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Appendix B Toxicology of Lead in Experimental Animals SELECTION OF A PROPER MODEL Ethical considerations dictate that potentially dangerous experiments involving toxic metals be carried out in animals, not man. Unfortunately, no one species of animal is a perfect model for man. Therefore, it is crucial to insure that the organs or systems known to be affected by the toxic metal in man are closely approximated in the experimental animal species. Many recent animal studies involving lead have sought to study lead's effect on the nervous system. If the results of these studies are to be used to predict comparable effects in young children, then the stage and rate of brain growth in the animals studies are of crucial importance. Neural Development in Humam; - Bobbing and Sands have described the quantitative growth and development of the human brain for the period from 10 weeks gestational age to seven postnatal years. The "growth spurt," the time of most rapid growth, begins in humans during mid- pregnancy. Three major components of the brain were examined to delineate the period of growth spurt. Glial replication and differentiation extends to at least the end of the first postnatal year and quite possibly beyond 18 months (see Fig. 3). Myelination continues into the third and fourth years (Fig. 4). Cerebellar growth is most rapid during the first 18 months of postnatal life (Fig. 5 and Fig. 6). Approximately 83 percent of the human brain growth spurt is postnatal.^5 Sutdies of malnutrition in human infants have shown that the brain is particularly vulnerable during the growth spurt. Klein and associates studied the relationship between starvation, caused by pyloric stenosis, and intelligence. Pyloric stenosis occurs between birth and three months of age, is surgically correctable and is not associated with any particular socioeconomic or cultural group. Klein found that the brief period of starvation in infancy, prior to surgery, had permanent effects on learning abilities and general adjustment, as measured 5-14 years later. Hertzig et al^. found reduced I.Q. levels in school-age boys who had been malnourished during the first two years of life. In humans, the initial exposure to lead in paint usually coincides with ambulation and so begins at 10-12 months postpartum, while exposure to lead from some canned nutrients may begin at or shortly after birth. Neural Development in Experimental Animals - Bobbing and associates have demonstrated the vulnerability of the developing brain to moderate hyponutrition in experimental animals.2 Hyponutrition occiiring during the growth spurt produced a permanent reduction in both body and brain weight as well as behavioral changes. Hyponutrition before this critical period had less severe effects on CNS development. 19

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The growth spurt in rats occurs during the first 25 days post- partum. Glial cell multiplication occupies the first half of this period. The second half extending to about the 25th postnatal day, is a period of rapid myelination. Dendritic authorization and synaptic connections are also occurring during this period, along with dramatic metabolic and neurochemical development and rapid cerebral growth. Demonstrable and permanent clumsiness is associated with cerebellar deficits caused by hyponutrition during the growth spurt." Figure 7 shows the velocity of human brain growth compared to the rat, pig and guinea pig. Striking differences are apparent in relation to the stage of brain development at time of birth. There is a dearth of information regarding the rate of brain growth in primates other than man. Nothing could be found concerning the rate of brain growth in the baboon which would have permitted a direct comparison of the CPSC studies with man. However, Portman and associates,76,77 in studying the. rhesus monkey, found that 70 percent of the adult brain weight was obtained by 165 days gestational age (mean age of birth). Allen and associates-5 produced obvious behavioral abnormalities in infant rhesus monkeys exposed to lead. No obvious behavioral abnormalities occurred in adolescent or adult mankeys exposed to a similar dose. These findings are consistent with the concept that the baboons in the CPSC studies were beyond the comparable period of growth spurt in young children. Absorption Factor in Relation to Age - Studies in both humans and animalsl3,31,43 indicate that the rate of absorption of lead from the gastrointestinal tract is greater in the young than in the adult. Studies in rats (See Tables Bl and Bl.a) illustrate the rapid decrease in absorption of lead as age increases from birth. The balance data of Alexander ^t al in children are too limited to permit any statement concerning possible differences in the rate of absorption during early childhood; however, the average absorption of dietary lead found in these children (53%) is substantially greater than the 5 to 10% absorption found in adults. Scientists seeking to evaluate the CNS effects of lead in human infants should select animals experiencing rates of brain growth and rates of intestinal absorption comparable to the human infant. •^ , £ Q Momcilovic and Kostial00 found that the uptake of lead in the brain of suckling rats was six to eight times greater than that found in the brain of the adult rat. Krigman et^ al, by adding PbCC>3 to the diet of the mother, induced a four-fold increase of lead in the brain of sucklings over the amount found in the mother.53,54 Total brain growth was inhibited and myelin production was reduced in the brain and in the sheath about the axons. Reduced amounts of galactolipids, cholesterol, plasma- logens and total phospholipids were observed in these animals. No data were reported for the lead content of milk or blood. SELECTION OF PROPER EXPERIMENTAL CONDITIONS Once the proper model has been selected to simulate a comparable rate of brain growth and comparable rate of intestinal absorption in 20

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man, the experimental conditions, particularly those relating to diet and method of lead administration, should be selected to simulate those conditions seen in man. Diet - Most experimental animals are fed optimal diets. Studies in rats47,64,92 reveal that dietary deficiencies of calcium, copper and iron increase the absorption of lead from the intestinal tract. Dietary deficiencies have been shown to exist in a significant number of American Children.1'98 In addition, fats and milk have been found ' to increase the absorption of lead in experimental animals. '' In two groups of rats of the same age, lead absorption decreased to a greater extent in the group switched from a milk diet to a dry dood diet, while those who continued to receive a milk diet showed only an age-related decrease in absorption (see Table I, p. 28). Experi- mental animals receiving dry feed diets containing all nutrient require- ments do not simulate a child's diet, which may contain both fats and milk and which may be inadequate in other respects. Method of Lead Administration - Administration of lead by injection in animals is the easiest method of determining the exact quantity administered, but does not simulate the method of exposure in man. It is difficult to determine the amount of lead ingested from loose feed, some of which the animal scatters about his cage. Studies designed to determine the rate of gastrointestinal absorption in relation to dose administered must contain a provision for accurately measuring the quantity of lead actually ingested by the animal. In order to make valid comparisons, the chemical form of lead administered to the animal should be the same as that ingested by a young child. Measures of Internal Dose - Many animal studies do not provide a measure of internal dose such as blood lead or tissue lead levels. Because of this, it is difficult to compare the results to human studies in which levels of lead in the blood are known. Measures of Subtle Metabolic^ or Functional Effects - Experiments designed to produce dramatic effects, such as death, are only the first step in demonstrating the toxic effects of lead. We do not believe that the absence of dramatic clinical symptoms at a particular dose level demonstrates the safety of that dose. Testing of animals for subclinical metabolic or functional effects, particularly those effects seen in the hematopoietic and neurologic systems, would be far more helpful in attempting to extrapolate the results of such studies to humans. RESULTS OF LEAD EXPOSURE IN YOUNG EXPERIMENTAL ANIMALS 13 The work of Brown appears to be an appropriate experimental model in terms of dose administered, age and neurodevelopmental stage. In addition, blood lead levels (Pb-B) were determined. Brown used suckling rats to investigate the vulnerability of the brain in relation to its developmental stage. Lead acetate was administered to the dam by gavage (35 mg/kg/day). The pups were dosed through the maternal 21

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milk either during days 1-10 or days ll-21. No further exposure to lead occurred throughout the study and the dose was controlled to prevent impaired physical growth. Suckling rats fed maternal milk dosed with lead during postnatal days 1-10 showed significantly slower learning compared to those fed maternal milk with equal doses of Pb during days ll-21. Learning ability in the ll-21 day group was not significantly different from controls. Blood lead levels were significantly higher in the 1-10 day group (45.8 yg Pb/dl) than in the ll-21 day group (20.4 yg Pb/dl), which did not differ significantly from controls (21.75 ug Pb/dl). The higher blood lead levels found in the younger rats suggest a higher absorption rate for Pb during the 1-10 day period than during the ll-21 day period. It is significant that learning deficits persisted at the 8-10 week level, even though Pb-B levels had returned to normal. The persistence of effect was also seen in lambs after Pb-B levels had returned .to normal. In an attempt to produce slowed learning in the ll-21 day group, Brown administered higher lead doses to the dams (35, 70 and 140 mg Pb/kg/day). Only when the dose was increased four-fold, did the ll-21 day group show slowed learning comparable to that found in the 1-10 day group. This indicates that the brain is still vulnerable at the latter stage of development, but that a much higher dose is required to produce the same effect as seen in the younger animal. Additional animal experiments show impaired CNS function due to lead. However, none contain the combination of appropriate dose, age, neuro-developmental status and blood or tissue levels seen in Brown's study. The most common information lacking is the blood lead concentration. Pentschew and Garro introduced an experimental model for studying the development of lead encephalopathy. '* At parturition, maternal rats were fed a diet containing 4 percent lead. The sucklings received maternal milk containing 45.9 ppm Pb. Paraplegia was observed in 90 percent of the young animals near the end of the suckling period (23-29 days) and 85-90% of the paraplegic animals died. There were no data on blood lead levels in the pups. Rosenblum and Johnson"-' used this model, but used mice fed smaller doses of lead than did Pentschew and Garro. These mice had a high mortality rate, retardation of growth, delayed eye opening, broad-based gate, poorly developed righting reflex and changes in vascular and glial cells. There were no data on the lead concentrations in maternal milk or suckling's blood. More recently, Michaelson and Sauerhoff, using a modification uf the lead-in-maternal milk feeding model, were able to produce hyperactivity, aggressiveness, tremors, and repetitive grooming behavior without extensive histopathology.65'66 The maternal milk contained approximately 25 ppm lead. No blood lead concentrations were reported. Both Golter and Michaelson,35 and Silbergeld and Goldberg,90 have experimentally produced hyperactivity using this same model. Golter and Michaelson found a slight increase in norepinephrine. Silbergeld and Goldberg's work linked hyperactivity to altered catecholamine metabolism. In addition, they applied current drug therapy, used in the diagnosis and treatment 22

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of hyperactive children, to their control and experimental animals. CNS stimulants (d- and _l-amphetamine and methylphenidate) suppressed hyperactivity, whereas phenobarbital increased the activity in the animals exposed to lead. The same drugs given to control animals produced the opposite effects. Chloral hydrate suppressed the activity in both groups. The suppressed activity from ^-amphetamine is similar to the response observed in some hyperactive children.90,91 There were no data on the lead concentration of the maternal milk or of the suckling's blood. Sobotka and Cook were able to demonstrate long-term behavioral deficits in neonatal rats administered oral doses of lead. Initially, the dose level did not produce obvious CNS disturbances. Feeding started at 3 days and continued through day 21 at dose levels of 9, 27 and 81 mg lead/kg body weight. Blood lead concentrations performed after 35 days showed 9 yg Pb/dl for control animals and 24 yg Pb/dl for t.hose receiving the highest dose. Activity in the high-dose lead group was decreased by administration of 3 mg amphetamine/kg body weight. We believe that properly designed animal studies, simulating conditions in human infants, are needed to identify the relationships between external dose (dose of lead administered), absorption rate (at various ages), internal dose (blood or tissue lead levels), vulnerability of the brain (at various ages), time between exposure and appearance of effect, and permanence or reversibility of effect. 23

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0J FIGURE 3. Velocity curves showing incremental rates of DNA (2 peaks, ), cholesterol (single peak, ) and fresh weight ( ) in whole human brain. Note the bimodel curve for DNA, representing neuroblast followed by glial multiplication. (From Dobbing.26 Bibl. "Nutr. Diet." 17:36-45, 1972. Publisher, S. Karger AG, Basel. Reprinted with per- mission. ) 30 o « 2O o -c u IO WHOLE BRAIN Birth 2468 Adult Aqe (years) FIGURE 4. Total cholesterol in whole brain -rlt—i o •j (From Dobbing and Sands. Arch. Dis. Child. 48:757-767, 1973. Ed.: Douglas Gairdner and Roger Robinson. Reprinted with permission.) 24

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KX> o > 75 "5 I so Brain brebrain .tern "crebcllum Birth Aqc (years) FIGURE 5. Comparative fresh weights of 3 brain regions during growth. Weights for forebrain, cerebellum, and stem have been calculated as a percentage of adult value, and smooth lines drawn by eye through the points. IOO I" o -50 41 25 1 Forebrain Stem Cerebellum Birth Aqe (years) FIGURE 6. Comparative values for total DNA-P, equivalent to to- tal numbers of cells (see text), in 3 brain regions. Values shown for forebrain, cerebellum, and stem have been calculated as a percentage of adult value, and smooth lines drawn through the points. (From Dobbing and Sands.27 Arch. Dis. Child. 48:757- 767, 1973. Ed: Douglas Gairdner and Roger Robinson. Reprinted with permission.) 25

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w X S gz O H-I A OH < " O OS (Q 3 3 S ^ g * 5 O fe O M H H _ ^55 2 « w * CJ s w Oc / Rat / % / ••..., ^x "V. *^ ^ v ** •*••• ^•s.^ " -.-r'rSRas 30 -20 AGE -10 A BIRTH 10 20 30 AGE FIGURE 7. Velocity of human brain growth (wet weight) compared with that in other species. Prenatal and postnatal age ex- pressed as follows: human in months; guinea pig in days; pig ------ in weeks; rat -.-.-.-.-.-. in days. 25 (From Dobbing. Pediatrics 53:2-6, Jan. 1974. Re- printed by permission.) 26

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•0 80 TO '•0 i» •rt, 40 i 3" SO ! 10 90 ISO TO 210 250 290 330 37O 4O 4SO 490 530 >2yn. GESTATIONAL. ACE (DAYS) FIGURE 8. The relationship of weight of parts of the brain to gestational age of rhesus monkeys. (From Portman et^ 43:197-213, 1972. permission.) . Brain Res. Reprinted by 27

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Table I The Effect of Milk on the Absorption of Lead Age of Rats Rats Receiving Milk Diet C°nt203 Rats* (days) (Percent203Pb absorbed) (Percent Pb absorbed) 9 65.01 (16) 71.50 (12) 15 70.46 (15) 65.28 (16) 25 57.47 (14) 6.75 (15) 37 52.72 (17) 2.50 (15) * Control rats received only milk until day 15, when they began eating their mother's food. Numbers in parentheses show the number of animals in each group. Adapted from Kostial, K., Kello, D., Jugo, S. and Gruden, N.: The effect of milk diet on toxic trace element absorption in rats. Pre- sented before the XVIII International Congress on Occupational Health, Brighton, England, Sept. 14-19, 1975.51 Table I-a Gastrointestinal absorption as a function of age (avg±SD) Age »F« «Sr mpb days % % % 13 100.2* 9.9 ( 4) ' 14 90.0±11.2 ( 8) 15 97.0± 9.4 (12) 84.8 ± 6.4 (7) 16 90.1* 8.0 ( 9) 83.3± 3.3 (6) 17 86.5 ± 9.7 ( 7) 79.4 ± 6.2 (8) 18 84.9* 3.6 (6) 19 71.1 ±13.7 (16) 20 78.4 ±15.1 ( 5) 73.1 ± 8.5 (5) 89.7* 6.7 (6) 21 72.5±31.5 ( 6) 22 32.1 ± 9.9 (18) 54.4*10.6 (7) 74.0±13.3 (6) 23 25 6± 12.1 (14) 24 35.6±15.0 (7) 42.4±11.9 (6) 25 26.6± 4.8 ( 4) 27 19.1 ± 4.4 ( 8) 36.9*10.2 (6) 29 23.1 ±13.5 ( 7) 32 15.2*12.3 (6) 33 9.7* 2.5 ( 8) 37 15.6± 4.6 ( 8) 39 15.3±11.8 (6) 89 + 4.7* 3.7 (14) 8.2± 8.1 (5) 16.0* 3.4 (3) 1 Number of animals in parentheses. (Adapted from Forbes and Reina.31 J. Nutr. 102:647-652, 1972. Re- printed by permission.) 28