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~7 Methodologic Issues of Extrapolation from Animal Studies to Human Toxicant Exposure This chapter briefly reviews some of the major approaches to relating animal findings In functional teratology to the assessment of potential human health haz- ards. The approaches include: Investigation of underlying mechan- isms of functional alterations observed . In animals. Investigation in animals of normal and abnormal development of functional end points that are comparable in humans. Direct comparison of functional ef- fects seen in animals and humans when data are available on both. Intrauterine exposures to some terato- genic agents have been linked to gross physical malformations in both humans and animals. Structural abnormalities are often profiled as syndromes, e.g., the fetal alcohol syndrome (Clarren and Smith, 1978) and the fetal hydantoin syndrome (Hanson et al., 1976~. Interest in people with subtle functional effects after low- dose exposures and people without overt anomalies has increased. Since Wilson ( 1973) included functional alterations in a list of possible effects of exposure to developmental toxicants, research in the subject has expanded greatly. All functional systems are theoretically at risk at some point in their development 289 and maturation. Only a few functional systems have been studied, and that situa- tion is changing (Kimmel and Buelke-Sam, 1981; Kavlock and Grabowski, 1983; Riley and Vorhees, 1986~. Unlike studies that evaluate multiple structural changes after exposures, studies of postnatal function typically evaluate effects in a single organ system or on a single end point, e.g., the CNS or immune deficiency. Additional complications are encoun- tered in cross-species comparisons of postnatal functional alterations, be- cause species often vary both in their responsiveness or susceptibility to toxic insult and in the manner in which they mani- fest toxicity. Examples of research aimed at overcoming those problems with each approach are discussed. INVESTIGATION OF UNDERLYING MECHANISMS One way of relating animal findings and human hazard is to evaluate underlying structural, biochemical, and physiologic correlates of overt functional changes seen in animals. The rationale is that the determination of the target and degree of toxicity produced by developmental exposure will yield information relevant to the human situation. Mirmiran and colleagues (1985) recently

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290 reviewed the relationships between be- havioral alterations observed in humans and animals and the underlying neurochemi- cal and electrophysiologic disturbances observed in rats that were exposed to phar- maceutical agents during development. Table 27-1 presents some of the findings. The immaturity of the blood-brain bar- rier and greater accumulation of many of these compounds in the developing brain make the fetal brain a major target of its mother's medication. Mirmiran et al. (1985) have shown that neonatal exposure of rats to clonidine, an antihypertensive agent, and clomipramine, an antidepres- sant that acts on Norepinephrine and sero- tonin neurotransmission, suppresses ra- pid-eye-movement (REM) sleep in the devel- oping rats. In adulthood, the offspring rats showed hyperactivity, hyperanxiety, reduced sexual behavior, disturbed sleep patterns, and smaller cerebral cortex. NEURODEVELOPMENTAL TOXICOLOaY STUDY OF COMPARABLE FUNCTIONAL END A second approach to determining the relationship of animal findings in post- natal functional studies situation is to select a to the human functional re- sponse that is comparable across species. A variety of potentially relevant end points are available, e.g., sleep pat- terns, neonatal vocalizations, and suck- ling patterns. The development of these end points and their sensitivity to toxic insult could be compared directly across species. The startle reflex is valuable in such an effort for several reasons: Startle can be elicited in all mam- mals, including humans. The startle reflex is mediated via simple neuronal circuits. TABLE 27-1 Sequelae of Developmental Exposure to Drugs in Humans and Animalsa - REM~ Sleep Relevant Effects in Effects in Deprivation Transmitter Drug Humans Animals Effects System Clonidine Smaller head Hyperactivity, + + + Norepinephrine circumference, delayed motor questionable development necrologic status, increased myoclonic jerks during sleep Diazepam Low Apgar score, Hyperactivity, + Gamma-amino- reluctance to decreased male isobutyric acid eat sexual behavior, decreased startle reflex Imipramine- Poor sucking, Hyperactivity, + + + Norepinephrine, lye agents irritability decreased male acetylcholine, sexual behavior, serotonin smaller brain Reserpine Anorexia, Smaller brain, + Norepinephrine, lethargy altered activity, dopami;ne, altered startle seroton~n reflex aData from Mirmiran et al., 1985. bREM=rapid eye movement.

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EXIRAPOLATIONFROMANIMAL STUDIES The startle reflex is modulated via several neurotransmitter systems. The startle reflex can be measured at early ages in many species. The startle reflex is quantifiable. Inhibitory or excitatory effects can be determined. Startle displays different types of plasticity. Davis ( 1984) has reviewed aspects of the mammalian startle reflex. It consists of a characteristic, very rapid sequence of muscular responses elicited by a sudden, intense stimulus. Under compar- able circumstances, the more intense the stimulus, the greater the response. The graded amplitude of the mammalian startle response can be detected in direct muscle recordings (e.g., electromyographic re- cordings from a limb or muscles involved in blinks) or in the output from transduc- ers that measure cage movements when whole-body startle is measured. A standard feature of this reflex is its very short latency; the response occurs only milli- seconds after the onset of the eliciting stimulus. Although the neural circuitry that medi- ates startle is at lower levels of the CNS, higher neural networks can modulate it. Nearly all defined neurotransmitter sys- tems interact to modulate the startle re- sponse (Fechter, 1974; Davis and Aghajani- an, 1976; Davis and Sheard, 1976; Handley and Thomas, 1979; Davis and Astrachan, 1981; Gallager et al., 1983; Holson et al., 1985~. In the spinal cord and facial motor nucleus, serotonin and norepinephrine increase auditory startle and glycine tonically inhibits it; it appears that GABA can also inhibit the response at this level. Supraspinally, dopamine and per- haps GABA receptor stimulation increases startle, and serotonin activation de- presses it. Startle is also modulated in several brain regions distant from the primary startle pathway itself. There- fore, the reflex can provide a sensitive indicator of function after toxicant ex- posure. Developmental insults that result in changes in a neurotransmitter system might be expressed as changes in the laten- cy, amplitude, or modification of the re- sponse. The type of change observed can 291 suggest which systems have been affected by exposure. Auditory startle has been used often in studies of animal developmental toxi- cology. Recently, automated procedures have been applied in such studies, thus allowing more specific characterization of changes in this reflex. Automated pro- cedures for stimulus presentation and data collection have yielded useful informa- tion for evaluating sensitization, habit- uation, prepulse inhibition, and reflex modification by prior associative learn- ing after toxicant exposure (Hoffman, 1984~. Startle thus represents a poten- tially powerful tool in developmental toxicology for investigating sensorimotor reactivity. The simplicity of the response and the plasticity displayed within it across animal species, including humans, suggest that specific efforts to inves- tigate the comparability of startle al- terations in animals and humans after de- velopmental insult are warranted. DIRECT COMPARISONS BETWEEN ANIMALS AND HUMANS A third approach to determining the rela- tionships among human and animal develop- mental toxicity is to compare observed effects when data are available for several species. Few human behavioral-teratology studies have been reported, and most ex- perimental behavioral-teratology studies have used rodents. The comparisons out- lined here reflect that situation. In addition, similarities and differences in design and conduct between experimen- tal and clinical research must be consid- ered in any comparison of results. The similarities between the two include the following: Physical growth and development are the most commonly measured end points. Several behavioral subsystems are assessed with a battery of functional tests. Experimental and control subjects are matched for maternal and environmental characteristics. The majority of studies are designed to provide descriptive information.

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292 In human studies, weight and motor devel- opment usually are measured for 1-2 years after birth. In rodent studies, weight is monitored repeatedly, most often throughout the duration of the study, and assessments of preweaning reflex develop- ment are often carried out as well. A battery of functional tests usually are used for neurobehavioral evaluation in both human and animal studies. The use of a single assessment technique that in- corporates multiple evaluations is most common in human research. The Apgar test (Apgar, 1953) is used routinely 1 and 5 minutes after birth; it consists of a 10- point scale based on five components: appearance (skin color), heart rate, la- tency of the cry reflex, muscle tone, and respiration. The Bayley scale of infant development (Bayley, 1969) is used common- ly for evaluation of older infants; it contains sensory, motor, verbal, and cog- nitive items, and results are summarized in motor and mental development scores. Neurobehavioral function in rodents is evaluated with a test battery that often includes assessment of reflex and sensori- motor development, activity level, and some evaluation of learning ability. Each category of function is evaluated with separate tests. Another similarity in study designs is the use of experimental and control subjects matched for maternal and environ- mental characteristics. In human studies, mothers are matched as closely as possible for age, parity, and nutritional and socio- economic status. In animal studies, mater- nal weight, parity, diet, and housing con- ditions routinely are controlled across groups. Both clinical and experimental studies designed to evaluate neurobehavioral outcomes after prenatal drug or chemical exposures provide primarily descriptive information. The methods permit a descrip- tion of functional deficits after insult, but not of underlying physiologic or neuro- chemical mechanisms responsible for the observed behavioral alterations. As noted above,-that situation is changing in animal studies. Multidisciplinary efforts can provide information on the mechanisms involved and thus might suggest types of intervention that could be effective in NEURODEVELOPMEI`ITAL TOXICOLOGY alleviating or improving clinical out- comes. Several basic differences in design and conduct between human and animal studies are common. They include differ- ences in the relative age range, in timing of administration of tests, in attempts at standardization, and in methods of re- porting results (Adams, 1986~. The relative age range studied is broader in much of the animal research than in human studies. Practically, it is very difficult to follow a prenatally exposed person for more than a year or two after birth. In the best of circumstances, clinical investigators can extend evalua- tions to 6 or 8 years of age. Funding, time requirements, and population mobili- ty and attrition all contribute to the difficulty. In contrast, rodent studies often include behavioral evaluations into early adulthood. Such a longitudinal ap- proach can be even more valuable if testing is identical across the age span studied. In human research, several functions usually are evaluated at a single age. Neurobehavioral function in rodents most often is evaluated through separate test- ing at different ages. Furthermore, unlike the results of multifunctional evaluations in humans, rodent performance across tests is not integrated into a sin- gle value or score. A greater effort is made in human than in animal studies to perform testing on infants in a comparable behavioral state, e.g., alert, drowsy, or asleep. Such con- trol contributes to both absolute response levels and decreased variability in be- havioral data collected in infants and children (Clifton and Nelson, 1976~. Ani- mal researchers at best attempt to control such factors by balancing time of day dur- ing testing across experimental groups. Developmental delays in physical, mo- tor, and cognitive end points are consid- ered more important in human than in animal studies. Such delays can be assessed only during particular periods of development. Their biologic meaning in rodents after prenatal exposures is not clear, and they have been viewed as problematic (Tilson and Wright, 1985~. One contributing factor might be the time disparity in postnatal developmental schedules between humans

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EXTRAPOLATIONFROMANIMAL STUDIES and rodents, i.e., months and years versus days. Finally, a characteristic difference between human and animal studies involves the method of reporting results. Clinical studies typically identify the incidence of behavioral dysfunction in individual control versus individual exposed sub- jects. Animal data usually are presented in terms of the presence or absence of group mean differences. Thus, the incidence of affected (and nonaffected) rodent offspring in the exposed group is not available. In the light of these differences in design, conduct, and reporting between human and animal behavioral-teratology studies, findings are discussed below if appropriate data were available for com- parison. The number of reported human studies was the limiting factor in the following brief overview; once those were identified, the animal literature was evaluated. In all cases, if human sub- system dysfunction was reported, corro- borative evidence was found in the animal data if a comparable end point had been eval- uated. In that manner, effects observed after developmental exposures to lead, mercury, PCBs, phenytoin, ethanol, and methadone are compared. Articles review- ing the spectrum of effects observed in humans and animals are cited in the follow- sing discussion. 293 Table 27-2 summarizes the comparability of neuromotor effects after exposure to particular toxicants. Delayed motor de- velopment has been reported in both humans and rodents exposed to lead (Rutter, 1980; Reiter, 1982), mercury (Reuhl and Chang, 1979), alcohol (Abel, 1980), and phenytoin (Hanson et al., 1976; Vorhees, 1983~. Cerebral palsy and seizure disor- ders have been reported in humans develop- mentally exposed to mercury, and motor dysfunction and increased susceptibility to seizure induction have been reported in rats and mice. Developmental exposures to PCBs have resulted in motor dysfunction in both humans (Jacobson et al., 1984) and mice (Tilson et al., 1979~. A prolonged neonatal abstinence syndrome with neuro- motor sequelae has been identified in human infants and rodents prenatally exposed to methadone (Hutchings, 1983~. The spe- cific motor alterations observed in humans and rodents were not always identical, but the normal behavioral repertoires of the two also are different. The data do indicate that the motor systems of humans and rodents are susceptible to disruption after developmental exposures to the agents in question. The clinical relevance of experimental data on cognitive functions is more dif- ficult to evaluate. Tests of human and animal cognitive abilities might evaluate very different functions; i.e., a rodent TABLE 27-2 Examples of Motor Dysfunction After Behavioral-Teratogen Exposuresa Agent Effects in Humans Effects in Rodents Lead Mercury PCBs Phenytoin Ethanol Methadone Delayed growth and motor devel- opment, motor incoordination, deficits in fine motor control Delayed growth and motor devel- opment, cerebral palsy, seizure disorders Depressed reflexes, delayed motor development Delayed growth and motor development Delayed growth and motor development Neonatal abstinence syndrome: tremors, sleep disturbances, hyporeflexia, irritability aData from Adams 1986. , Delayed growth and motor development Delayed growth and motor development, ataxia, seizure susceptibility Neuromotor weakness, poor balance Delayed growth and motor development Delayed growth and motor development Neonatal abstinence syndrome: hyperactivity, lability of state, sleep disturbances

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294 brain is not capable of the many complex functions evaluated in human assessments. Techniques used to assess cognitive func- tion measure responses that are modified by sensory and motivational processes, as well as motor capabilities. However, performance on such tests can provide use- ful information concerning the postex- posure integrity of underlying systems that contribute to an animal's or chills ability to learn, process, store, and re- trieve relevant information. Table 27- 3 summarizes some of the cognitive def- icits noted after developmental insult. Reduced general intelligence, as measured on standardized tests, has been found in some children who were exposed prenatally to lead (Needleman et al., 1979), mercury (Harada, 1976), ethanol (Streissguth et al., 1984), and phenytoin (Hanson et al., 1976~. The IQs of those children are often less than 70. Mental retardation is one of the most serious results of exposure to the agents in question. Attentional deficits have been reported in some chil- dren prenatally exposed to lead, mercury, and ethanol. Experimental studies have indicated impairments in visual recogni- tion memory in infants exposed to PCBs (Jacobson et al., 1985) and increased reac- tion times during a vigilance task in eth- anol-exposed children. The animal literature indicates im- paired learning and memory abilities in rodents after developmental exposures to the agents. Performance deficits on avoidance tasks have been reported after NEURODEVELOPMENTAL TOXICOLOGY exposures to lead (Kimmel et al., 1978), mercury (Spyker et al., 1972), PCBs (Tilson et al., 1979), ethanol (Abel, 1980), and phenytoin (Vorhees, 1983~. Results of water-maze tasks have indicated impaired function in rodents exposed to mercury (Spyker et al., 1972), ethanol (Abel, 1980), and phenytoin (Vorhees, 1983~. Hughes and Sparber (1979) found that pre- natal mercury exposure disrupted operant performance. It is interesting that prenatal metha- done exposure does not appear to alter cognitive performance in either humans or animals (Hutchings, 1983~. The fact that both humans and animals showed no effects on cognitive performance supports the utility of experimental-teratology data in assessing toxic effects. Sensory/perceptual processes have not been carefully evaluated in most be- havioral-teratology studies (Adams and Buelke-Sam, 1981; Ison, 1984~. As shown in Table 27-4, clinical case reports have suggested that some persons exposed in utero to ethanol (Clarren and Smith, 1978) or to phenytoin (Hill et al., 1974) have unspecified hearing defects. Visual impairments have been reported in some children exposed in utero to phenytoin (Wilson et al., 1978~. However, specific sensory functions have not been evaluated in animals that have been exposed prenatal- ly to alcohol or phenytoin. Vorhees (1983) reported delayed development of auditory responsiveness in rats treated with pheny- toin prenatally. TABLE 27-3 Examples of Cognitive Dysfunction After Behavioral-Teratogen Exposuresa Agent Effects in Humans Effects in Rodents Lead Decreased general intelligence, decreased attention span, impaired verbal ability PCBs Phenytoin Ethanol Mercury Decreased general intelligence, decreased attention span Impaired visual recognition memory Decreased general intelligence Decreased general intelligence, decreased attention span, delayed reaction time Methadone None reported Impaired learning ability on passive avoidance and T-maze tasks Learning deficits on many tasks Impaired learning on avoidance tasks Impaired spatial learning on water-maze tasks Learning deficits on many tasks None reported aData from Adams, 1986.

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EXIRAPOLATIONFROMANIM41L STUDIES TABLE 274 Examples of Sensory/Perceptual Processing Dysfunction After Behavioral-Teratogen Exposuresa Agent Effects in Humans Effects in Rodents 295 Lead Decreased visual acuity, altered brain Decreased visual acuity, altered brain electrophysiologic response to visual electrophysiologic response to visual stimulation stimulation Mercury Unspecified hearing defects, altered Increased reactivity to auditory startle stimuli reactivity to visual and auditory stimuli PCBs None specifically evaluated None specifically evaluated Phenytoin Unspecified hearing defects and visual Delayed development of auditory startle impairments in some cases response Ethanol Unspecified hearing defects in some None specifically evaluated cases Methadone Deficits in visual, auditory, and Hyperreactivity to aversive stimuli tactile perception, but no specific sensory deficits aData from Adams, 1986. Decreases in visual acuity have been reported to occur in children (Rummo et al., 1979), rats (Fox et al., 1977, 1979; Fox and Wright, 1982), and monkeys (Bushnell et al., 1977) after exposure to inorganic lead. Altered electrophysio- logic brain activity in response to visual stimulation has also been found in lead- exposed children (Otto et al., 1981, 1982, 1985; Otto and Reiter, 1983) and rats (Fox etal.,1979~. Fetal exposure to methylmercury has been reported to produce postnatal altera- tions in reactivity to visual and auditory stimulation in humans (Harada, 1976, 1977~. Studies done on prenatally exposed rats have shown increased reactivity to auditory stimuli (Buelke-Sam et al., 1985~. Wilson et al. (1979) reported that chil- dren prenatally exposed to methadone had deficits in visual, auditory, and tac- tile perception, but these were inter- preted as resulting from poor attentional and strategic processing abilities, rath- er than from specific sensory deficits. Lodge (1976) reported alterations in brain electrophysiologic responses to visual stimuli and hypersensitivity to auditory stimulation in children exposed to metha- done before birth. The integrity of sen- sory functioning has not been specifically evaluated in studies carried out in ro- dents, but hypersensitivity to aversive stimulation has been reported by Hutchings (1983~. In nearly all the cases outlined above, the clinical problem was identified before the development of animal models to explore such toxicity. The literature of animal behavioral teratology has expanded great- ly in recent years and suggests that a num- ber of additional drugs and chemicals war- rant clinical investigation. However, the potential value of animal data in pre- dicting human hazard cannot be determined fully until clinical studies to look for behavioral dysfunctions identified in experimental animals are designed and conducted. DATA INTERPRETATION We have discussed many problems that contribute to the difficulty of evaluating the relevance of animal data in identifying potential health hazards in developing humans. The first three issues that follow bear on the interpretation of both human and animal data; the last two are related to problems in cross-species extrapola- tion of results. The first problem centers on determining whether behavioral-teratology findings are a result of primary developmental toxi- city or are secondary to maternal toxicity or to primary toxicity produced in other organ systems, e.g., liver or kidney. The heart of the issue is the relative suscep- tibility of the developing organism (whether human or animal) to toxic insult. If postnatal dysfunction only accompanies maternal toxicity, such information might be of value in alerting clinicians to moni-

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296 tor such pregnancies more closely. If postnatal functional deficits are ob- tained in the absence of overt toxic signs, the potential for selective developmental toxicity must be considered in human risk assessment. Genetics might play a large role in sus- ceptibility or expression of postnatal dysfunction. Genetic makeup could predis- pose the parents or offspring to greater sensitivity to a toxicant. Toxicants could produce postnatal dysfunction via genetic mechanisms, or such dysfunction could be transmitted to later generations (cf. Fujii et al., 1987; Stoetzer et al., 1987~. Thus, whether developmental-toxicology studies are performed with inbred strains of mice or in the highly diverse human and whether one or both parents have been ex- posed to a toxicant are important consider- ations in evaluating and interpreting postnatal functional data. The postnatal environment can have an impact on developmental toxicity, maxi- mizina or minimizing the expression of NEURODEVELOPMENTAL TOXICOLOGY enrichment might contribute to the mani- festations of toxicity. Once those issues are considered, two additional aspects must be dealt with in determining the relevance of animal data to the human situation. The first concerns the disparity in timing of organ-system development across species. The rat gesta- tion period covers approximately 3 weeks, and CNS development, including cell dif- ferentiation and migration, continues into the immediate neonatal period. More CNS development occurs in humans during the 9-month gestation period, although completion of histogenesis does not occur until well after birth. Agent exposure is timed specifically in animal studies, and care is taken to standardize doses within treatment groups. Such control usually is not available in human studies, and retrospective investigations often rely on maternal reporting of exposure to drugs and when it occurred. Postnatal development schedules also differ between humans and animals. Post- damage. In human studies, the role of ma- natal development through puberty in a ternal socioeconomic status is great in rat requires approximately 6 weeks. It that regard, as well as contributing to takes years to reach that stage in humans. Overall prenatal and perinatal status. Thus, prenatal and postnatal disparities In animal research, controlling litter in timing must be accounted for, as well size is one means of standardizing this as the variations in agent exposures during factor. In both types of research, the the comparative process. degree of environmental experience and