Lessons for Neurotoxicology fromSelected Model Compounds:SGOMSEC Joint ReportDeborah C. Rice,1 Ana Maria Evangelista de Duffard,2Ricardo Duffard,2 Anders Iregren,3 Hiroshi Satoh,4and Chiho Watanabe41Toxicology Research Division, Banting Research Centre, Tunney'sPasture Ottawa, Ontario, Canada; 2Laboratorio de ToxicologiaExperimental, Facultad de Ciencias Bioquimicas y Farmaceuticas,Universidad Nacional de Rosario, Suipacha Rosario, Santa Fe, Argentina;3Division of Psychophysiology, National Institute of Occupational Health,Solna, Sweden; 4Department of Environmental Health Sciences, TohokuUniversity School of Medicine, Sendai, Japan

The ability to identify potential neurotoxicants depends upon the characteristics of our testinstruments. The neurotoxic properties of lead, methylmercury, polychlorinated biphenyls, andorganic solvents would all have been detected at some dose level by tests in current use,provided that the doses were high enough and administered at an appropriate time such as

during gestation. The adequacy of animal studies, particularly rodent studies, to predict intakelevels at which human health can be protected is disappointing, however. It is unlikely that theuse of advanced behavioral methodology would alleviate the apparent lack of sensitivity of therodent model for many agents. Environ Health Perspect 1 04(Suppl 2):205-215 (1996)

Key words: lead, methylmercury, neurotoxic potential, organic solvents, polychlorinatedbiphenyls, screening protocols, tiered testing

This paper on model compounds addressesthe lessons that may be extracted from theintensive investigation over the last 15 yearsor so of selected agents (lead, methylmer-cury) or classes of agents (polychlorinatedbiphenyls [PCBs], solvents) identified as

This joint report was developed at the Workshop onRisk Assessment Methodology for NeurobehavioralToxicity convened by the Scientific Group onMethodologies for the Safety Evaluation of Chemicals(SGOMSEC) held 12-17 June 1994 in Rochester,New York. Manuscript received 1 February 1995;manuscript accepted 17 December 1995.

Address correspondence to Dr. Deborah C. Rice,Toxicology Research Division, Banting ResearchCentre 220201, Tunney's Pasture, Ottawa, OntarioKlA OL2, Canada. Telephone: (613) 957-0967. Fax:(613) 941-0659. E-mail: drice@bcadl.food.hwc.ca

Abbreviations used: PCBs, polychlorinatedbiphenyls; IQ, intelligence quotient; WISC, WechslerIntelligence Scale for Children; FOB, functional obser-vation battery; SCOB, schedule-controlled operantbehavior; U.S. EPA, U.S. Environmental ProtectionAgency; CAR, conditioned avoidance response; RfD,reference dose; NOAEL, no observed adverse effectlevel; LOAEL, lowest observed adverse effect level;GD, gestational day; DRH,

potent neurotoxicants by environmental orindustrial exposure of human populations.Such recognition in each case promptedextensive research in animal modelsto characterize effects and to predict poten-tial neurotoxic end points in humans.Epidemiological investigation focused oncharacterizing effects in the human popula-tion and identifying no-effect levels. Muchof the research on the effects of lead,methylmercury, and PCBs has focused onidentifying behavioral and other neurotoxiceffects produced as a result of developmen-tal exposure, while most of the research onsolvents has focused on exposure in adults.

There are a number of questions thatmay be asked regarding what we havelearned from these neurotoxic agents. Howwas neurotoxicity identified in the humanpopulation? How well are the effects char-acterized in humans, and how confident arewe of our estimates of the intake or bodyburden necessary to produce effects? Whatdo we know about the nature of effects inadults versus those in the developing

organism? How good are animal models inidentifying the neurotoxic potential of theseagents? How well do effects in animals pre-dict effects in humans? And finally, howwell do the estimates of safe levels based onanimal models actually predict what weknow about intake necessary to producehuman neurotoxicity?

Characterization of EffectsLead

The recognition of lead as a neurotoxicantarose initially in the ancient world wherethe classic signs of lead poisoning-colic,constipation, pallor and palsy-were recog-nized by both the Greeks and Romans.This recognition reemerged in the 17thcentury and was brought to public atten-tion periodically thereafter. Occupationalexposure to lead still poses a threat to thehealth of workers, resulting in peripheralneuropathy and deficits in attention andcognitive function (1,2). Early in the 20thcentury, it was recognized that childrenrepresent a particularly vulnerable popula-tion, with exposure potentially resulting inencephalopathy and death. Despite thisrecognition, lead was used widely in paintand other industrial products and added togasoline, ensuring worldwide and persis-tent distribution. Over the next severaldecades, physicians continued to reportuntoward effects as a result of lead expo-sure in children. As early as the 1940s therewas a recognition that permanent neuro-logical damage could result from exposureto lead at levels that had never producedovert signs of toxicity. In 1979, a landmarkstudy by Needleman and colleagues inBoston (3) reported decreased intelligencequotient (I) and an increased incidenceof distractibility and inattention in middle-class children with no identifiable source oflead exposure. The conclusion to be drawnfrom this study was that environmentalsources of lead were producing intellectualimpairment in children at levels that hadcome to be regarded as normal.

In the last decade and a half, there hasbeen intense research into the health effectsof lead in children and developing animals,such that probably more is known aboutthe health effects of lead than any othernoncarcinogenic environmental contami-nant (4-8). The result in the United Stateshas been that, over the last two decades, theblood level of lead considered safe for chil-dren has rapidly decreased to the present

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level of 10 jpg/dl. Prospective studies inBoston, Cincinnati, Port Pirie, and NewZealand, as well as a number of cross-sec-tional studies, have demonstrated deficitsin IQ as a function of increased body bur-den of lead after control for potential con-founding variables. A 10 pg/dl increase inblood lead level is associated with about a3 point deficit in IQ according to a recentmeta-analysis (7). These effects have beendocumented from infancy through early-to-middle school age; historic lead levels haveoften been a better predictor of deficit thanconcurrent blood lead levels. Assessment ofbehavior by teachers or parents has revealedshort attention span, increased distractibil-ity, hyperactivity, and problems in follow-ing sequences of directions as a function ofincreased blood lead levels. Not surpris-ingly, deficits in school performance arealso associated with an increased body bur-den of lead. Epidemiological studies inchildren have revealed deficits on vigilancetasks and increased reaction time, which mayreflect increased distractibility and/ordecreased attention span. Lead-exposed chil-dren also engage in perseverative behavior,continuing to respond in inappropriate ways.

The early animal research in the 1970sfocused largely on the effects of exposure tohigh doses of lead on simple learning prob-lems. Studies in which rats were prenatallyexposed generally revealed lead-induceddeficits, while postnatal or adult exposuregenerally produced no impairment (9). Asbehavioral methodology was refined, how-ever, it became clear that the prenatalperiod was not the only period sensitive tolead-induced impairment. Lead researchusing animal models over the last 15 yearshas revealed lead-induced impairment atincreasingly lower doses and on a widerange of behavioral tasks. Much of themore sophisticated work in rodents wasperformed at the University of Rochesterby Cory-Slechta and colleagues using apostweaning exposure paradigm (8,10).Extensive research has also been performedin two species of macaque monkeys, therhesus and cynomolgus (crab-eating), inthree different laboratories (8). Exposurewas prenatal, postnatal, or lifetime in vari-ous experiments. Robust deficits have beenobserved in monkeys exposed only prena-tally or only postnatally and tested yearsafter cessation of exposure, as well as inmonkeys with low blood lead levels exposedover a lifetime. Consistent effects have beenobserved on complex tests of learning andmemory. Analysis of error patterns responsi-ble for lead-induced deficits has consistendy

revealed increased distractibility, persevera-tion, inability to inhibit inappropriateresponding, and inability to change responsestrategy as hallmarks of developmental leadexposure in both rats and monkeys.

An interesting parallel in methodologiesbetween the experimental and epidemiologi-cal literature addressed the effect of leadexposure on the ability to change responsestrategies from an established pattern to anew one. This issue was assessed in monkeysusing a series of discrimination reversal tasksand in children using the Wisconsin CardSort Test. The test in monkeys requiredthem to learn a simple visual discrimination;once they learned the task, the formerly cor-rect stimulus became the incorrect one, andvice versa (11,12). A series of such reversalswas performed. Then the rules werechanged in a different way: the relevantstimulus dimension changed from form (tri-angle vs cross) to color (red vs green). Thetriangle and cross were still superimposedon the colors; however, each appeared onthe red or green in a balanced design. Themonkey was required to learn to ignore the(formerly relevant) forms and to attend tothe colors. After a series of reversals on thistask, the relevant stimulus dimension waschanged from color to form and the mon-key was required to switch strategies again.In the Wisconsin Card Sort Test, the 10-year-old child was required to pick the cardthat went with a set of examples presentedby the investigator (13). The relevant stim-ulus class could be color, number, or suit.The experimenter changed the relevantstimulus dass at a number of points duringthe experiment. Thus, both of these testsrequired the subject to change an establishedresponse strategy and to figure out that anew rule was in effect. Both lead-exposedchildren and monkeys were impaired intheir ability to do so: they stayed with anold strategy that was no longer useful.

Recent epidemiological studies suggestthat children with blood lead levels as lowas 10 jig/dl are impaired relative to chil-dren with lower blood lead levels (8). Arecent meta-analysis concluded that therewas no evidence for a threshold for lead-induced deficits down to a blood lead levelof 1 pg/dl (7). These data are consistentwith data from monkeys in which a groupwith blood lead levels of 11 pg/dl wereimpaired on a number of tasks comparedto controls with blood lead levels below 5pg/dl (8). Behavioral impairment has beenobserved in rats with blood lead levels of20 pg/dl (14). A no-effect dose has notbeen observed in either the monkey or rat

studies. It is probable that the very strongevidence from both the experimental andepidemiological studies concerning the dele-terious effects of lead were necessary for thedecision of the Centers for Disease Controland Prevention in the United States to setthe current action level for children at10 ,ug/dl. Lead levels have decreased inNorth America as a result of the removal oflead from gasoline in the 1970s, althoughleaded paint in old houses continues to pre-sent a hazard. In many other countries, leadis still allowed in gasoline and paint, andaverage blood lead levels in children arehigher than they are in North America.

Blood lead levels in children typicallypeak at about 2 years of age and decreasethereafter. Of course, exposure to additionalsources of lead may result in a temporaryincrease in lead body burden at any timeduring childhood. It is therefore importantto determine whether these peaks in bloodlead levels have lasting consequences. Itwas known as early as the 1940s that overtlead toxicity could produce permanentbehavioral sequelae in children (8). Datafrom the modern prospective studies, inwhich behavioral performance at 5 to 10years of age often correlated best withblood lead levels early in life, around theage of the peak in blood lead levels, aresuggestive of long-lasting impairment,although the continued exposure to leadmakes interpretation difficult. Experimentsin which monkeys were exposed either inutero only or postnatally for a year or lesshave demonstrated clear deficits on a num-ber of behavioral tasks when monkeys wereadults (8). Thus, experimental research, inwhich there is the opportunity to controlexposure conditions, was able to address animportant issue that cannot be addresseddirectly epidemiologically.

The animal literature has also providedclarification to the epidemiological litera-ture with regard to an issue that has provedcontentious: the control of confoundingvariables in epidemiological studies. It iswell known that IQ is affected by manyvariables: e.g., parental IQ, socioeconomicstatus, maternal ingestion of various drugsduring pregnancy (including tobacco andalcohol), obstetric complications, and birthweight. In many of the lead studies, someof these variables, particularly socioeco-nomic status and maternal IQ, were highlycorrelated with children's blood lead levels.In most studies, adjusting for variouspotential confounders decreased the statis-tical significance of the effect of lead. (Thiswas not always the case, however. In the

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prospective study in Boston, for example,socioeconomic status was higher in chil-dren with higher blood lead levels so thatlead effects were more significant aftercovariate adjustment.) The argument hasbeen made that effects attributed to leadwere in fact due to some other unidentifiedfactor that also covaried with lead levels.This assertion ignores the rather substantialanimal literature reporting lead-induceddeficits at roughly the same blood lead lev-els as those reported in children. The resultsfrom animal studies are not plagued bysuch potential confounders; subjects arerandomly assigned to exposure groups, andall variables except exposure to lead are keptas constant as possible. The congruence ofeffects thus provides reassurance that theresults of the epidemiological studies arenot misleading.

The recognition of the potential for aprofound effect on the population to beproduced by a small effect has been high-lighted by the decision to expose millionsof children to lead. Generally, the effect oflead only accounts for a few percent of thevariance on measures of IQ since IQ isinfluenced by many factors. It has beenargued that the effects of lead are thereforeunimportant. However, a 5 point decre-ment in IQ, identified in many prospectivestudies, will have a catastrophic effect onthe characteristics of the population. It willresult in a decrease in the number of peo-ple with IQs above 130 by more than halfwhile concomitantly increasing those withIQs below 70, resulting in substantial eco-nomic and other consequences (15).

MethylmeyMethylmercury is known as one of themost hazardous environmental pollutants,largely due to endemic disasters such asMinamata disease in Japan and methylmer-cury poisoning in Iraq (16). In bothtragedies, infants exposed in utero wereseverely affected, even though their moth-ers may have had minimal symptoms ofmethylmercury poisoning. Thus, the devel-opmental toxicity of methylmercury hadbecome a focus of both human and animalstudies. Even today there are several popu-lations that are exposed to methylmercury-througir substantial fish consumption.Effects of in utero exposure to methylmer-cury in infants born to these populationsare currently being evaluated.

Minamata disease was first identifiedamong people living along Minamata Bayin Kyushu, Japan. The source of methyl-mercury was effluent from a chemical

company where mercury was used as acatalyst. As a result of bioconcentration inthe food chain, high concentrations ofmethylmercury accumulated in fish andshellfish. Abnormal gait, dysarthria, ataxia,deafness, and constriction of the visual fieldwere the main symptoms. Cats living in thevillagers' homes showed signs of motorimpairment similar to those manifested inhumans. The early epidemiological investi-gation concluded that an unidentified toxicagent in fish and shellfish was responsible.It took almost 3 years to identify the causalagent by experimental pathology, clinicalstudy, and chemical analyses of environ-mental samples. The report 3 years laterfinally concluded that "organic mercury ismost suspected" (16).

Methylmercury easily crosses the pla-centa, and the developing brain can beseverely affected by the compound. In theMinamata tragedy, affected infants mani-fested severe disease resembling cerebralpalsy. Mental retardation, cerebellarataxia, primitive reflex, dysarthria, seizure,and pyramidal signs were also observed.Because of the severity of signs in theinfants, the typical symptoms such as con-striction of the visual field could not beexamined. The mothers of these childrenhad seemed healthy at the time of parturi-tion, although they developed symptomslater. Therefore, it was considered thatthe fetus is particularly vulnerable tomethylmercury neurotoxicity.

In a subsequent episode in Iraq, peoplewere exposed to methylmercury as a resultof distribution of seed grain treated with amethylmercury fungicide. Rural people usedthe grain to make bread. The total numberof official victims was 6530 including 459deaths. Observed symptoms induded pares-thesia, malaise, ataxia, constriction of visualfields, and hearing impairment. Babiesexposed in utero to methylmercury wereinvestigated for physical and mental develop-ment. A scoring system of examinationresults was adopted in the investigation.Although individual scores exhibited vari-ability, a dose-response relationship wasobserved between effects, such as retardationof walking and neurological signs, andmaternal hair mercury concentration. Fromthese analyses, an estimated lowest effectlevel was determined. Delays in speechdevelopment have also been observed follow-ing developmental methylmercury exposurein this population, although the possiblecontribution ofhearing deficits is unknown.

In a study of234 Cree children 12 to 30months of age in Canada, the mother's peak

hair level was used as the index of exposure(17). Assessment of several neurologicalmeasures, in addition to the Denver develop-mental scale, revealed only abnormal musdetone or reflexes and only in boys. There wasnot a dear dose-dependent relationship.

In a population-based study in NewZealand (18,19), mothers consumed fishon a frequent basis. Assessment on theDenver development scale revealed abnor-mal or questionable results at a higherfrequency in 4-year-old children ofmothers with hair levels of >6 pg/g dur-ing pregnancy compared to a matchedcontrol group. When these children were 6to 7 years old, an average maternal hairconcentration of 15 pg/g was associatedwith a poorer performance on the WechslerIntelligence Scale for Children (WISC).However, the number of children tested inthis study was small (60-70).

Methylmercury is also a potent neuro-toxicant in animals. Emphasis has beenplaced on characterization of developmen-tal neurotoxicity, although toxicity in adultanimals has also been demonstrated. Spykeret al. (20) demonstrated that mice exposedto methylmercury in utero showed impairedswimming ability. Their result firstconfirmed fetal methylmercury poisoning inan animal model, although the impairmentwas less severe compared to fetal Minamatacases and was subtle before the mice wereforced to swim. Subsequently, a large num-ber of animal studies were performed usingvarious methods; thus, a much wider rangeof effects has been examined in animalstudies compared to the human studies.

As was the case for lead, considerableresearch on neurotoxicity produced bymethylmercury has been performed in mon-keys, presumably in response to the episodesof human poisoning. Research in adultmonkeys replicated the constriction ofvisualfields and other visual deficits observed inadult humans as a result of methylmercuryexposure (21); these findings of sensory sys-tem deficits have been extended in develop-mentally exposed monkeys in which visual,auditory, and somatosensory deficits havebeen observed (22). Early developmentalexposure at high doses also produces thepattern of cerebral palsy and severe visualdeficits including blindness (23,24)observed in humans.

Research in monkeys has also addressedthe issue of cognitive impairment as aresult of developmental methylmercuryexposure. In utero exposure resulted inimpairment of visual recognition memory(25) and retarded object permanence

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performance (26) when monkeys weretested during infancy. Interestingly, thissame cohort of monkeys demonstratedfacilitated performance on a delayed spatialalternation task when tested as adults (27).In utero plus postnatal exposure failed toproduce deficits on a discrimination rever-sal task during both the infant and juvenileperiods (28), even in a monkey with clearmotor signs of methylmercury intoxication.It may be that the monkey does not providea good model of the gross cognitive impair-ment observed after high-dose develop-mental exposure in humans.

Methylmercury also produces neurotoxi-city in rodents, despite some differences inthe neuropathology and pharmacokineticsbetween rodents and primates (includinghumans). The pattern of neuropathologicaldamage in the brain is different in rodentsthan in species with deep sulci; specifically,methylmercury produces preferential dam-age to deep sulci such as calcarine fissure(subserving visual function) in adult humans(29), a pattern which is replicated in pri-mates (30,31) but not in rodents. However,the pattern of damage after developmentalexposure is more diffuse in all species (29).There are also significant toxicokinetic dif-ferences between species, including bloodand whole-body half-times, and brain:bloodratios (32). The rat is anomalous in having avery high red blood cell:plasma ratio, whichundoubtedly contributes to the very lowbrain:blood ratio of 0.06, compared to ablood:brain ratio of 2 to 5 for monkeys andhumans (22). The most obvious effect ofmethylmercury in adult rodents is motordeficit (33-36); reports of effects on activityare conflicting (37,38). In utero exposure athigh doses reliably produces deficits inmotor function. Very high doses may resultin a decrease in locomotor activity (16),while lower doses often produce no effect(39-41). Both positive and negative resultshave been obtained on simple learning tests(16,22). The sensory system damage that isa hallmark of methylmercury toxicity inhumans may not be produced by moderateexposure in rodents based on indirectevidence such as auditory startle responseand visual discrimination performance,although high-dose in utero exposure pro-duces changes in visual evoked potentials(22) or blindness (29). Direct assessment ofauditory thresholds revealed no deficits inmethylmercury-exposed rats (39).

Polychiorinated BiphenylsPolychlorinated biphenyls (PCBs) are a fam-ily of chlorinated hydrocarbons containing

209 different isomers (congeners). Theirmajor use was as a dielectric in transformersand capacitors, although they had otherindustrial uses as well. They were in wide-spread use from the 1930s until the 1970s;although PCBs were banned in the UnitedStates in the 1970s and subsequently else-where, they are presently a worldwide pollu-tion problem. Residues persist in air, soil,water, and sediment and can be detected inbiological tissue in most residents of indus-trialized countries. The chemicals are storedin fat and are not readily excreted except inbreast milk.

In 1968 a tragic epidemic occurred inJapan as a result of contamination of riceoil with PCBs and small amounts of othercontaminants. Infants born to mothers whoconsumed the contaminated oil had darkpigmentation of the skin, low birth weight,early eruption of the teeth, and swollengums and eyelids (42). In another incidentin Japan, affected children had hypotonicreflexes, were dull and apathetic, and hadlow IQs (43). Adults ingesting high levelsof contaminated oil suffered chloracne,numbness and weakness of limbs, anddecreased peripheral nerve conductionvelocities. However, the developing fetuswas much more sensitive than the mother.Children born to mothers exposed to ahigh, acute dose of PCBs in Taiwan havebeen followed for at least 6 years (44).These children exhibited delayed develop-mental milestones, deficits in intellectualfunctioning, and other behavioral prob-lems. These effects were observed at expo-sure levels that produced overt signsincluding gum hypertrophy, deformed orpigmented nails, chloracne, hyperpigmen-tation, and hair loss.

An extensive prospective study involvedMichigan children born to women whoconsumed fish from Lake Michigan(45-50). Reduced birth weight and headcircumference were associated with con-sumption of contaminated fish. Fishconsumption by the mothers was also asso-ciated with lower scores on the Brazeltonneonatal development scale in the infants.Decreased visual recognition memory inthis same set of infants at 7 months of agewas associated with both maternal fish con-sumption and cord serum PCB levels (47);this task is reasonably predictive of IQmeasured at school age. There was no asso-ciation with postnatal exposure throughnursing. Decreased weight and poorershort-term memory at 4 years of age wasalso associated with cord but not concur-rent PCB levels (48-49). These measures

were not associated with concurrent bloodlevels of PCBs, polybrominated biphenyls,lead, or dichlorodiphenyltrichloroethane(DDT). Hypoactivity was associated withconcurrent blood PCB levels (49).A prospective cohort of breast-fed

infants was followed for 60 months in aNorth Carolina study (51-54). Mothershad no known excessive exposure to PCBs.Higher in utero PCB exposure, as assessedby maternal milk fat PCBs, was associatedwith hypotonicity and hyporeflexia; therewas no association with birth weight or headcircumference (54). Higher transplacentalbut not postnatal PCB exposure was associ-ated with lower scores on the Bayley Scale ofIntelligence at 6 and 12 months of age (51).There was no association between eitherprenatal or concurrent PCB levels and out-come on intelligence tests at 3 to 5 years.

Numerous developmental studiesin both rodents and monkeys have demon-strated neurotoxicity as a result of PCBexposure. A recent review (55) summarizedchanges in activity levels, impaired neuro-logical development, and impairment onsimple learning tasks in rodents whosedams were exposed to various commercialPCB mixtures, often with dosage regimensthat did not produce increased mortality ordecreased weight in the pups. Dams wereapparently unaffected. A series of studies inrhesus monkeys was performed at theUniversity of Wisconsin. Maternal expo-sure to Arodor 1016 at doses approximating0.007 or 0.028 mg/kg/day (56,57) begin-ning prior to breeding and continuing untilinfants were weaned at 4 months of ageresulted in hyperpigmentation in infants inboth dose groups and decreased weight inthe high-dose group (57). Impairment on alearning and memory task was alsoobserved in the high-dose group duringinfancy. Further testing of cognitive func-tion when these monkeys were juvenilesrevealed no impairment (56,57). In studieswith Aroclor 1248, female monkeys wereexposed to 0 or 1.0 ppm PCBs in the diet3 days a week, or 0.5 ppm in the feed dailybeginning prior to breeding and continu-ing until offspring were weaned at 4months of age. Additional groups offemales exposed to 2.5 ppm produced anumber of sets of offspring: concurrentwith exposure or in which exposure in themothers ceased 1.0, 1.5, or 3.0 years priorto breeding. Concurrent exposure to2.5 ppm resulted in reduced birth weight(58) and deficits in discrimination reversallearning (59). These monkeys were hyper-active when young (59) and hypoactive at

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44 months of age (60). Monkeys exposedconcurrently to 0.5 ppm were hyperactiveat 12 months of age (61). The group bornto mothers 1.0 year after cessation ofexposure to 2.5 ppm showed facilitatedperformance on a shape discrimination-reversal task (56), which the authors inter-preted as a deficit in the treated group'sability to learn the irrelevance of the shapecue on a previous task. The performance ofthe 1.0 ppm group was not impaired onthis task. Monkeys born to mothers 1.5 or3.0 years after cessation of exposureto 2.5 ppm PCBs were impaired on aspatial alternation task at 4 to 6 years ofage (57). The mothers did not exhibitsigns of neurotoxicity.

There is excellent correspondencebetween the effects of developmental PCBexposure in the monkey and that observedin humans, including learning deficits,changes in activity, and hyperpigmenta-tion. The rodent also provides a goodmodel for effects in humans, with changesin activity levels and learning deficitsobserved as a result of perinatal exposure.It is not possible to determine whetherbehavioral processes underlying observeddeficits are the same in animal modelscompared to humans because the issue hasnot been addressed.

Predicted Ability ofStandard Neurotoxicity Teststo Detect NeurotoxicityThere is a question central to the theme ofthis review: Knowing what we know today,if another agent that has effects similar tothose of these model neurotoxicants weresubmitted to a governmental agency formarketing, would its neurotoxic potential bedetected? Can we feel secure that the arsenalof methodology available to the experimen-tal behavioral toxicologist today woulddetect these agents as neurotoxicants? Howwell would these methodologies predicteffects observed in humans? Would themethods generally required by regulatoryagencies detect these agents as neurotoxic,or would their neurotoxic potential bemissed? One of the difficulties in attempt-ing to answer these questions is thatdifferent testing strategies are required bydifferent governments, different agencieswithin the same government, or for differ-ent classes of agents depending uponchemical structure or intended use. Insome instances, observation of adult animalswould provide the only opportunity todetect neurotoxicity. Under other protocols,reproductive studies are required. For thepurposes of the present exercise, protocolsrequiring acute exposure in adults, longerterm exposure (e.g., 28 days) in adults, or

reproductive/developmental exposure wereconsidered. It was assumed that rats or micewould be the experimental model, andeffects reported in both species were consid-ered. For each type of protocol, an attemptwas made to determine whether effects havebeen observed on end points included in thefunctional observation battery (FOB), motoractivity, simple tests of learning/memory, orschedule-controlled operant behavior(SCOB). These tests have been recom-mended by various agencies as appropriateunder various conditions. The results of thisanalysis are presented in Table 1.

One conclusion that may be drawnfrom this analysis is that all of the agentswould have been identified as neurotoxic atsome dosage level in some exposure proto-col. It is probable that neither lead norPCBs would have been identified as neu-rotoxicants following short-term adultexposure, while methylmercury would beidentified on the basis of motor deficits.On the other hand, lead, PCBs, andmethylmercury would have been identifiedas developmental neurotoxicants at highdoses on the basis of screening proceduressuch as the FOB, although the effects ofthese agents on other measures at lowerdoses is a little less clear.

Behavioral analysis in rats exposeddevelopmentally to lead has reliably

Table 1. Predicted ability of standard tests to detect neurotoxicity of model agents.

Lead Methylmercury PCBs Solvents

Adult acuteFOB No motor effect (62) No neurological signs (668-68) Noneb Yesc

Motor impairment (63)(3 days injection)

Locomotor activity ? Yes (69) None YesSimple learning/SCOB None (98,63) No (67) None Yes

Adult longer-term (e.g., 28 days)FOB No effect after 15 weeks (38) Landing foot splay, weakness, Noneb ?

irritability (36,66,68-70)Locomotor activity No effect until 6 weeks (38) None, rat (38) None Yesc

Yes, mouse (69)Simple learning/SCOB None (98,38) CAR, rat, effect after 9 weeks (38) None ?

No effect avoidance, rat (71)Developmental (reproductive)FOB High dose, paraplegia, Cerebral palsy, spasticity, Neurological signs, Yes/nod

tremors (64) seizures, at high doses (298,40,72) impaired incline screen,spinning (538)

Locomotor activity Both increase Negative (39-41) Increase or decrease (53) ?and decrease (64,65) Positive (based on 6 labs) (73)

Simple learning/SCOB Numerous effects (9a) Effect on water maze, Avoidance, water maze, ?avoidance (298,73) learning, memory (538,74,75)No effect on learning, memory (39,74) Fl (76)

No effect, RAM (77)Abbreviations: FOB, functional observation battery; SCOB, shedule-controlled operant behavior; CAR, conditioned avoidance response; FIfixed interval; RAM, radial arm maze.'Review article. bNo mention of neurotoxicity in numerous studies. CExpected at high doses as a result of narcotic effects. dExpected as a result of known effects. 'Expectedeffect, depending on agent.

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revealed deficits in learning and perfor-mance at blood lead levels that are environ-mentally relevant (8,10,14). Such effectswere detected in some instances on com-plex tasks using very sophisticated dataanalysis. However, lead-induced changeshave also been detected using intermittentschedules of reinforcement. Such behav-ioral methodology is a standard part of thearsenal of behavioral tests available to thebehavioral pharmacologist and toxicologist.It must be pointed out that these are notscreening procedures, however; theyrequire automated equipment and opti-mally a computer to control the experi-ment and collect data on line. If simpletests of learning that do not necessarilyrequire automated equipment are consid-ered, lead-induced deficits were revealed ingeneral after prenatal exposure only, evenat high doses. At very high doses, prenatalexposure resulted in overt signs of toxicityin the pups including nervous systemlesions and paralysis. It is clear, then, thathigh-dose effects in rodents mimic thoseobserved following high-dose developmen-tal exposure in humans. It is significant,however, that the intraperitoneal route ofexposure in general resulted in negativeresults (9). It was necessary for the expo-sure route to be the same as that of humanexposure, i.e., oral. It is also important topoint out that, while effects on suchscreening tests as locomotor activity weresometimes (but not always) positive, resultswere inconsistent, with both increased anddecreased locomotion observed. It mightbe tempting to conclude in such circum-stances, if the agent in question were notalready known to be neurotoxic, that sucheffects did not reflect neurotoxicity.

It is clear that perinatal exposure tomethylmercury at high doses produces overtneurological effects that would be detectedon the FOB. Methylmercury generally pro-duced decreased locomotor activity in pupswhose dams were exposed to high doses,while more recent studies at lower doseshave been negative. The effects ofmethylmercury on simple tests of learningare equivocal, with both positive and nega-tive results reported (22). Methylmercuryreliably increased auditory startle in a collab-orative study of six laboratories, with incon-sistent effect on a discrimination task (73).No effects of developmental methyl-mercury exposure were observed on a bat-tery developed at the U.S. EnvironmentalProtection Agency (U.S. EPA). This bat-tery included T-maze alternation, auditorystartle, and olfactory discrimination (39).

In a European collaborative study, therewere no effects on accuracy on visual dis-crimination reversal and spatial delayedalternation tasks, although changes in audi-tory startle were observed (74). Changes inlatency to respond and failure to respondwere observed, which would not necessarilybe interpreted as cognitive deficits.

Developmental exposure to PCBsproduces neurotoxic effects in rodents withgood reliability. High doses result inneurological signs and impaired motordevelopment, which would presumably bedetected on an FOB. PCB exposure alsoproduces changes in locomotor activity,although both increased and decreasedactivity have been observed. High dosesalso result in impairment on simple learn-ing tasks such as active avoidance andwater-maze performance. Effects have beenobserved on SCOB (76) and delayed alter-nation performance (77) but not on radialarm-maze performance (77). It seems clearthat PCBs would have been identified asneurotoxic to the developing organismbased on studies in rodents.

The ability of adult exposure paradigmsto detect neurotoxicity in these three agentsis less clear. Industrial exposure to lead inadult humans results in motor effects, aswell as in psychiatric and cognitive distur-bances following long-term exposure. It isgenerally recognized that the adult rodent isextremely resistant to lead-induced neuro-toxicity (62). Administration of very highdoses for a number of days may producehind-limb weakness while repeated expo-sure to lower doses produced no effect onvarious measures included in the FOB. A15-week exposure to high doses resulted indecreased body weight and motor activity,with no effect on body temperature, gripstrength, negative geotaxis, startle, or condi-tioned avoidance response (CAR) (38).Effects became apparent 6 weeks after expo-sure started, so they would not have beendetected in a 28-day study. Attempts todemonstrate impaired learning as a result ofadult exposure have been largely negative(9). Other effects such as nephrotoxicity areapparent at doses lower than those neededto produce overt neurological signs. It istherefore unlikely that lead would have beenregulated as a neurotoxicant on the basis oftests in adult rodents.

Methylmercury reliably produces grossneurological signs and changes in othermeasures of the FOB following repeatedexposure in adult rodents; however, resultsafter a single administration of methylmer-cury have been negative. Locomotor activity

has been found to be affected in one study(69). There has been little research on theeffects of methylmercury on learning tasksin the adult rodent, but effects that havebeen observed may be attributed to sensoryor motor impairment (38).

For PCBs, little or no research hasspecifically addressed the issue of neurotox-icity as a result of exposure in adultanimals. However, it is clear that otherorgan systems are more sensitive to PCBtoxicity than the nervous system. Theeffects of PCBs in adult rodents at highdoses include changes in body weight andimpairment of liver, kidney, and immunefunction. Perusal of dozens of papersrevealed no mention of overt neurotoxiceffects. It is also clear that reproductive anddevelopmental neurotoxicity are producedat doses that do not result in any overt tox-icity in the mothers. It seems reasonable toassume, then, that if only adult exposurewere used to assess PCB toxicity, thenervous system would not have beenidentified as a target.

The recognition that solvents repre-sented a hazard at levels that did notproduce narcosis arose from long-termindustrial exposure. Therefore animalresearch focused on effects in adult ani-mals. Screening tests would certainly detectthe fact that solvents produce narcosis, andresults of tests of motor activity or learningwould be confounded by this effect, partic-ularly at high doses. Little research hasbeen performed on the effect of solventson development.

Congruence ofExposure Levels at WhichNeurotoxicity Is Observed inHumans and AnimalsAn additional issue worth addressing is thecorrespondance between the dose levels atwhich neurotoxicity has been observed inanimals and the estimated intakes thatproduce neurotoxic effects in humans forthese model agents. The protection pro-vided by the ways in which animal data areused in the risk assessment process to pro-tect human health may also be scrutinized.For this exercise, the rules by which refer-ence doses (RfDs) are derived by the U.S.EPA will be used, since these or similarprocedures are currently used by otheragencies as well. The central question isthis: From the results of the FOB, locomo-tor activity, and effects on simple learningtests or performance on SCOB, would thederived RfDs protect against neurotoxicity

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Table 2. Comparison of doses required to produce neurotoxicity in humans and rodents and calculated RfDs (mg/kg/day).Human Rodent

Compounds Exposure NOAEL Rfd NOAEL Rfd

Lead Adult Occupational exposure ? 5.2 5.2 x 10-2Developmental 5 x10-3a 5 x104-1 x10-3 1(LOAEL)- >100 1iD3- >1

Methylmercury Adult 3 x10-3 3 x 10 0.7-1.6 7 x 10-3-1 .6 x 10-2Developmental 7 x10D4-1.2 x10-3 7 x105-1.2 x104 5 x10-3- >2 5x10-5- >2 x10-2

PCBs Adult ? No effectDevelopmental 10-5 104 0.2-5 2x10-3-10-2

"Based on estimated 50th percentiles for intake from food by children 2 years of age. Data from Beloian (78).

in humans? The conclusions are summa-rized in Table 2.

Lead produces neurotoxicity in adultrodents after repeated but not acute expo-sure, but only at doses that produce mor-tality or significant weight loss. The lowestobserved adverse effect level (LOAEL) fordecreased motor activity was 7.5 mg/kg,with a no observed adverse effect level(NOAEL) of 5.2 mg/kg (38). In general,learning has been found to be unaffectedby lead exposure in adult rodents (9). Theintake necessary to produce neurotoxiceffects in adult humans has not beenquantified. More information is availableon the effects of developmental exposure tolead. The studies in which behavioraleffects were detected at the lowest dose inrodents were performed by Cory-Slechtaand colleagues using a post-weaning expo-sure paradigm (8). Neurotoxicity wasdetected at a dose of approximately 1 mg/kg(10), three orders of magnitude higher thanthe average lead intake by children,although assessment was not performed atlower doses in the rat studies. In those stud-ies, effects were demonstrated in perfor-mance on schedules of reinforcement andcomplex learned behavior using sophisti-cated methodology. If a factor of 1000 isconsidered to be an appropriate safety fac-tor-the procedure used for agents forwhich there is a LOAEL but not a NOAELfrom animal data-the RfDs based on theanimal data are in good agreement withestimates of human intake. Most studies,however, particularly earlier ones, detectedneurotoxicity at much higher doses; insome studies doses over 100 mg/kg yieldednegative results (9). RfDs based on thesesimple learning tests would yield RfDsgreater than 1 mg/kg, which clearly greatlyunderestimates the toxicity of lead to thedeveloping organism.

For methylmercury, the present U.S.EPA RfD of 0.3 pg/kg/day is based onparesthesias in adults. In a 15-week studyin rats (38), 1.4 mg/kg/day producedeffects on motor function after 3 months

of exposure while 0.7 mg/kg/day producedno effect. A safety factor of 100 would yieldan allowable intake of 7 pg/kg/day.Landing foot splay in the mouse wasaffected after exposure to 2.7 mg/kg/day forless than 28 days, while 1.6 mg/kg/day pro-duced impairment after more than 60 days(36). An RfD based on a 28-day exposurewould be 16 pg/kg/day. RfDs generatedfrom these two studies are one to twoorders of magnitude above the RfD basedon adult human data. With respect to thedevelopmental effects of methylmercuryexposure, Stern (79) derived a referencedose of 0.07 pg/kg/day based on develop-mental neurotoxicity in humans, while theAgency for Toxic Substances and DiseaseRegistry has determined a minimum risklevel of 0.12 pg/kg/day based on the samedata (80). It must be reiterated that thedevelopmental data at this point consist ofrelatively crude end points, unlike the veryextensive body of data on subtle behavioraldeficits that exists for lead. Most rodentstudies reported NOAELs between 50 and>2000 pg/kg/day depending on the studyand end point examined (22). Tests thatwould typically be used in an assessmentbattery yielded values at the high end ofthis range. For example, in an interlabora-tory study involving six laboratories (73),effects were observed at 6 mg/kg adminis-tered on gestation days (GD) 6 to 9 whileeffects were minimal or absent at 2 mg/kgon simple tests of activity and learning. Inanother study in which rats were dosedfrom GD 6 to 15, no effect was observed at1 or 2 mg/kg using a testing battery devel-oped at the U.S. EPA, including severaltests of learning and activity (39). RfDsgenerated from these two studies would be10-2 mg/kg/day or higher, two to threeorders of magnitude greater than the RfDcalculated from the human data. In con-trast, a study in which rats were required toperform on a differential reinforcement ofhigh rate (DRH) schedule, which requiredthe animal to emit a specific number ofresponses within a specified time, detected

effects at a much lower level than otherstudies (81). A dose of 10 pg/kg during GD6 to 9 produced effects, with a NOAEL of5 pig/kg. With a safety factor of 100, theallowable intake would be 0.05 pg/kg/day,which is in very good agreement with esti-mates based on the human data. It is highlyunlikely that an allowable intake formethylmercury in humans would have beenbased on a single apparently anomalousrodent study, however. The experimentaldesign of most of these studies in whichdams were dosed for only several days dur-ing pregnancy also presents problems inextrapolating the rodent data to humans.

While perinatal exposure to PCBs reli-ably produces a variety of behavioral effectsin rodents, the doses at which these effectshave been identified are considerably higherthan those at which untoward effects appar-ently occur in humans. In their review,Tilson et al. (55) calculated RfDs based onrodent, monkey, and human data. RfDsfrom the human data were calculated to beapproximately 10-5 to 10-6 mg/kg/daybased on behavioral data, using a safety fac-tor of 10 below the estimated NOAEL.RfDs from the monkey data, based onmotor activity and impairment on learningand memory tasks, were in the range of10-5 mg/kg/day, which is in good agree-ment with the human estimates. RfDs frommost measures of developmental toxicitybased on the rodent data were approxi-mately 10-2 mg/kg/day; the most sensitiveindicator was motor activity, which yieldedan RfD of 10-3 mg/kg/day based on aNOAEL of 0.2 mg/kg/day and dividing bya safety factor of 100. This is three orders ofmagnitude higher than the estimates ofintake that would protect against develop-mental neurotoxicity in humans. While theassumptions used to calculate the humanRfDs may have resulted in an underestima-tion of the dose required to produce effects,it is doubtful that adjustments in the calcu-lations would result in a change in the RfDby three orders of magnitude. Moreover, thecalculations based on monkey data, which

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are not subject to the same uncertainties inintake estimates, are in close agreement withthe estimates based on the epidemiologicalstudies. It therefore appears that the rodentdata underestimate the intake that wouldprotect against neurotoxicity in humans byabout three orders of magnitude, based oncurrently accepted practices of estimatinghuman risk from animal data.

An alternative strategy would be to baseallowable intakes on body burden ratherthan dose. This would have the advantage ofat least partially circumventing differences intoxicokinetics between humans and animalmodels. The most relevant comparisonwould be levels in target organ tissues, i.e.,the nervous system. For example, in theirreview Burbacher et al. (29) point out thatthere is good congruence between signs ofmethylmercury toxicity between smallmammals, primates, and humans based onbrain mercury levels. Of course, such com-parative data are not available for mostagents. A readily accessible compartment isthe blood compartment. For lead, cleareffects in rats have been observed in experi-ments by Cory-Slechta and colleagues atblood lead levels of approximately 20 pg/dl(14); lower levels have not been studied. Arecent meta-analysis of the epidemiologicalliterature showed no evidence of a thresh-old for cognitive deficits produced by leaddown to blood lead concentrations of1 lig/dl (7). If one safety factor of 10 wereeliminated for interspecies extrapolationbecause a measure of body burden wasbeing compared directly between rats andhumans, the allowable blood lead concen-tration based on these rodent data wouldbe 0.2 pg/dl, which is in good agreementwith the epidemiological conclusions.Blood levels associated with neurotoxicityare unknown for either methylmercury orPCBs in the rodent. However, methyl-mercury blood levels in the rat wouldundoubtedly be extremely misleading,given the huge difference in blood:brainratios between rat and human. Inaddition, most current government regu-latory protocols do not require measuresof body burden; therefore, such datawould be unavailable from animal studies.Moreover, if the agent being tested were anew chemical to which humans had notbeen exposed, relevant data from humansand particularly from children would alsobe lacking. The strategy of basing allow-able intakes in humans on comparison ofbody burden between human and animalmodels is therefore not at all practical fornew agents.

ConclusionsThere are a number of conclusions that maybe drawn from the exploration of effects inthese model compounds. First, it is clearthat the degree to which the effects ofassessment of specific functions in animalspredict effects in humans depends on boththe agent and the developmental period atwhich exposure occurs. Developmental leadexposure in humans is characterized bydeficits in IQ and other problems of cogni-tive functioning. Cognitive deficits havebeen reliably demonstrated in animal mod-els including rodents and primates. In fact,the experimental and epidemiological liter-atures show remarkable congruence withregard to the behavioral processes underly-ing these deficits. The ability of effects inadult animals to predict effects producedby lead in humans is disappointing. It isdoubtful that the peripheral neuropathyand changes in cognitive function pro-duced by occupational exposure would bepredicted on the basis of rodent studies.While chronic lead exposure may producechanges in locomotor activity in rodents,studies of neurological function and learn-ing have been almost universally negative.Methylmercury produces severe neurologi-cal impairment in humans as a result ofadult exposure; these effects are replicablein animal models only after repeated expo-sure. One of the hallmarks of methylmer-cury poisoning in adults is constriction ofvisual fields and other visual effects. Thisprobably would not have been predicted byrodent tests because of differences in thepattern of pathological damage betweenhuman and rodent brain and because ofimportant differences in the visual systemsbetween humans and rodents. Such effectshave been demonstrated in monkeysexposed as adults, however. Developmentalexposure to methylmercury in humansresults in neurological impairment at highlevels of exposure and developmental delayand possibly cognitive impairment at lowerexposure levels. Developmental exposure inanimals clearly replicates the neurologicaldysfunction observed in human infants.On the other hand, results of tests of cog-nitive function in both rodents and mon-keys is conflicting (22). DevelopmentalPCB exposure produces behavioral delaysor cognitive deficits in children. In goodagreement with these findings, deficits ontests of learning and changes in activityhave been demonstrated as a result of PCBexposure in both rodents and monkeys.Adult monkeys chronically exposed toPCBs develop the classic signs of PCB

toxicity in humans, including chloracne,hair loss, and swelling of the eyelids(82,83); however, whether PCB exposurein adult monkeys also produces the pares-thesias and weakness reported in humanshas not been addressed. Neurotoxicity hasnot been reported in adult rodents as aresult ofPCB exposure.

The ability of animal studies to predictintake levels at which human health wouldbe protected is less encouraging. It is clearfrom comparison of the human and rodentdata that results from rodent studies oftenvastly underestimated intakes at whichneurotoxicity was observed in humans. ForPCBs, the difference in the estimatedacceptable intake between human androdent developmental data is 3 to 4 ordersof magnitude, while for methylmercury thedifference is two orders of magnitude orgreater for most studies. For lead, deficitswere revealed on activity and simple learn-ing tests at doses that would also result inallowable intakes much higher than thoseat which cognitive impairment has beendemonstrated in children. However, datafrom one laboratory using sophisticatedanalyses of behavior on operant schedulesof reinforcement detected impairment atlevels that would result in derivation of anRfD that would clearly indicate thathuman health was not protected at intakelevels of many children in industrial soci-eties. One conclusion that may be drawnfrom this analysis is that current methodsof calculating acceptable intakes based onanimal data, exemplified for the sake ofdiscussion by current practices in theUnited States, are insufficient to protectthe human population against behavioraltoxicity. It may be argued that agents suchas lead, methylmercury, and PCBs repre-sent worst case scenarios because theseagents have been released into the environ-ment in huge quantities, are not degradedenvironmentally, accumulate in the foodchain, and/or have very long biologicalhalf-lives in humans. Therefore the degreeto which the lessons from these potentneurotoxicants may be extrapolated toother agents needs to be interpreted withsome caution. However, the clarification ofthese issues for new agents would requireextensive biological and environmentaltesting. It seems unlikely that such issueswould be satisfactorily resolved for pro-posed new chemicals before their approvaland use. (For example, it was argued thatlead would not accumulate in the environ-ment when industry proposed adding leadto gasoline.)

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It might also be suggested that sophis-ticated behavioral testing, including devel-opmental testing, be required for allchemicals suspected of producing neuro-toxicity. A tiered approach has been sug-gested by a number of national andinternational agencies whereby detection ofneurotoxicity at high doses would triggerassessment at lower doses using moresophisticated methodology. While such astrategy would undoubtedly aid in thecharacterization of effects as well as result

in detection of neurotoxicity at lowerdoses, it is unlikely that this strategywould provide sufficient protection in allcases. For example, numerous reproduc-tive studies using reasonable end pointsgenerated NOAELs as much as five ordersof magnitude above those estimated fromhuman data (Table 2). It is doubtful thatany test done in rodents, no matter howsophisticated, would lower the doseat which impairment was detected by suchan amount.

In conclusion, neurotoxicity would havebeen detected for all of these model agentsonly if both developmental and adultassessments had been performed. In addi-tion, doses at which effects were observed inrodents were often several orders of magni-tude higher than those at which effects wereactually observed in humans. Whetherthese agents would have been approved foruse would ultimately depend on decisionsmade subsequent to the detection of thefact that these agents were neurotoxic.

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