, 1977; Grabsky, 1961; King, 1982; Knox and Nelson, 1966; Satran and Dodson, 1963; Weisenberger, 1977~. Moreover, although children appear to be the high-risk group in the general population, solvent abuse can occur in the occupational setting as well, and many of the case studies reporting persistent CNS effects have involved adults whose initial experience with the euphoric effects of organic solvents occurred in the occupational setting. ~ . .. . . , . ~ . . ~ . Systematic animal studies of the relative a ruse potential of differ- ent organic solvents have not yet been conducted; however, animal models for examining the stimulus properties of these chemicals have been described. Nonhuman primates, for example, will self-administer inhaled vapors (Wood, 1979~. Further, in studies using rodents, drug discrimination procedures have been used to evaluate the stimulus

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168 BEVERLY M. KULIG properties associated with acute intoxication (Overtop, 1984~. Rees et al. (1987) have shown the similarity in stimulus properties of toluene and barbiturates. If similar results are found with other solvents that are abused by humans, it may be possible to develop a systematic framework for evaluating the abuse potential of industrial and com- mercial compounds. The issue of abuse is so different from the usual concerns of toxi- cology, neuropathology, and occupational medicine that one may question whether this unique behavioral property of solvents belongs in the toxicological picture of risk assessment. When one considers, how- ever, that compounds producing acute euphoric effects may lead to repeated near-lethal (and sometimes lethal) exposure situations that result in irreversible brain damage, an evaluation of which solvents possess the potential for abuse would seem warranted. CHRONIC SOLVENT EFFECTS ON THE PERIPHERAL NERVOUS SYSTEM The Role of Animal Studies The effects of organic solvents on the peripheral nervous system provide some of the strongest evidence for the potential of these compounds to produce irreversible nervous system damage, and animal studies have played an important role in both helping to identify the causative agent in outbreaks of human disease (Allen, 1980) and elu- cidating the underlying mechanisms of action. In what has now be- come an almost classic example of neurotoxicological detective work, animal studies, initiated following an outbreak of peripheral neuropathy in a plastics coating plant in Columbus, Ohio in 1973, not only helped identify methyl n-butyl ketone (MnBK) as the causative agent, but also demonstrated the role of the solvent methyl ethyl ketone in causing the outbreak. Further, animal experimental studies helped clarify the role of the gamma-dike/one pathway in the neurotoxicity of MnBK, identified hexacarbons as a general class of potential neurotoxic agents, and stimulated research into the pathological processes involved in dying back neuropathies (see Spencer and Schaumberg, 1980~. In addition, human data together with animal experimental studies have identified solvents other than those involved in the gamma- diketone pathway as toxic to peripheral nerve. Carbon disulfide, for example, is a metabolic poison which produces a wide array of effects including psychosis and peripheral neuropathy in man and lesions in the brain and peripheral nerve in experimental animals (Wood, 1981~. Exposure to trichloroethylene (TCE), a compound used extensively

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 169 in degreasing operations, was originally thought to be responsible for causing cranial neuropathies. However, when TCE is exposed to light or heat, it breaks down easily into dichloroacetylene. Animal studies helped establish the neurotoxicity of dichloroacetylene, and it is this compound that is now generally recognized as the causative agent in producing TCE-induced trigeminal neuropathy (Spencer, 1985~. More recently, 2-tert-butylazo-2-hydroxy-5-methylhexane (BHMH), a solvent used in the manufacture of polyester-based plastics, was removed from the market when central and peripheral nervous sys- tem dysfunction developed in workers after several weeks of exposure (Horan et al., 1985~. Although premarket testing demonstrated the acute CNS toxicity of this compound in rodents, no chronic neurotoxicity studies were carried out. Following the outbreak of human disease at a manufacturing plant in Texas, animal studies were undertaken which showed that dermal exposure to BHMH produced functional signs of peripheral neuropathy within three weeks of exposure (Spencer et al., 1985~. In addition, neuropathological studies indicated that BHMH exposure produced axonal degeneration of the optic tracts, ascending and descending spinal tracts, and peripheral nerve (Spen- cer et al., 1985~. Although BHMH is a six-carbon straight-chain structure, its decomposition does not seem to involve the gamma-dike/one pathway (Horan et al., 1985) as with n-hexane or methyl n-butyl ketone. It is, however, a potent neurotoxic agent both in animals and in humans. What, of course, is particularly unfortunate in the case of BHMH is that the human disease resulting from exposure to this compound need not have occurred if adequate premarket animal testing had been conducted. Neurobehavioral Methods for Screening Sensory and Motor Effects Although morphological evidence of nervous system changes has historically been the accepted endpoint in determining neurotoxicity, there is increasing interest in quantitative functional measures that could be used in conjunction with neuropathological evaluations for screening purposes (Buckholtz and Panem, 1986~. In the United States, for example, the U.S. Environmental Protection Agency, under the Toxic Substances Control Act, has published guidelines for the use of behavioral methods in neurotoxicity testing (USEPA, 1985), and at an international level, the World Health Organization (WHO) is currently sponsoring various activities in the field of neurotoxicity (WHO, 1986~. Because of the well-documented potential of organic solvents and other industrial compounds to affect sensory and motor function in

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 4 - 2 Go 1 o 173 Controls CS2 7s CS2 22s CS2 700 ppm rA Exposure 0 6 12 18 24 30 36 42 WEEK OF EXPERIMENT FIGURE 7 Effects of carbon disulfide on the latency of the compound nerve action potental measured from the caudal nerve during and following 36 weeks of exposure. was also affected beginning in Week 9 of exposure (Figure 5) and was accompanied by deficits in coordinated movement (Figure 6~. Furthermore, the behavioral evidence for disturbed peripheral nerve function was supported by electrophysiological changes in peripheral nerve conduction velocity (Figure 7~. For all measures, CS2-induced changes persisted well beyond the end of the exposure period, indi- cating that these effects were chronic in nature. In order to evaluate possible structural changes in these animals, neuropathological ex- aminations were conducted by I. B. Cavanagh at the University of London at the termination of the study. Results indicated swollen axons and nerve fiber degeneration in the 700-ppm group in the sci- atic, tibial, and caudal nerves as well as in the spinocerebellar tracts and the superior colliculus. Taken together, these data demonstrate the ability of currently available neurobehavioral methods to quantify the progressive development of chemically induced changes in peripheral nerve function and to study the relationship between neurofunctional changes and the morphological changes. In addition to providing information regarding the time course of effects, the repeated testing of chronically exposed animals can also be used to operationally define appropriate time

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174 BEVERLY M. KULIG points for conducting morphological and chemical investigations. Further, the ability to quantitate progressive changes in nervous system function during a time in which no observable signs of dysfunction are evident is also an important consideration in the use of these methods for screening new compounds. Decisions regarding exposure schedules and the total duration of exposure, especially in inhalational studies, are often based on practical or rule-of-thumb considerations. How- ever, there is nothing inherent in a 90-day exposure study with a particular exposure schedule to ensure the frank expression of ob- servable signs of neurotoxicity or easy-to-identify light-microscopical changes. In such cases, small, but reliable, quantitative neurofunctional changes may be the only indication that closer examination of the compound using different exposure schedules and a longer duration of exposure is warranted. Despite the advantages of neurobehavioral testing, an examination of control baseline performance in the CS2 study demonstrates some of the considerations that must be taken into account in designing neurobehavioral methods for use in chronic exposure experiments. For example decreased levels of behavior such as that seen in tests of spontaneous activity resulting from repeated testing or perhaps combined with the effects of aging in nonexposed control animals can lead to a floor effect and diminish the usefulness of the test in the latter stages of prolonged studies. Conversely, difficulties with caudal nerve conduction time measurements are more likely to occur during the early stages of exposure. Caudal nerve conduction time improves with age until 150-300 days after birth, and remains relatively stable until an advanced age when it again shows signs of prolongation (Schmelzer and Low, 1987~. Because most chronic exposure studies begin when animals are young adults, the first months of exposure correspond to the time of greatest improvement in conduction velocity, making detection of compound-related effects during the early stages of exposure difficult to detect. The need to consider age-related changes is not unique to neurofunctional approaches to neurotoxicity evalua- tion because morphological and neurochemical changes can also be expected to occur. There is, however, a need in the further develop- ment of neurobehavioral methods to evaluate the long-term operating characteristics of any given test in order to better understand its strengths and limitations in the chronic exposure situation. CHRONIC TOXIC ENCEPHALOPATHY In addition to their effects on peripheral nerve, organic solvents have also been shown to produce irreversible effects on brain func-

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 175 lion. Examination, for example, of CS2-poisoned workers conducted during the first 40 years of this century when occupational exposures were apparently very high, demonstrated the potential of this compound to produce severe manic-depressive psychosis and other signs of CNS dysfunction (see Wood, 1981~. In later studies using psychological test instruments for quantifying the degree of psychological changes, Hanninen (1971) demonstrated the ability of these techniques to describe the range and pattern of cognitive and affective changes associated with exposure in workers with symptoms of CS2 poisoning. In addition, compared to nonexposed control subjects, exposed workers with no subjective symptoms or clinical signs of overexposure also showed changes in psychological test performance. The finding of psychological changes in the absence of overt signs and symptoms together with epidemiological studies indicating that occupational exposure to carbon disulfide in the viscose rayon industry was associated with higher rates of suicide (Mancuso and Locke, 1972), was particularly disturbing because it raised the possibility that organic solvents as a general class of industrial compounds could produce cognitive and affective changes of a significant nature which were virtually undetectable by clinical methods and unrecognizable by the exposed person himself as being associated with chemical exposure. As a consequence, an increasing number of studies were initiated to examine the psychological functioning of workers in different industries who were exposed to different types of organic solvents. In general, results from these studies repeatedly have shown a higher incidence of subjective complaints related to CNS effects in solvent-exposed workers, changes in objective measures of psychological functioning, and in some cases, a higher prevalence of EEG abnormalities and reduced peripheral conduction velocities (see Baker and Fine, 1986; WHO, 1985~. In a series of studies examining persons occupationally exposed to mixed solvents, for example, painters and other occupational groups have been identified as being at risk for developing irrevers- ible changes in brain function based, at least in part, on the results of behavioral evaluations (Arlien-Soborg et al., 1979; Bruhn et al., 1981~. As a result of the growing number of cross-sectional occupational studies demonstrating solvent-related neurofunctional changes and the number of case reports of chronic encephalopathy produced by solvent abuse, two workshops were convened to develop internationally acceptable diagnostic criteria applicable to solvent-induced CNS dis- ease (Cranmer and Goldberg, 1986; WHO, 1985~. Because toxic encephalopathy produced by nervous system poisons has been recognized as a clinical entity for many years, diagnostic criteria based on the DSM-III classification of mental disorders (American Psychiatric As-

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76 BEVERLY M. KULIG sociation, 1980) served as a basis for differentiating three different levels of psychological impairment produced by chronic neurotoxic overexposure. The mildest level of impairment, termed "organic af- fective syndrome," was defined as one in which chronic chemical exposure was accompanied by subjective complaints of fatigue, mild memory and concentration difficulties, and affective changes. The term used to describe the second level of impairment was "mild chronic toxic encephalopathy"; it includes both subjective neurotoxic symp- toms and sustained changes in personality or mood, as well as defi- cits in performance on formal neuropsychological testing. Finally, the third level of solvent-induced toxic encephalopathy refers to a severe neuropsychiatric condition characterized by global deterioration of intellectual and emotional functioning such as that described in the turn of the century literature for carbon disulfide poisoning or in present-day case studies of solvent abuse (Cranmer and Goldberg, 1986; NIOSH, 1987; WHO, 1985~. Problems in the Interpretation of Human Studies The ability of drugs and chemicals to produce toxic encephalopathy is widely recognized, and there is little disagreement regarding the potential of high-level exposure to lead, thallium, alcohol, or drugs to produce severe signs of central nervous system poisoning that may be irreversible or only slowly reversible. The potential of organic solvents to produce persistent changes in brain function, however, has become, for a number of reasons, an issue of considerable controversy. First, from the discussion above, it is obvious that acute effects can have important consequences for behavioral functioning. However, it is often difficult or impossible to design occupational studies in which the possibility of acute solvent effects contributing to changes in psychological performance has been ruled out. Given the fact that the test instruments sensitive to the effects of acute solvent exposures in the experimental exposure situation are often the same as those that are sensitive in detecting changes in psychological functioning in cross-sectional occupational studies (Gamberale, 1985), a differen- tiation of acute neurotoxicant effects from mild toxic encephalopathy based on the selection of the test instrument does not seem feasible. Moreover, occupational environments often contain many differ- ent organic solvents, and workers are not necessarily aware of the level and type of their present or previous occupational exposures. Thus, conclusive proof as to the identification of the causative agents producing psychological changes based on the results of these studies is often difficult to obtain.

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 177 Another issue in both the conduct and the interpretation of occu- pational behavioral studies is the selection of appropriate nonexposed control subjects who are suitably matched on the basis of age, sex, educational level, socioeconomic level, and other demographic vari- ables known to affect psychological test performance. When such variables are taken into account in the statistical analyses of group differences of exposed and nonexposed workers, some studies have indicated that originally large differences in psychological test results can become borderline or disappear altogether (Baker et al., 1988; Cherry et al., 1985~. Problems stemming from the use of inappropriate control data are illustrated by a recent report by Gade and his colleagues (Gade et al., 1988~. In this study, solvent-exposed workers were examined with clinical psychological tests and diagnosed as having solvent-induced chronic toxic encephalopathy. However, the original evaluation of these patients was apparently made without reference either to population norms for the determination of within-subject profiles of cognitive deficits or to estimates of premorbid levels of functioning, despite the fact that both types of information are necessary for a valid neuropsychological evaluation (Lezak, 1983~. When these patients were retested at a later date and compared with nonexposed persons of similar age and education, no differences in psychological test performances could be seen. As a result, the authors were forced to revise their earlier diagnoses of solvent-induced dementia and to conclude that the poor test performance of these patients was related not to solvent exposure, but to the lower level of intelligence and education in their subject sample. It is apparent from the discussion above that if appropriate con- trols are used, well-designed neurobehavioral studies can be used to evaluate acute neurotoxicant effects, to monitor the safety of workers exposed to known neurotoxic agents, and to identify possible occupational hazards. However, it is doubtful whether cross-sectional studies at the human level can or should be used as screening tools for the initial identification of compounds that possess CNS neurotoxic properties. In all fairness to the behavioral toxicologists and neuropsychologists working at the human level, they have received little help from their counterparts at the animal level in addressing the issues surrounding the possible adverse effects of long-term solvent exposures on cognitive functioning. In part, this may be due to the difficulty most psychologists working at the animal level have in evaluating and interpreting the sometimes diffuse effects reported in the human literature. However, it is more likely due to the lack of adequate test instruments for examining the effects of chemical exposures on learning, memory,

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178 BEVERLY M. KULIG and emotional functioning which can be applied to the chronic expo- sure situation. Animal Models of Cognitive Effects Although the study of learning and memory has occupied the in- terest of psychologists for many years, some of the paradigms developed to study memory function, such as one-trial passive avoidance learning, are obviously unsuitable for repeated evaluation of cognitive changes in chronic exposure studies. There are, however, techniques described in the literature which, with further study, may provide useful approaches to examining those behavioral processes that would seem to be most likely affected by exposure to centrally acting neurotoxic agents. Heise (1983), for example, has described several discrete-trial operant procedures involving delayed response and delayed comparison paradigms, which can be easily acquired by normal rats and can be used repeatedly to assess changes in memory. Recent studies employing radial arm maze techniques (Peele and Baron, 1988) also indicate that repeated acquisition paradigms may prove useful in assessing memory changes. Even with the further development of behavioral methods to address more fully the issue of possible cognitive changes accompanying solvent exposure, it still remains to be seen whether these methods can provide sufficiently stable control baselines such as those needed for long- term studies. Moreover, if agent-related effects can be measured on cognitive performance in the absence of clear-cut neuropathological changes in appropriate brain structures, it will be necessary to seek possible underlying mechanisms of action either at the neurochemical level or with morphological techniques more sensitive than those that are used routinely for neurotoxicity assessment. One approach that may prove fruitful is the combined study of neurobehavioral and neurochemical changes accompanying long-term solvent exposure. Investigators at the Karolinska Institute examining the effects of low-level (80 ppm) toluene exposure, for example, have recently reported reductions in catecholamine turnover rates in rat striatum and increased catecholamine levels in hypothalamus (Fuxe et al., 1982), as well as changes in central receptor binding properties (Fuxe et al., 1987) during subchronic exposure. Studies with styrene have also demonstrated an effect on catecholamine function (Husain et al., 1980), and a common mechanism at the neurochemical level has been proposed (Mutt) and Franchini, 1987) based on the ability of dopamine to condense nonenzymatically with solvent metabolites from different chemical groups. Whether such neurochemical effects are acute or chronic in nature and whether they can be directly related to measurable neurofunctional changes have not yet been studied. However,

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NEUROTOXIC EFFECTS OF ORGANIC SOLVENTS 179 efforts to examine the role of possible changes in transmitter function in producing central neurofunctional effects would appear to offer a promising approach. CONCLUSION There seems to be a growing acceptance in toxicology of animal neurofunctional methods for use in screening for neurotoxicity. The development of neurobehavioral methods for assessing motor and sensory function which has occurred in the last 10 years, the growing empirical data base demonstrating both the sensitivity and the appli- cability of these methods to chronic studies, and the increasing possi- bility of designing studies to examine neurofunctional changes along with the underlying neurochemical and neuromorphological changes that accompany them, will continue to provide evidence for the im- portance of neurobehavioral methods in the identification and further understanding of the actions of chemicals on the nervous system. There are, however, many industrially and commercially used or- ganic solvents already on the market about which little or no infor- mation regarding their potential effects on the nervous system is available (McMillan, 1987~. Apparently, even the setting of occupational expo- sure limits to avoid acute, intoxicating effects on the nervous system has eluded an experimental basis. Moreover, psychologists working at the human level have been virtually left on their own to identify neurotoxic agents in the workplace and to sort out, as best they can, the complex issues surrounding chronic human exposures. The sub- ject matter of many of these issues is not the domain of classical toxicology or neuropathology, it is uniquely behavioral in nature. Despite the fact that the potential for chemicals to alter memory, learning, and performance or to produce addiction may not be issues of primary concern in toxicity screening, they are nonetheless important considerations in evaluating the risks to human health associated with long-term chemical exposures. With a better understanding of the issues faced by investigators working at the human level and a greater collaboration with scientists working at the cellular and sub- cellular levels, behavioral toxicologists may be able to supply the necessary methods and conceptual framework to bridge the rather formidable gap that has evolved in neurotoxicity risk assessment. REFERENCES Allen, N. 1980. Identification of methyl n-butyl ketone as the causative agent. Pp. 834-845 in Experimental and Clinical Neurotoxicology, P. S. Spencer and H. H. Schaumberg, eds. Baltimore: Williams and Wilkins.

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