Several studies have reported a greater retention of manganese in the neonate than in adults. In a study of the nutritional requirements for manganese in pre term infants, Zlotkin and Buchanan ( 1986) showed that 99% of the manganese given intravenously for 6 days was retained. Mena ( 1969) observed that healthy adults absorb 3% of ingested manganese. Lonnerdäl et al. ( 1987) showed that manganese uptake from brush border membranes was higher in 14 day old rats than in 18 day old rats. Although Rehnberg et al. ( 1985) found that younger animals had a slower distal intestinal transit time than older animals ( potentially contributing to a higher proportional uptake), Bell et al. ( 1989) showed that the uptake rate was similar in preand post weanling animals suggesting that age dependent differences in manganese retention were not due to immature intestinal transport mechanisms.

Dorner et al. ( 1989) studied retention of manganese in breast fed infants compared to preterm (~ 34 36 weeks gestational age) or full term ( 2 17 weeks postgestational age) infants fed cow's milk formulas. This study is unique in that it analyzed potential differences in infant 6 3 Manganese   February 2003 development on the intake and retention of manganese from different dietary sources. The authors observed that full term breast fed infants retain approximately 41% of ingested manganese from breast milk ( containing 6.2 µ g Mn/ L). Manganese intake in the formula fed infants ( 14.2 µ g/ kg, full term and 15.0 µ g/ kg, pre term) was high relative to that of breast fed infants ( 1.06 µ g/ kg). Formula fed infants also retained a higher absolute amount of manganese from their diet compared to breast fed infants ( 0.06, 2.8, and 0.43 µ g/ kg retained in pre term formula fed, full term formula fed, and breast fed, respectively). These data indicate that the percentage of manganese retained between the different food sources is not comparable; a higher percentage of ingested manganese from breast milk is retained by the infant. Nevertheless, formula fed babies retain a larger total amount of manganese, due to the greater amount of manganese present in the formula ( 77 99 µ g/ L). The data also indicate that pre term infants had an active excretory capacity for manganese obtained from formula, as compared to full term infants.

Because human breast milk contains low levels of manganese ( 4 10 µ g/ L; Arnaud and Favier, 1995; Collipp et al. 1983; Dorner et al. 1989), it is suggested that the neonates' propensity to retain greater amounts of manganese was an adaptive mechanism to insure that sufficient amounts were available to the developing animal. Regardless of the mechanism ( e. g., increased uptake and/ or decreased elimination), results from human and animal studies suggest increased manganese retention in the neonate. Neurological development in the rat is incomplete at birth, suggesting that there may be differential susceptibility to excess levels of manganese during this critical developmental period. Although much of the nervous system is complete at birth in humans, there is evidence that some discrete neurological functions undergo further development after birth. The developmental stage in humans that is exactly comparable to the pre weanling age in rats is unclear. Although results from animal data suggest that elimination rates reach adult levels by the age of weaning, the comparable period in human development at which manganese uptake and elimination reaches that of an adult is unknown.

Factors that Affect Absorption in Humans Bioavailability of ingested manganese is an important issue in assessing the health hazard of manganese. Multiple factors have been reported to affect the absorption of manganese by humans, including chemical form, age, dose, route of exposure, and presence or deficiency of other dietary components ( Greger, 1999; Greger and Snedeker, 1980). Thomson et al. ( 1971) and Gibbons et al. ( 1976) reported that the divalent form of manganese is absorbed most efficiently. However, the efficiency of absorption also varies for different manganese salts. In this regard, Bales et al. ( 1987) reported that manganese chloride was more efficiently absorbed than the sulfate or acetate salts.

Presence of other dietary components may influence the absorption of manganese. Calcium, for example, may inhibit the absorption of manganese. McDermott and Kies ( 1987) suggested that this inhibition results from the influence of calcium on GI tract pH. Manganese is more readily absorbed as the Mn( II) form. As the pH rises, conversion to the less absorbable Mn( III) and Mn( IV) forms is favored, and uptake is decreased. Alternatively, calcium and manganese may compete for common absorption sites. The extent to which calcium effects on absorption influence net manganese balance is uncertain. However, Spencer et al. ( 1979) did not 6 4 Manganese   February 2003 observe any significant effect of dietary calcium levels ( from 200   800 mg/ day) on manganese balance in healthy males.

A strong association between dietary iron and manganese uptake has been noted in several human studies. Thomson et al. ( 1971) observed that iron deficiency increased manganese absorption. Davis and Greger ( 1992) reported that women consuming increased levels of nonheme iron experienced decreased levels of serum and urinary manganese. Finley et al. ( 1994) observed that serum sodium ferritin concentration was negatively associated with manganese absorption in young women consuming a manganese adequate diet.

Finley ( 1999) demonstrated that iron status ( assessed as serum concentrations of sodium ferritin) may also affect manganese absorption and retention. Absorption ( determined by regression of whole body 54Mn counts) was assessed in women aged 20 to 45 years who were categorized as having high ( upper 10% of normal range, mean values 68 to 69 : g/ L) or low ( lower 10% of normal range, mean values 8.7 to 8.9 : g/ L) serum ferritin levels. Absorption was determined under conditions of high ( 9.5 mg Mn/ day) or low ( 0.7 mg Mn/ day) dietary manganese intake. Within a diet group, individuals with low ferritin absorbed 3 to 5 fold more manganese ( as a percentage of dose) than individuals with high ferritin. Manganese absorption was greatest in women with low serum ferritin concentrations consuming the low manganese diet. The level of dietary manganese had no significant effect on absorption in women with high ferritin concentrations.

Phytate, a component of plant protein, may also interfere with manganese absorption. Davies and Nightingale ( 1975) observed a decrease in manganese retention in the presence of phytate. This result was attributed to the formation of a stable complex between manganese and phytate in the intestinal tract. Bales et al. ( 1987) reported that cellulose, pectin, and phytate reduced the plasma uptake of manganese in human subjects. These data suggest that the presence of these components contributes to the decreased bioavailability of manganese from vegetarian diets. However, Schwartz et al. ( 1986) found no significant correlation between phytate intake and manganese absorption in healthy males.

Ruoff ( 1995) conducted a literature review to determine the relative bioavailability of manganese from water versus food. The calculated ratio following evaluation of a wide variety of exposure scenarios in non fasted subjects was 1.4. However, the difference in absorption between the two media was not statistically significant. The ratio for fasted subjects was 2.0, indicating that the absorption from drinking water is twice that from foods when the water is consumed in the absence of partially digested foods in the gastrointestinal tract. A study that directly measured the absorption of radiolabelled manganese from various manganese rich plant foods given to adult men and women after an overnight fast reported a significantly greater percent absorption of MnCl 2 from water compared to manganese absorption from lettuce, spinach, sunflower seeds, or wheat ( Johnson et al., 1991). In addition, different diets may have different levels of constituents that affect manganese absorption. The greater levels of phytates, tannins, oxalates, and fiber in vegetarian diets, for instance, are expected to have an inhibitory effect on manganese uptake from the gastrointestinal tract. Johnson et al. ( 1991) reported mean percent absorption values from lettuce and spinach of 5.20 and 3.81%, respectively, and from 6 5 Manganese   February 2003 sunflower seeds and wheat of 1.71 and 2.16%, respectively. Mean percent absorption values from MnCl 2 dissolved in water only ( controls) ranged from 7.74 to 10.24%.

Animal Studies There are studies using 54Mn labeled manganese to estimate absorption by animals. However, these studies measured the apparent absorption, not true absorption., because feeding radioactive isotopes of manganese does not eliminate the problem that absorbed manganese is very rapidly excreted through bile into the feces ( Malecki et al., 1996). Thus, it is impossible to separate non absorbed manganese from secreted manganese without elaborate study designs. When investigators used elaborate methodology in which 54Mn bound to albumin was injected intraportally, true manganese absorption was calculated to be 8.2%, and 37% of the absorbed manganese was excreted into the gut ( Davis et al., 1993).

Greenberg et al. ( 1943) administered a single oral dose containing 0.1 mg of 54Mn labeled manganese ( as chloride) to rats, and estimated that 3   4% was absorbed from the intestine. Pollack et al. ( 1965) administered a single oral dose of 54Mn as chloride with 5 : mol ( 0.27 mg Mn) stable carrier to fasted rats and reported 2.5   3.5% absorption 6 hours after administration. In separate studies, Rabar ( 1976) and Kostial et al. ( 1978) administered a single oral dose of 54Mn as chloride, carrier free, to post weaning non fasted rats and reported 0.05% absorption 6 days after administration. This low absorption value may reflect either loss of absorbed manganese through fecal excretion, or the fact that the rats were not fasted ( U. S. EPA, 1984).

Cikrt and Vostal ( 1969) showed that manganese is likely to be absorbed from both the small and large intestine in rats. Factors reported to influence manganese absorption in animals include dose, chemical form, and age. With respect to dose, Garcia Aranda et al. ( 1983) studied the intestinal uptake of manganese in adult rats and concluded that saturation of the absorptive process occurred at higher levels of intake. Keen et al. ( 1986) observed that when suckling rats were fed 0.5 mL of infant formula containing 5 or 25 mg Mn/ mL, retention of manganese decreased at the higher concentration.

Tissue levels of manganese may be influenced by the form of manganese administered in the diet. Komura and Sakamoto ( 1991) administered manganese in soluble ( manganese acetate or manganese chloride) and relatively insoluble ( manganese dioxide or manganese carbonate) forms to male ddY mice. Weight gain was reduced in animals receiving the more soluble forms. Manganese levels in the liver and kidney appeared to be higher in animals fed manganese acetate or manganese carbonate. The statistical significance of these apparent differences was not determined.

Keen et al. ( 1986) demonstrated a strong effect of age on intestinal manganese uptake and retention. Sprague Dawley rat pups were fasted overnight and then intubated with 0.5 mL of human milk containing 0.005 mg 54Mn/ mL. Manganese retention was highest ($ 80%) in pups less than 15 days old. In older pups ( 16   19 days old), the average retention was 40%. Keen et al. ( 1986) also administered infant formula to rat pups. Soy formula typically contains a much higher level of Mn than does human milk. The amount of manganese retained in 14 day old rat 6 6 Manganese   February 2003 pups was 25 times higher in animals given soy formula when compared with pups receiving human milk.

Chan et al. ( 1987) demonstrated that developmental stage has a significant influence on the absorption of manganese. Manganese absorption decreased in rat pups from age 9 days to 20 days. The observed decrease in manganese absorption was correlated with a switch in the site of maximal absorption. The duodenum was more active in manganese uptake in younger rats, while the jejunum became more important as the animals matured.

Little is known about the factors that determine the bioavailability of ingested manganese in animals. Chan et al. ( 1982, 1987) reported differences in the concentration and chemical form of manganese found in different milk sources. Human milk contained only 0.008 ± 0.003 mg Mn/ L, while bovine milk, infant formula and rat milk contained 0.030 ± 0.005, 0.073 ± 0.004, and 0.148 ± 0.018 mg Mn/ L, respectively. However, absorption of manganese by suckling rats from these four types of milk was comparable, suggesting that total concentration may not always be a reliable indicator of bioavailable manganese. Chan et al. ( 1982) determined that the chemical form of manganese in infant formula is very different from that in human or cows' milk. Human and cow's milk contain two and three manganese binding proteins, respectively. All manganese in milk from these sources is protein bound, while the manganese in infant formulas is in the form of soluble salts. The degree to which the association of manganese with protein influences absorption is unknown, but is likely to be important.

Lönnerdal et al. ( 1987) reported that age, manganese intake and dietary factors affect manganese absorption and retention in rats. Retention is very high during the neonatal period and decreases considerably with age. Decreased absorption with age apparently results from a combination of decreased intestinal absorption and increased excretion in the bile. In young rat pups, the bioavailability of manganese from various milk sources varied, with greater absorption occurring from human and cow's milk formula than from soy formula. These differences were less pronounced in older pups.

Several studies have explored the interrelationship among manganese, cobalt, and iron uptake. Thomson et al. ( 1971) reported that iron and cobalt compete with manganese for the same absorption sites. Competition was proposed to occur during uptake from the lumen into mucosal cells and in the transfer from mucosa into other compartments. Rehnberg et al. ( 1982) administered dietary Mn 3 O 4 ( 450, 1,150, or 4,000 mg/ kg Mn) to young rats. These authors amended the basal diets with varying levels of iron, and demonstrated that iron deficiency promoted the intestinal absorption of manganese. Conversely, manganese absorption was inhibited by large amounts of dietary iron. Gruden ( 1984) demonstrated that 3 week old rat pups given a high concentration of iron ( 0.103 mg Fe/ L) in cow's milk absorbed 50% less manganese than pups receiving the control milk ( 0.005 mg Fe/ mL). This difference was not observed in rats tested at 8, 11, 14, or 17 days of age, suggesting that the inhibition of manganese absorption by iron has a rapid onset during the third week of life. 6 7 Manganese   February 2003 6.2 Distribution Human Studies Manganese is a normal component of human tissues and fluids. Information about the distribution of manganese in humans is generally derived from post mortem analyses of various organs and tissues. The patterns observed in these analyses reflect the body and organ burden of a lifetime intake of manganese. Cotzias ( 1958) and WHO ( 1981) reported a total of 12 20 mg manganese in a normal 70 kg man. Sumino et al. ( 1975) reported an average of 8 mg among 15 male and 15 female cadavers with an average weight of 55 kg.

The highest concentrations of manganese in the body of persons without excessive exposure are found in the liver, kidney, pancreas, and adrenal glands. Intermediate concentrations occur in the brain, heart and lungs ( Table 6 1) ( ATSDR, 2000). The lowest concentrations of manganese are observed in bone and fat. Some data suggest that tissues rich in mitochondria ( for example, liver, kidney, and pancreas) contain higher levels of manganese ( Kato, 1963; Maynard and Cotzias, 1955).

Manganese levels have been determined in human serum and blood. Serum concentrations in healthy male and female subjects in Wisconsin were 1.06 : g/ L and 0.86 : g/ L, respectively ( Greger et al., 1990; Davis and Greger, 1992). Blood and serum levels of manganese in healthy subjects living in the Lombardy region of Italy were 8.8 ± 0.2 : g/ L and 0.6 ± 0.014 : g/ L, respectively ( Minoia et al., 1990).

A variety of factors have been reported to influence manganese levels in blood and blood fractions. Hagenfeldt et al. ( 1973) found variations in plasma manganese concentrations in women and suggested that the variation may be due to hormonal changes. Horiuchi et al. ( 1967) and Zhernakova ( 1967) found no difference in the concentration of manganese in the blood of men and women. Slight seasonal ( lower during summer and autumn) and diurnal ( lower during the night) variations in blood manganese concentrations have also been reported ( U. S. EPA, 1984).

Three studies have addressed manganese distribution within human organs. Perry et al. ( 1973) investigated manganese concentrations in different sections of the liver and found little variation. Larsen et al. ( 1979) and Smeyers Verbeke et al. ( 1976) studied the regional distribution of manganese in the brain and reported the highest concentrations in the basal ganglia.

Studies by Schroeder et al. ( 1966) and Widdowson et al. ( 1972) indicate that placental transfer of manganese occurs in humans. Manganese levels in fetal and newborn tissues were reported to be similar to adult levels, with the exception of higher concentrations observed in fetal bone. 6 8 Manganese   February 2003 Table 6 1. Normal Manganese Levels in Human and Animal Tissues.

Tissue Tissue concentrations (: g Mn/ g wet weight)

Humans Rats Rabbits A B C D Liver 1.68 1.2 2.6   2.9 2.1 Pancreas 1.21 0.77  1.6 Adrenals 0.20 0.69 2.9 0.67 Kidney 0.93 0.56 0.9   1.0 1.2 Brain 0.34 0.30* 0.4 0.36 Lung 0.34 0.22  0.01 Heart 0.23 0.21  0.28 Testes 0.19 0.20 0.4 0.36 Ovary 0.19 0.19  0.60 Muscle 0.09 0.09  0.13 Spleen 0.22 0.08 0.3 0.22 Fat  0.07

Bone ( rib)  0.06

Pituitary   0.5 2.4 Adapted from ATSDR ( 2000) A Tipton and Cook ( 1963) B Sumino et al. ( 1975) C Rehnberg et al. ( 1982) D Fore and Morton ( 1952) * Average of cerebrum and cerebellum  No data Animal Studies Knowledge of manganese distribution patterns in animals was initially derived from parenteral exposure studies which facilitated the use of radioactive manganese as a tracer. The distributions of parentally ( injected) and orally administered manganese are very different. Cellular uptake of manganese is affected by the way in which manganese is transported in the plasma. Injected manganese ( and probably inhaled manganese as well), which is transported by transferrin, is more apt to accumulate in the brain and cause toxicity than orally administered manganese, which is transported from the gut to the liver by albumin ( Andersen et al., 1999; Davis et al., 1993). Davis et al. ( 1993) demonstrated that the distribution pattern of albumin 6 9 Manganese   February 2003 bound, but not transferrin bound, intraportally injected manganese was similar to that of orallyadministered manganese.

Kato ( 1963) and Maynard and Cotzias ( 1955) suggested that mitochondria rich tissues such as liver, kidney, and pancreas contain higher levels of manganese. Distribution studies in mice, rats, and monkeys have subsequently identified liver, kidney, and endocrine glands as primary sites of manganese accumulation following parenteral exposure. Kato ( 1963), for example, investigated distribution in mice using radiolabeled manganese. High levels of radioactive manganese were found in the liver, kidneys, and endocrine glands, with lesser amounts detected in brain and bone. Dastur et al. ( 1969) administered an intraperitoneal dose of radioactive manganese to rats, and subsequently found the highest concentrations of labeled manganese in suprarenal, pituitary, liver, and kidney tissue. In general, these results are in agreement with the patterns of manganese distribution observed in human tissues.

Dastur et al. ( 1971) observed a similar pattern of distribution in monkeys exposed to manganese by intraperitoneal injection. The highest concentrations of manganese were found in the liver, kidney and endocrine glands, as observed in rodents. Following treatment, manganese levels in the central nervous system decreased more slowly than levels in other tissues. Suzuki et al. ( 1975) injected monkeys subcutaneously with manganese, and subsequently found increased tissue concentrations of manganese in endocrine and exocrine glands ( thyroids, parotids, and gall bladder) and in the nuclei of cerebral basal ganglia. Newland et al. ( 1989) noted substantial accumulation in the pituitary gland of Macaca fascicularis and Cebus apella monkeys at low cumulative doses.

Several studies have addressed regional distribution of manganese in the brain following parenteral exposure. Newland and Weiss ( 1992) investigated distribution of manganese in the brain of monkeys. Three Cebus monkeys received multiple intravenous doses of 5 or 10 mg/ kg of manganese chloride over the course of 450 days. Magnetic resonance imaging revealed darkening of the globus pallidus and substantia nigra. This result is consistent with accumulation of manganese in these regions.

Scheuhammer and Cherian ( 1981) reported the distribution of manganese in male rat brain tissue with and without intraperitoneal exposure to 3 mg Mn/ kg as manganese chloride. In unexposed rats, the highest concentrations of manganese were found in the hypothalamus, colliculi, olfactory bulbs, and midbrain. In treated rats, all brain regions showed an increase in manganese concentration, and the highest manganese concentrations were observed in the corpus striatum and corpus callosum.

Autissier et al. ( 1982) reported that rats given a daily intraperitoneal dose of 10 mg/ kgday manganese chloride for 4 months showed significant increases in the accumulation of manganese in the brain. This dose was equivalent to 4.4 mg Mn/ kg day. The study showed a 359% increase in the concentration of manganese in the brain stem, a 243% increase in the corpus striatum, and a 138% increase in the hypothalamus.

The tissue distribution of manganese appears to be affected by co exposure to other metals. Shukla and Chandra ( 1987) exposed young male rats to lead ( 5 mg/ L in drinking water) 6 10 Manganese   February 2003 and/ or manganese ( 1 or 4 mg/ kg, by intraperitoneal injection) for 30 days. Exposure to individual metals resulted in accumulation in all brain regions. Co exposure to lead and manganese resulted in increased levels of both metals, particularly in the corpus striatum. Administration of manganese alone led to dose dependent increased levels in liver, kidney and testis. Co exposure to lead further increased manganese accumulation in liver. The authors concluded that the interaction of metals can alter tissue distribution of manganese, and that adverse health effects may result from co exposure to even low levels of metals.

The chemical form in which manganese is injected may influence the subsequent tissue distribution of manganese. Gianutsos et al. ( 1985) demonstrated that blood and brain levels of manganese in mice are increased following intraperitoneal injection of manganese chloride, manganese oxide, or methylcyclopentadienyl manganese tricarbonyl ( MMT). However, MnCl 2 administration resulted in more rapid accumulation and ultimately higher levels of blood and brain manganese. It was suggested that the differences seen among the three manganese compounds result from the oxide and MMT forms being more hydrophobic. Hydrophobicity may cause formation of a depot at the site of injection that retards absorption. Gianutsos et al. ( 1985) also demonstrated that the exit of manganese from the brain is a slower process than its entry, resulting in a long retention period and potential accumulation. A single injection of 0.4 mEq Mn/ kg resulted in a significant increase (> 2 fold) in brain levels within 1   4 hours. The increased levels were maintained for at least 21 days. Brain manganese levels were especially sensitive to a repeated dose regimen. Much greater accumulation occurred when the dose was divided into 10 injections given every other day as compared with a single injection. This observation may help explain the slow onset of manganese neurotoxicity: acute exposure results in other organs serving as the primary target, while chronic exposure results in gradually increasing brain levels with subsequent neurotoxicity.

Distribution of manganese has also been investigated in oral exposure studies. Chan et al. ( 1981) administered 278 mg/ L manganese chloride in drinking water to rats for two years. At the termination of the study, these investigators found a 31% increase in manganese concentration in the brain and a 45% increase in the liver relative to control values. Assuming a body weight for rats of 0.35 kg and water consumption of 0.049 L/ day, the average daily dose of manganese in this experiment was equivalent to 17 mg/ kg day.

Some oral exposure data suggest that developmental stage may influence the distribution of manganese. The brain, for example, may be a site for preferential accumulation of manganese in neonates. Kostial et al. ( 1978) observed that rat pups showed a greater accumulation of manganese in the brain, but not in the liver, than did their mothers. The data of Rehnberg et al. ( 1980, 1981, 1982) indicate that the neonatal brain reaches higher concentrations of manganese than other tissues. The authors suggested that this pattern reflects a response to a nutritional need for manganese in the developing brain.

Kontur and Fechter ( 1985) demonstrated placental transfer of manganese in Long Evans rats exposed via drinking water throughout gestation. Transfer was limited, with only 0.4% of the administered manganese accumulating in a single fetus. Neonatal pups of exposed dams had significantly increased levels of manganese in the forebrain. However, the increase was not associated with any overt signs of toxicity. 6 11 Manganese   February 2003 Komura and Sakamoto ( 1993) investigated the subcellular distribution of Mn and the binding characteristics of Mn to brain protein in male mice following administration of different forms of manganese. Four different manganese compounds ( MnCl 2° 4H 2 O, Mn( CH 3 COO) 2° 4H 2 O, MnCO 3, or MnO 2) were administered in the diet at a concentration of 2,000 mg Mn/ kg for 12 months. Each treatment group included 6 male mice. The control group received a diet containing approximately 130 mg Mn/ kg ( form not specified). Assuming a food factor of 0.13, the control and treatment dietary levels correspond to approximately average daily doses of 17 and 260 mg Mn/ kg day, respectively. Cerebral cortex concentrations of Mn were significantly higher in mice receiving the relatively insoluble compounds MnCO 3 and MnO 2 than in controls. The subcellular distribution of manganese in the striatum and the gel chromatographic profiles of manganese were similar for all tested manganese compounds.

Roels et al. ( 1997) reported that repeated gavage dosing of rats ( once weekly for 4 weeks) with 24.3 mg Mn/ kg ( 5% of the dose, or 1.22 mg/ kg, was assumed to be absorbed by the study authors) as MnCl 2 resulted in significantly increased concentrations of the metal in blood ( 68%) and brain cortex ( 22%) compared to saline controls but did not significantly increase striatal or cortex Mn concentrations. Similar administration of MnO 2 at the same dose level did not result in significant increases of Mn in blood or any brain tissue. Further studies indicated that Mn from MnCl 2 was absorbed much more rapidly and reached a higher peak concentration in the bloodstream of the dosed rats than did MnO 2. The peak Mn blood level following gavage dosing of MnCl 2 was roughly twice that of the oxide and was reported 1 hour post dosing, while that of MnO 2 was not reported until 144 hours post dosing ( Roels et al., 1997). These data indicate that administered manganese can be distributed into the brain and the kinetics of uptake and partitioning depend on the chemical form of the manganese compound.

6.3 Metabolism As a metallic element, manganese does not undergo metabolic conversion to other products. However, manganese has the potential to exist in several oxidation states in biological systems. Circumstantial evidence from the study of manganese containing enzymes and from electron spin trapping experiments suggests that manganese undergoes conversion from Mn( II) to Mn( III) within the body ( ATSDR, 2000). The conversion from Mn( II) to Mn( III) appears to be catalyzed by the " globulin protein ceruloplasmin ( Andersen et al., 1999). This reaction may be enhanced by the high affinity of the iron transporting protein transferrin for Mn( III).

A small fraction of absorbed manganese is present as the free ion. However, manganese readily forms complexes with a variety of organic and inorganic ligands. The complexes formed include 1) low molecular weight complexes with bicarbonate, citrate or other ligands; 2) an exchangeable complex with albumin; and 3) tightly bound complexes with proteins such as transferrin and " 2 macroglobulin. In addition, manganese can assume a structural role in metalloproteins such as mitochondrial superoxide dismutase, pyruvate decarboxylase, and liver arginase. Manganese also plays a catalytic or regulatory role in enzymatic reactions involving select hydrolases, dehydrogenases, kinases, decarboxylases and transferases. 6 12 Manganese   February 2003 6.4 Excretion The primary route for elimination of manganese is to the feces through bile, as demonstrated in several animal studies ( Weigand et al., 1986; Davis et al., 1993; Malecki et al., 1996). Fecal manganese concentration reflects both unabsorbed manganese and biliary secretion of absorbed manganese.

Human Studies The primary route for elimination of manganese is via the feces. Fecal manganese concentration reflects both unabsorbed manganese and biliary secretion of absorbed manganese.

Price et al. ( 1970) determined the excretion pattern for preadolescent girls consuming 2.13 to 2.43 mg Mn/ day. Approximately 1.66 to 2.23 mg Mn/ day was excreted in the feces. In contrast, only 0.01 to 0.02 mg/ day was excreted in the urine. Results from other studies confirm the importance of the fecal pathway for excretion. WHO ( 1981) and Newberne ( 1973) reported that human excretion of manganese in urine, sweat, and milk is minimal. The normal level of manganese found in urine of humans has been reported to be 1   8 : g/ L, but values as high as 21 : g/ L have also been reported ( U. S. EPA, 1984). Greger et al. ( 1990) reported urinary excretion levels of 7.0 and 9.3 nmol Mn/ g creatine/ day ( 0.38 and 0.51 : g Mn/ g creatinine/ day) for healthy men and women, respectively. Urinary excretion of manganese was not responsive to oral intake levels of manganese ( Davis and Greger, 1992).

A number of studies have addressed the kinetics of manganese excretion. Humans who ingested tracer levels of radioactive manganese excreted the tracer with whole body retention half times of 13 to 37 days ( Mena et al., 1969; Davidsson et al., 1989b; Sandström et al., 1986). Sandström et al. ( 1986) gave volunteers a single oral dose of radioactive manganese and reported a mean biologic half life value of 13 days ( range 6   30 days) for 14 subjects monitored on postexposure days 5   20, and a mean half life of 34 days ( range 26   54 days) for 6 subjects monitored on post exposure days 20   50. Two additional subjects received manganese intravenously and experienced a much slower turnover.

Mahoney and Small ( 1968) investigated the clearance of intravenously injected MnCl 2 by humans. These investigators observed a biphasic clearance pattern, with a rapid phase that lasted 4 days and a slow phase that lasted 39 days. Schroeder et al. ( 1966) reported a whole body turnover rate in healthy adults of about 40 days, with a total body manganese content of about 20 mg.

Cotzias et al. ( 1968) injected manganese intravenously and reported values for biological half time of 37.5 days in healthy subjects, 15 days in healthy miners, and 28 days in subjects with chronic manganese poisoning. These researchers also found that clearance by healthy subjects averaged 25 days from the liver, 54 days from the head, and 57 days from the thigh, as measured by external counting with a collimator. In healthy miners, liver clearance averaged 13 days; head clearance averaged 37 days; and thigh clearance averaged 39 days. Subjects with 6 13 Manganese   February 2003 chronic manganese poisoning cleared manganese from the liver in 26 days, from the head in 62 days, and from the thigh in 48 days.

Finley ( 1999) demonstrated that iron status ( assessed as serum concentrations of sodium ferritin) may affect manganese excretion. Biological half life ( determined by regression of whole body 54Mn counts) was assessed in women aged 20 to 45 years who were categorized as having high ( upper 10% of normal range, mean values 68 to 69 : g/ L) or low ( lower 10% of normal range, mean values 8.7 to 8.9 : g/ L) serum ferritin levels. Biological half life was determined under conditions of high ( 9.5 mg Mn/ day) or low ( 0.7 mg Mn/ day) dietary manganese intake. Subjects with low ferritin status consuming the low manganese diet had a mean biological halflife that was more than twice the value determined for high ferritin status subjects consuming the same diet ( 36.6 days versus 17.0 days). There was no effect of ferritin status on mean half life for subjects consuming the high manganese diet ( 13.0 and 11.8 days for low and high ferritin status groups, respectively).

Animal Studies No studies of excretion following oral administration of manganese in animals were identified.

Greenberg and Campbell ( 1940) reported that 90.7% of a 1 mg intraperitoneal dose of radiolabeled manganese ( 54Mn) was found in rat feces within 3 days. In a subsequent study, Greenberg et al. ( 1943) found that 27.1% of a 0.01 mg intraperitoneal dose of radiolabeled manganese and 37.3% of a 0.1 mg dose were collected in rat bile within 48 hours. Tichy et al. ( 1973) administered a 0.6 : g dose of manganese chloride to rats and reported that 27% was excreted into the bile within 24 hours.

Klaassen ( 1974) demonstrated that bile is the main route of manganese excretion, and that biliary excretion represents a major homeostatic mechanism for manganese levels in the body. This investigator administered intravenous doses of 0.3, 1.0, 3.0, or 10.0 mg Mn/ kg to rats, rabbits and dogs. Urinary excretion was low. As the dose increased, the excretion of manganese into the bile also increased. The concentration of manganese in bile was 100 to 200 times higher than in plasma at the three lower doses. However, at the 10 mg dose there was no further increase in excretion of manganese into the bile. A maximum excretion rate of 8.5 : g Mn/ min/ kg was attained, suggesting that a saturable active transport mechanism may exist ( U. S. EPA, 1984).

Britton and Cotzias ( 1966) and Suzuki ( 1974) found that an increase in dietary intake of manganese decreased biologic half times. Studies also indicate that the biologic half time of manganese in the brain of rats, mice and monkeys is longer than the half life in the body ( Suzuki, 1974; Dastur et al., 1969, 1971).

In developmental studies of manganese excretion, neonatal mice, rats, and kittens were found to rapidly accumulate manganese without excreting it during the first 18 days of life ( U. S. 6 14 Manganese   February 2003 EPA, 1984). In contrast, when lactating rats and cats were given excessive doses of manganese in drinking water (> 280 mg/ L), their offspring initiated excretion before the 16th day of life.

Although human and animal evidence indicates that most manganese is excreted to the feces in bile, alternative routes for manganese excretion also exist. Experiments conducted by Bertinchamps and Cotzias ( 1958), Kato ( 1963), and Papavasiliou et al. ( 1966) demonstrated direct excretion of manganese through the intestinal wall. This route is most evident in the presence of biliary obstruction or following high doses of manganese. Bertinchamps et al. ( 1966) and Cikrt ( 1973) reported that in rats excretion of manganese occurred through the intestinal wall into the duodenum, jejunum and terminal ileum. Burnett et al. ( 1952) demonstrated that manganese excretion by dogs also occurs via the pancreatic juice. Other potential sources of fecal manganese include intestinal secretions and the manganese present in sloughed off intestinal microvillus cells. The fraction of total excretion attributable to these alternative pathways has not been reported, but is expected to be relatively small when compared to biliary secretion. 7 1 Manganese   February 2003 7.0 HAZARD IDENTIFICATION 7.1 Human Effects 7.1.1 Case Reports General Population A number of investigators reported the toxicity of total parenteral ( TPN) manganese in humans, especially on changes in brain MRI scans ( Ejima et al., 1992; Fell et al., 1996; Mirowitz and Westrich, 1992). These studies emphasize the difference in the effect of oral and parenteral manganese. When administered parenterally, manganese bypasses the typical excretory mechanisms in the gastrointestinal tract and liver and accumulates in the brain ( Mirowitz and Westrich, 1992).

In addition, there are a limited number of case reports describing the outcome of exposure following accidental or intentional ingestion of manganese from potassium permanganate, a strong oxidizing agent. Unspecified toxic effects were reported following ingestion of 2.4 mg/ kg day potassium permanganate ( 0.83 mg Mn/ kg day) by a woman of unknown age and health status. This information was reported in a 1933 French study cited in NIOSH ( 1984), and was not available for review. Dagli et al. ( 1973) described a case in which oral ingestion of a 300 mg dose of potassium permanganate ( 104 mg Mn) resulted in extensive damage to the distal stomach and pyloric stenosis. Mahomedy et al. ( 1975) described two cases of methemoglobinemia following ingestion of an unspecified amount of potassium permanganate which had been prescribed by African tribal healers. Development of methemoglobinemia likely reflects the chemical oxidation of heme iron.

Holzgraefe et al. ( 1986) reported neurological effects in an adult man who ingested approximately 1.8 mg/ kg day of potassium permanganate ( 0.62 mg Mn) for 4 weeks. A syndrome similar to Parkinson's disease developed after about 9 months. However, data in this study are reported to be insufficient to establish causation ( U. S. EPA, 1993). Bleich et al. ( 1999) published a 14 year follow up of this case report. Most of the symptoms originally noted ( including rigor, muscle pain, hypersomnia, increased libido, sweating, fatigue, and anxiety) had improved, and the study authors noted that there appeared to be no evidence for progression of the parkinsonian syndrome as described by others ( Huang et al., 1998).

Additional case reports suggest the potential for manganese toxicity following oral exposure, but are difficult to assess quantitatively. One report involved a 59 year old male who was admitted to the hospital with classical symptoms of manganese poisoning, including dementia and a generalized extrapyramidal syndrome ( Banta and Markesbery, 1977). The patient's serum, hair, urine, feces, and brain were found to have manganese " elevated beyond toxic levels." No source of manganese exposure was identified for this individual. Exposure may have resulted from the use of large quantities of vitamin and mineral supplements for 4 to 5 years. No quantitative data were provided in this report. 7 2 Manganese   February 2003 Manganese intoxication was described in a 62 year old male who received total parenteral nutrition that provided 2.2 mg of manganese ( form not stated) daily for 23 months ( Ejima et al., 1992). This level corresponds to a dose of approximately 0.023 mg Mn/ kg day for a 70 kg adult. The patient's whole blood manganese concentration was elevated. The patient exhibited dysarthria, mild rigidity, hypokinesia with masked face, a halting gait, and severely impaired postural reflexes, and the diagnosis of this condition was parkinsonism. Assuming an average absorption of roughly 5% of an oral dose, the intravenous dose of 2.2 mg Mn/ day would be approximately equivalent to an oral intake of 40 mg Mn/ day ( U. S. EPA, 1993).

Sensitive Populations Individuals with impaired liver function or bile flow may represent potentially sensitive subpopulations for manganese exposure. For example, Hauser et al. ( 1994) reported changes in brain MRI scans in liver failure patients which were identical to those observed in cases of manganese intoxication. The patients ( n= 3) examined exhibited bilateral signal hyperintensity in the globus palladi and substantia nigrae in T1 weighted MRI and increased blood manganese levels but had no history of increased exposure to manganese. Hauser et al. ( 1994) postulated that impaired elimination of normal dietary manganese could result in manganese intoxication. Devenyi et al. ( 1994) described a case study of an 8 year old girl with Alagille's syndrome, an autosomal dominant disorder characterized by neonatal cholestasis, intrahepatic bile duct paucity, and end stage liver disease. The patient exhibited a stable peripheral neuropathy, and for a period of 2 months exhibited episodic, dystonic posturing, and cramping of her hands and arms. Whole blood manganese level was elevated ( 27 : g/ L, in contrast to a normal range of 4 to 14 : g/ L), and cranial T1 weighted magnetic resonance imaging ( MRI) revealed symmetric, hyperintense globus pallidi and subthalamic nuclei. These findings were interpreted as indications of manganese toxicity. Following liver transplantation, the patient's manganese levels returned to normal, her neurological symptoms improved, and MRI results appeared normal. The interpretation of this series of events was that: 1) progressive liver dysfunction resulted in inadequate excretion of manganese into the bile, 2) subsequent accumulation of manganese resulted in neurotoxicity, and 3) liver transplantation restored biliary excretion and alleviated the symptoms.

Manganese has been identified as a possible etiologic agent in the occurrence of neurological symptoms associated with hepatic encephalopathy ( a brain disorder associated with chronic liver damage). Medical evidence supporting an etiologic role has been summarized by Layrargues et al. ( 1998). Patients with chronic liver disorders such as cirrhosis experience a high incidence of extrapyramidal symptoms resembling those observed in cases of occupational manganism. Manganese concentration increases in the blood and brain of patients with chronic liver disease and these changes are accompanied by pallidal hyperintensity on T1 weighted MRI. Autopsy data from ten patients who died in hepatic coma indicate that manganese levels are 2 to 7 fold higher in the globus pallidus of cirrhotic patients when compared to the general population. Liver transplantation normalizes the pallidal MR signals and results in the disappearance of extrapyramidal symptoms. Conversely, transjugular intrahepatic portosystemic shunting ( a procedure which increases the systemic availability of manganese) intensifies the pallidal MR signal and results in deterioration of neurological function. 7 3 Manganese   February 2003 7.1.2 Short term Studies General Population Kawamura et al. ( 1941) reported health effects resulting from the ingestion of manganese contaminated well water by 25 individuals. The source of contamination was identified as leachate from approximately 400 dry cell batteries buried near the drinking water well. Chemical analysis also revealed high levels of zinc in the well water. The length of exposure to manganese was estimated to be 2 to 3 months. The concentration of manganese in the well was approximately 14 mg Mn/ L ( as Mn 3 O 4) when analyzed 7 weeks after the first case appeared. This level corresponds to a dose of approximately 28 mg Mn/ day ( assuming a daily water intake of 2 L), or 0.5 mg Mn/ kg day ( for a 60 kg adult). When reanalyzed 1 month later, the manganese concentration had decreased by about 50%. Based on these measurements, retrospective extrapolation suggests that the initial exposure level may have been 28 mg Mn/ L or higher. Assuming a daily water intake of 2 L, and an additional manganese intake from food of at least 2 mg/ day, this represents a dose of at least 58 mg Mn/ day. This intake of manganese is about 25 to 32 times the level considered to be safe and adequate by the Food and Nutrition Board ( IOM, 2002). Assuming a body weight of 60 kg for an adult, this intake level corresponds to a dose of 0.93 mg Mn/ kg day from drinking water. No information on dietary intake was available.

Health effects reported by Kawamura et al. ( 1941) included lethargy, increased muscle tonus, tremor and mental disturbances. Out of 25 people examined, 15 had symptoms. Five cases were considered severe, 2 cases were categorized as moderate and 8 cases were described as mild. The most severe symptoms were observed in the elderly. Younger people were less affected, and symptoms of intoxication were absent in young children ( age 1 to 6 years). Three deaths occurred, including one from suicide. Upon autopsy, the concentration of manganese in the brain of one person was found to be 2 to 3 times higher than concentrations measured in two control autopsies. Extreme macroscopic and microscopic changes were seen in the brain tissue, especially in the globus pallidus. The authors also reported elevated levels of zinc in the well water, but concluded that the zinc appeared to have no relation to the observed symptoms or tissue pathology. This conclusion was largely based on the observation of morphological changes in the corpus striatum which are characteristic of manganese poisoning, but are not a feature of zinc poisoning.

While toxicity in the Kawamura et al. ( 1941) study is attributed to manganese, several aspects of the observed health effects are inconsistent with traits of manganism observed in humans following chronic inhalation exposure. Inconsistencies include the rapid onset of symptoms and rapid progression of the disease. Two adults who came to tend the members of one family developed symptoms within 2 to 3 weeks. The course of the disease was very rapid, progressing in one case from initial symptoms to death in 3 days. Some survivors recovered prior to significant decreases in the manganese concentration of the well water which resulted when the batteries that caused the contamination were removed from the site. This pattern contrasts with the longer latency period and irreversible damage caused by inhalation exposure to manganese. These observations may represent differences in the pharmacokinetics of ingested 7 4 Manganese   February 2003 versus inhaled manganese, but there is little information to support this conclusion. Although these individuals were clearly exposed to high levels of manganese, it is possible that additional factors contributed to the observed effects ( U. S. EPA, 1993; ATSDR 2000).

Sensitive Populations Study data for sensitive populations were not identified in the materials reviewed for preparation of this document.

7.1.3 Long Term and Epidemiological Studies General Populations Kondakis et al. ( 1989) conducted an epidemiologic study of manganese in drinking water in northwest Greece. Three areas with different levels of manganese in the drinking water supply were chosen for this study. Area A had manganese concentrations of 3.6 to 14.6 : g/ L, Area B had concentrations of 81.6 to 252.6 : g/ L, and Area C had concentrations of 1,800 to 2,300 : g/ L. The total population in the study areas ranged from 3,200 to 4,350 people. The study included only individuals over the age of 50 drawn from a random sample of 10% of all households. The sample sizes were 62, 49, and 77 for areas A, B, and C, respectively. The study authors reported that " all areas were similar with respect to social and dietary characteristics," but few details were provided. Kondakis et al. ( 1989) determined whole blood and hair manganese concentrations in samples collected from study participants. The mean concentration of manganese in hair was 3.51, 4.49 and 10.99 : g/ g dry weight for areas A, B and C, respectively. Concentrations in hair differed significantly between areas C and A ( p < 0.001). No significant differences in whole blood manganese levels were observed among the three areas. However, manganese concentration in blood is not considered to be a reliable indicator of manganese exposure ( U. S. EPA, 1993).

Kondakis et al. ( 1989) also administered a neurological examination which evaluated the presence and severity of 33 symptoms ( e. g., weakness/ fatigue, gait disturbances, tremors, dystonia) in all subjects. The results of the neurological examination were expressed as a composite score. A higher neurological score indicated an increased frequency and/ or severity of the 33 evaluated symptoms. Results for the three geographic areas are summarized in Table 7 1. Mean scores for both sexes combined were 2.7 ( range 0   21) for Area A; 3.9 ( range 0   43) for Area B; and 5.2 ( range 0   29) for Area C. The authors indicated that the difference in mean scores for Area C versus Area A was statistically significant ( Mann Whitney Test, z = 3.16, p = 0.002, for both sexes combined), suggesting neurologic impairment in people living in Area C. In a subsequent analysis, logistic regression indicated a significant difference between areas A and C when both age and sex were taken into account ( Kondakis, 1990). 7 5 Manganese   February 2003 Table 7 1. Mean Neurological Scores of Residents in Three Areas of Northwest Greece with Different Levels of Manganese in Drinking Water ( range is given in parentheses).

Subject Area A ( 3.6   14.6 : g Mn/ L) Area B ( 81.6   252.6 : g Mn/ L) Area C ( 1,800   2,300 : g Mn/ L)

Males 2.4 ( 0   21) 1.6 ( 0   6) 4.9 ( 0   29)

Females 3.0 ( 0   18) 5.7 ( 0   43) 5.5 ( 0   21)

Both 2.7 ( 0   21) 3.9 ( 0   43) 5.2 ( 0   29)

Source: Kondakis et al. ( 1989)

Limitations to the Kondakis study have been noted by ATSDR ( 2000). These include: 1) lack of clearly detailed descriptions of neurological signs and symptoms that reportedly increased following manganese exposure, and 2) failure to describe procedures for avoiding bias when evaluating subjective neurological scoring parameters. An additional shortcoming of this study is the lack of quantitative exposure data ( U. S. EPA, 1996a). The individuals examined by Kondakis et al. ( 1989) also consumed manganese in their diet. The initial estimate of dietary intake was 10 to 15 mg/ day based on high intake of vegetables ( Kondakis, 1990). This figure was subsequently revised to an estimate of 5 to 6 mg Mn/ day ( Kondakis, 1993), but data were not provided to substantiate this estimate. Lack of dietary intake and water consumption data prevents determination of a quantitative dose response relationship for manganese toxicity in this study. Nevertheless, this study raises concern for adverse neurological effects at estimated doses that are not far from the range of essentiality ( U. S. EPA, 1996a).

Although conclusive evidence is lacking, some investigators have linked increased intake of manganese with violent behavior. Gottschalk et al. ( 1991) found significant increases in the level of manganese in the hair of convicted felons ( 1.62 ± 0.173 ppm in prisoners compared with 0.35 ± 0.020 ppm in controls). The study authors suggested that " a combination of cofactors, such as the abuse of alcohol or other chemical substances, as well as psychosocial factors, acting in concert with mild manganese toxicity may promote violent behavior." The number of potential variables indicates that caution should be exercised in interpretation of these data.

Results from studies of an Aboriginal population in Groote Eylandt have been cited as additional evidence for a relationship between elevated manganese exposure, violent behavior, and adverse health effects. The soil on this Australian island is exceptionally high in manganese ( 40,000 to 50,000 mg/ kg), and the fruits and vegetables grown in the region are reported to contain elevated concentrations of manganese. High alcohol intake, anemia, and a diet deficient in zinc and several vitamins ( Florence and Stauber, 1989) may contribute to increased uptake and retention of manganese. The proportion of arrests in this native population is the highest in Australia, and high incidences of stillbirths and congenital malformations, as well as a high occurrence of Parkinson like neurobehavioral syndrome, have been observed ( Cawte and Florence, 1989; Kilburn, 1987). Clinical symptoms consistent with manganese intoxication are present in about 1% of the inhabitants. Quantitative data on oral intake have not been reported, 7 6 Manganese   February 2003 but elevated concentrations of manganese have been determined in the blood and hair of the Aborigines ( Stauber et al., 1987). However, Stauber et al. ( 1987) did not find a correlation between hair levels of manganese and the severity of neurological symptoms in individuals.

A study of the neurologic status of the Aborigines in Groote Eylandt identified two general syndromes. One syndrome is characterized by muscle atrophy and weakness, while the other is characterized by ataxia and oculomotor disturbances ( Kilburn, 1987). Although an association of adverse health effects with elevated manganese exposure is suggested by these observations, the small population of Groote Eylandt and the difficulty in defining an appropriate control population have prevented the identification of statistically significant trends ( U. S. EPA, 1993).

Several of the studies above utilized hair analysis as a method for estimating exposure to manganese. ATSDR ( 2000) has outlined several potential limitations to the use of hair analysis. The normal cycle of hair growth and loss restricts its usefulness to a period of a few months following exposure. External contamination of hair by dye, bleaching agents, or other materials may result in values which are not representative of absorbed doses. The affinity of manganese for pigmented tissue may result in variation of manganese concentration with hair color.

Goldsmith et al. ( 1990) investigated a Parkinson's disease cluster within southern Israel. The prevalence of the disease was increased among persons 50 to 59 years old, suggesting an early onset of the disease. Well water and soils in the region reportedly contained high levels of manganese, although no quantitative data were provided. In addition, the manganese containing fungicide Maneb was commonly used in the area. However, several factors limit the use of this study for evaluation of the human health effects of excess manganese exposure. Lack of environmental concentration data prevented reliable estimation of exposure rates. Potentially confounding factors included the high levels of aluminum, iron, and other metals in the soil and water, and the use of the herbicide paraquat in the area ( ATSDR, 2000). Paraquat is structurally related to N methyl 4 phenyl 1,2,3,6 tetrahydropyridine ( MPTP), a piperidine derivative which causes irreversible symptoms of parkinsonism in humans.

Vierrege et al. ( 1995) investigated the neurological impact of chronic manganese exposure via drinking water in a cross sectional study of two proband cohorts in rural northern Germany. The study population was drawn from the county Herzogtum Lauernburg in the northernmost province of Germany. This region is characterized by agricultural and forestry activities but no steel or mining industry. Many of the residents of this area draw their drinking water from wells, and by law, the well water is routinely monitored for chemicals and bacteria. A survey was conducted in 1991 and was combined with a cross sectional investigation of a randomly selected group of right handed residents aged 40 years or older who had used their wells as the primarily source of drinking water for a minimum of 10 years ( range 10 to 40 years). Complete documentation of manganese monitoring results for six years prior to the investigation was required for study eligibility. Participants were assigned to two groups on the basis of manganese concentration in their well water. Group A included individuals who continually ingested well water containing between 0.300 and 2.160 mg Mn/ L. Group B included individuals whose well water manganese concentration had never exceeded 0.050 mg/ L. 7 7 Manganese   February 2003 Detailed information on medical history, employment history, diet, alcohol consumption, drug use and smoking was collected by interview. Individuals in Group A were matched to individuals in Group B with respect to age, sex, nutritional habits, and drug intake. Criteria for exclusion from the study included history of employment in the steel industry, adherence to dietary restrictions, history of CNS relevant drug use, diabetes mellitus, history of stroke, or treatment for psychiatric disorders. Conditions that could affect performance on the neurological assessment tests ( neurorthopedic impairment of hand finger function or poor vision) were also grounds for exclusion from the study.

A total of 164 eligible subjects was identified. Of these, 49 subjects were excluded for failure to meet the health or water monitoring criteria. Group A included 41 subjects ( 21 male and 20 female) with a mean age ( ± standard deviation) of 57.5 ± 10.3 years ( range 41 to 84 years). Group B included 74 subjects with a mean age of 56.9 ± 11.8 years ( range 41 to 86) years. No dietary differences were evident between the two groups. Neurological status was assessed by experienced personnel blinded to the group status of the subjects. Each participant was evaluated for neurotoxicological symptoms by use of a modified German version of a standardized symptoms list. Signs of parkinsonism were evaluated by the Columbia University Rating Scale ( CURS). Fine motor ability ( each hand) was assessed using a conventional apparatus (" Motorische Leistungsserie," MLS) and application of aiming, steadiness, line pursuit, and tapping tests. Manganese status was evaluated by determination of manganese in blood. The concentration of manganese in hair or nails was not determined.

The results of neurological evaluations are summarized in Table 7 2. There were no significant differences between groups for the mean item scores on the standardized symptoms list or the CURS. MLS test results were obtained for 36 group A subjects ( 18 male and 18 female, mean age 56.4 ± 8.4 years, range 41 to 72 years) and 67 Group B subjects ( 35 men and 32 women, mean age 55.1 ± 9.9 years, range 41 to 72 years). Results of participants older than 72 years were not included in the statistical analysis of MLS data because normative information from the general population have an upper age limit of 72 years. No significant differences were observed between groups for any test when results were standardized to age corrected values. Mean blood manganese concentrations were 8.5 ± 2.3 : g/ L and 7.7 ± 2.0 : g/ L for groups A and B, respectively. The blood manganese values did not differ significantly and both fell within the normal range for the general ( non occupationally exposed) population. Separate analyses for possible confounding factors did not reveal differences in clinical or instrumental test outcomes related to high or low consumption of alcohol, mineral water, coffee, tea, tobacco, vegetables, or fruit. Where cases of parkinsonism ( n = 3) were encountered in this study, they occurred in the low exposure group ( Group B) and were considered to be typical Parkinson's disease and thus unrelated to manganese exposure. The authors of this study concluded that there was no evidence of an association between consumption of high concentrations of manganese in well water and neurological impairment ( including those suggestive of parkinsonism).

Three potential limitations related to the ecologic design of this investigation were noted by Vieregge et al. ( 1995). First, the investigators could not control for possible migration of subjects with manganese induced neurological disorders from the study area prior to the investigation. However, Vieregge et al. ( 1995) stated on the basis of inquiries and general experience in the region that a migration effect was unlikely to be significant. 7 8 Manganese   February 2003 Table 7 2. Mean Neurological Scores of Residents in Germany Exposed to Different Levels of Manganese in Well Water.

Assessment Measure Exposure Group Group A ( High) Group B ( Low)

Neurotoxicological Symptom Questionnaire Item Number 3.2 ± 3.0a 3.9 ± 3.1 CURS Parkinsonism Item Number 1.2 ± 1.0 1.7 ± 2.0 MLS Aiming Duration ( sec) 104.8 ± 9.1b 102.9 ± 10.0 MLS Steadiness Errors ( number) 103.9 ± 103.9 103.1 ± 7.9 Duration of errors ( sec) 100.8 ± 10.6 100.2 ± 10.5 MLS Line Pursuit Errors ( number) 106.4 ± 7.6 106.6 ± 8.0 Duration of errors ( sec) 102.3 ± 8.1 103.1 ± 10.6 Total duration ( sec) 104.3 ± 12.6 100.7 ± 15.5 MLS Tapping Rate ( number) 103.1 ± 7.2 103.9 ± 10.5 a Mean ± standard deviation b MLS test results are for right hand Second, although possible confounding by several dietary items or groups was evaluated and found to be non evident, confounding effects of nutrition ( particularly in subjects working outside their home residence) could not be completely excluded. Finally, blood manganese levels are thought to primarily reflect current body burden of manganese rather than exposure.

Iwami et al. ( 1994) reported that the incidence of motor neuron disease ( MND) in a small town in Japan was positively correlated with a significantly increased manganese concentration in local rice and a low magnesium concentration in the drinking water. This study, however, did not provide good estimates of overall exposure to manganese in either the control population or the population with MND.

Adverse neurological effects ( decreased performance in school and in neurobehavioral exams of the WHO core test battery) were reported in 11 to 13 year old children who were exposed to excess manganese through ingestion of well water and from wheat fertilized with sewage water ( He et al. 1994; Zhang et al. 1995). The exposed group consisted of 92 children pair matched to 92 controls from a nearby area. The groups were matched for age, sex, grade, family income level, and parental education level; further, both groups lived on farms. The average manganese concentration of the drinking water of the exposed group was 0.241 mg/ L compared to the control level of 0.04 mg/ L. Although the study authors had drinking water data 7 9 Manganese   February 2003 from a 3 year period, it was not clear how long the children were exposed prior to the study. Further, the exposure data were not well characterized; therefore it was not possible to establish a cause effect link between ingestion of excess manganese and preclinical neurological effects in children.

Sensitive Populations Study data for sensitive populations were not identified in the materials reviewed for preparation of this document.

7.1.4 Beneficial Effects Manganese is a naturally occurring element that is required for normal physiological functioning in all animal species ( U. S. EPA, 1996a). Manganese plays a role in bone mineralization, metabolic regulation, protein and energy metabolism, protection of cells from oxidative stress, and synthesis of mucopolysaccharides ( ATSDR, 2000). Many of these roles are achieved by participation of manganese as a catalytic or regulatory factor for enzymes, including hydrolases, dehydrogenases, kinases, decarboxylases and transferases. In addition, manganese is a structural component of the metalloenzymes mitochondrial superoxide dismutase, pyruvate carboxylase, and liver arginase. Additional information on the biochemical and nutritional roles of manganese in human health is available in Wedler ( 1994) and Keen et al. ( 1999). At present, the optimal levels of oral manganese exposure have not been well defined for humans ( Greger, 1999).

Overt signs of manganese deficiency have been demonstrated in multiple animal species ( Keen et al., 1999). Biochemical effects observed in manganese deficient animals include alterations in carbohydrate, protein, and lipid metabolism. Physiological outcomes associated with deficiency include impaired growth ( Smith et al., 1944), skeletal abnormalities ( Amdur et al., 1944; Strause et al., 1986), impaired reproductive function in females, and testicular degeneration in males ( Boyer et al., 1942). The molecular basis for these effects has not been established with certainty, but may be related to the participation of manganese in numerous enzymatic reactions. In addition, the effect of manganese deficiency on mitochondrial superoxide dismutase activity has functional consequences. Manganese deficient rats experience more oxidation of mitochondrial membranes of the heart and more formation of conjugated dienes than manganese adequate rats ( Malecki and Greger, 1996). In another study, Gong and Amemiya ( 1996) observed ultrastructural changes suggestive of oxidative damage in the retinas, of rats fed a manganese deficient diet for 12 to 30 months.

Manganese is ubiquitous in human foods, and outright manganese deficiency has not been observed in the general population. However, observations reported by Doisy ( 1973) and Friedman et al. ( 1987) indicate that manganese is an essential element for humans. Doisy ( 1973) reported a decreased level of clotting proteins, decreased serum cholesterol, reddening of black hair, retarded growth of hair and nails, and scaly dermatitis in a subject inadvertently deprived of manganese. Friedman et al. ( 1987) administered a manganese deficient diet to seven men for 39 days. Five of the seven subjects exhibited dermatitis at the end of the manganese deficient 7 10 Manganese   February 2003 period. The development of dermatitis was attributed to decreased activity of manganeserequiring enzymes that are required for skin maintenance. The symptoms cleared rapidly when manganese was restored to the diet.

7.2 Animal Studies This section presents the results of manganese toxicity studies in animals. The first four subsections provide study results by duration of exposure. In general, acute studies are those which address exposure durations of 24 hours or less. Short term studies have exposure durations greater than 24 hours but less than approximately 90 days. The exposure duration of subchronic studies is typically 90 days, and chronic studies are those in which exposure is longer than 90 days. Some studies may fall into more than one exposure category since they measure impacts over several exposure periods. The discussion of acute, short term, subchronic and chronic studies summarizes observed toxicological effects on all body systems. The remaining subsections of Section 7.2 provide toxicological data related to specific organ systems and types of endpoints, including neurotoxicity, developmental and reproductive toxicity, and carcinogenicity.

7.2.1 Acute Toxicity Oral Exposure LD 50 values determined for selected manganese compounds are summarized in Table 7 3. Oral LD 50 values among the water soluble manganese compounds ranged from 400 to 475 mg Mn/ kg for manganese chloride, and from 379 to 810 mg Mn/ kg for potassium permanganate. An LD 50 of 836 mg Mn/ kg was reported for manganese acetate.

Age may be a factor in susceptibility to acute manganese toxicity. Kostial et al. ( 1978) found that MnCl 2 produced the greatest oral toxicity in the youngest and oldest groups. Roth and Adleman ( 1975) proposed that the increased susceptibility of older rats may result from a decrease in adaptive responsiveness, which is characteristic of the aging process. Increased susceptibility of younger rats may reflect high intestinal absorption and body retention of manganese.

Parenteral Exposure Manganese compounds administered by parenteral routes generally result in mortality at lower doses. LD 50 values for the intraperitoneal route ranged from 14 to 64 mg Mn/ kg. Franz ( 1962) and Bienvenu et al. ( 1963) conducted comparative intraperitoneal toxicity studies, and found that manganese is less toxic than many other metals. Jonderko ( 1965) found increased serum calcium and decreased inorganic phosphorous in rabbits exposed intramuscularly to 3.5 mg Mn/ kg. Details on the compound and the duration of exposure were not available. 7 11 Manganese   February 2003 Table 7 3. LD 50 Values for Manganese Compounds.

Compound Species Route LD 50 ( mg Mn/ kg) Reference Manganese acetate rat oral 836 Smyth et al. ( 1969)

Manganese chloride rat oral 425 Shigan and Vitvickaja ( 1971)

rat oral 475 Kostial et al. ( 1978)

rat oral 410 Holbrook et al. ( 1975)

mouse oral 450 Shigan and Vitvickaja ( 1971)

guinea pig oral 400 Shigan and Vitvickaja ( 1971)

rat i. p. 38 Franz ( 1962); Holbrook et al. ( 1975)

mouse i. p. 56 Franz ( 1962); Holbrook et al. ( 1975)

mouse i. v. 16 Larsen and Grant ( 1997)

Manganese dioxide rat oral 2,197 Holbrook et al. ( 1975)

Manganese sulfate mouse i. p. 44 Bienvenu et al. ( 1963)

Manganese sulfate, tetrahydrate mouse i. p. 64 Yamamoto and Suzuki ( 1969)

Manganese nitrate mouse i. p. 56 Yamamoto and Suzuki ( 1969)

Methylcyclopentadienyl manganese tricarbonyl ( MMT) rat oral 10 Hanzlik et al. ( 1980)

rat oral 12 Hinderer ( 1979)

rat oral 12 Hysell et al. ( 1974)

mouse oral 48 Hinderer ( 1979)

Potassium permanganate mouse oral 750 Shigan and Vitvickaja ( 1971)

rat oral 379 Smyth et al. ( 1969)

rat oral 750 Shigan and Vitvickaja ( 1971)

guinea pig oral 810 Shigan and Vitvickaja ( 1971)

i. p. = intraperitoneal i. v. = intravenous 7 12 Manganese   February 2003 Baxter et al. ( 1965) measured physiological parameters in manganese treated rats weighing 100 to 550 g. Measurements were made 1 to 72 hours after subcutaneous administration of 5 to 150 mg of manganese as MnCl 2 in saline. Levels of hemoglobin, hematocrit, and mean corpuscular volume were significantly increased in rats receiving 150 mg Mn/ kg. A measurable response in these parameters occurred at 50 mg Mn/ kg, while the peak increase in these parameters occurred at 12 and 18 hours after dosing. The maximum response occurred at 170 to 300 mg Mn/ kg. Necrotic changes were noted in hepatic tissue 18 hours after a single dose of 170 mg Mn/ kg.

Pancreatic endocrine function is affected by acute manganese exposure. Baly et al. ( 1985) injected rats intraperitoneally with 40 mg Mn/ kg. Manganese injection resulted in a decrease in plasma insulin levels, an increase in plasma glucose levels, and a transitory increase in glucagon concentration.

Larsen and Grant ( 1997) administered a single intravenous dose of 150, 200, 300, or 400 : mol/ kg manganese chloride in saline to male mice ( 5/ group). These doses correspond to 8.2, 11, 16, and 22 mg Mn/ kg, respectively. These study authors reported an LD 50 value of 300 : mol/ kg ( 16 mg Mn/ kg).

7.2.2 Short Term Studies Oral Exposure Matrone et al. ( 1959) orally administered 2,000 ppm manganese as MnSO 4 CH 2 O to 6 month old anemic rabbits for 6 weeks. The investigators also administered 125 ppm Mn as MnSO 4 CH 2 O to anemic newborn pigs for 27 days. In each case, the investigators observed decreased hemoglobin content in the blood of treated animals. Hemoglobin depression in baby pigs fed up to 2,000 ppm manganese was overcome by a dietary supplement of 400 ppm iron.

Kimura et al. ( 1978) provided rats with diets supplemented with 564 mg/ kg of manganese as MnCl 2 for 3 weeks. Assuming a food consumption factor of 0.05 above the dietary background, this corresponds to a daily dose of 28 mg Mn/ kg day. The study authors reported that brain serotonin levels were decreased in manganese treated rats. Monoamine oxidase activity was unchanged, but L amino acid decarboxylase activity in the brain was decreased by manganese treatment. Histopathological analysis of the brain was not conducted. Blood serotonin levels were increased in treated rats, and this change was accompanied by decreased blood pressure.

Shukla et al. ( 1978) administered a dose of 16 mg MnCl 2 C4H 2 O/ kg ( 4.4 mg Mn/ kg) in drinking water ( dose calculated by investigators) to rats for 30 days and evaluated the effect on hepatic enzyme activity. Treated rats revealed significantly decreased succinic dehydrogenase, alcohol dehydrogenase, and $ amylase activity when compared with controls. In contrast, manganese exposure resulted in significantly increased activities of monoamine oxidase ( MAO), adenosine triphosphatase, arginase, glutamate pyruvate transaminase (= alanine 7 13 Manganese   February 2003 aminotransferase, or ALT), ribonuclease, glucose 6 phosphatase, and " amylase activity in the livers of treated rats.

Hietanen et al. ( 1981) also studied the effect of manganese on hepatic and extrahepatic enzyme activities. Male Wistar rats were exposed to 0.5% Mn ( as MnCl 2) in the drinking water for 1, 4, or 6 weeks. Assuming an average body weight of 0.35 kg and average water consumption of 0.049 L/ day ( U. S. EPA, 1986d), this corresponds to an exposure of 0.7 mg Mn/ kg day. Changes in the activity of several enzymes, including aryl hydrocarbon hydroxylase, ethoxycoumarin O deethylase, and epoxide hydrase, were observed at 1 week but not at 6 weeks. Enzyme activities were increased in the liver, and decreased in the intestines and kidney.

In a 14 day oral exposure study, NTP ( 1993) administered diets containing 0, 3,130, 6,250, 12,500, 25,000, or 50,000 ppm manganese sulfate monohydrate to F344 rats ( 5/ sex/ dose). All rats survived the exposure period. Statistically significant differences in manganese treated rats included reduced body weight gain ( 57% decrease) and final body weight ( 13% decrease) in the high dose males when compared to the control group. Decreased leukocyte and neutrophil counts and reduced liver weight were observed in high dose males and females. The high dose groups also exhibited diarrhea during the second week of the study. Manganese concentrations in the livers of animals receiving the 50,000 ppm diet were more than twice those of the controls. The NOAEL and LOAEL values based on decreased weight gain ( males) and hematological changes were approximately 650 and 1,300 mg Mn/ kg day, respectively.

NTP ( 1993) also administered diets containing 0, 3,130, 6,250, 12,500, 25,000, or 50,000 ppm manganese sulfate monohydrate to B6C3F 1 mice ( 5/ sex/ dose) for 14 days. However, study animals were poorly randomized at the beginning of the study, and no effects clearly attributable to manganese exposure were identified.

Parenteral Exposure Singh et al. ( 1974; 1975) administered 6 mg Mn/ kg day ( as MnSO 4° 4H 2 O) intraperitoneally to male IRTC rats for 25 days. Histopathological analysis of the livers revealed mild congestion of central veins and sinusoids, and some focal necrosis in treated animals.

Scheuhammer and Cherian ( 1983) reported toxic effects in the pancreas resulting from intraperitoneally injected manganese. The exposure duration was 30 days. Adverse effects included a pancreatitis like reaction. The authors suggested that this reaction was potentiated by the presence of manganese in the peritoneal cavity, and would not occur as readily with manganese administered by the oral route.

Khandelwal et al. ( 1984) administered 6 mg Mn/ kg day ( as MnCl 2° 4H 2 O) intraperitoneally to male IRTC rats for 28 days. Activity of succinic dehydrogenase and cytochrome oxidase in liver tissue were decreased after 28 days of manganese treatment.

Khan et al. ( 1997) administered 16 mg/ kg day MnCl 2° 4H 2 O in saline intravenously to male beagle dogs ( 3/ group). Treatment duration was up to 4 hours/ day for 4 days. Two of the three dosed animals were in moribund condition, and were sacrificed for ethical reasons ( one on 7 14 Manganese   February 2003 day 3 and one on day 4). The third treated dog died on day 4. Symptoms prior to death included vomiting, diarrhea, tremors, lethargy, reduced food intake, reduced blood pressure with reflex tachycardia, and severe hepatotoxicity.

7.2.3 Subchronic Studies Oral Exposure Mitochondria rich organs, such as the liver and pancreas, are hypothesized to be most affected by excess manganese exposure. Wassermann and Wassermann ( 1977) reported ultrastructural changes of the liver cells in rats exposed to 200 mg/ L of manganese chloride in their drinking water for 10 weeks. Assuming water consumption of 0.049 L/ day and an average body weight of 0.35 kg ( U. S. EPA, 1986d), this level of exposure corresponds to an average daily dose of approximately 12 mg Mn/ kg day. Increased metabolic activity was inferred from an increased amount of rough endoplasmic reticulum, the occurrence of multiple rough endoplasmic cisternae and prominent Golgi apparatus, and large Golgi vesicles filled with osmiophilic particles in the biliary area of the liver cell. The authors attributed this apparent increase in metabolic activity to biochemical processes related to the nutritional requirement for manganese, and homeostatic processes triggered by increased exposure. They noted that other observed liver effects, including the presence of glycogenosomes in the biliary area, groups of collagen fibers in the Disse's spaces, and degenerative changes in some centrilobular liver cells, may either be direct toxic phenomena or secondary responses to the effect exerted by manganese on other target tissues. ATSDR ( 2000) evaluated these data and designated 12 mg Mn/ kg day as the NOAEL in this study.

Carter et al. ( 1980) exposed young, iron deficient rats to 400 to 3,550 ppm Mn as Mn 3 O 4 for 32 weeks ( route not specified). Manganese treatment resulted in decreased hemoglobin levels.

Leung et al. ( 1982) administered 1,000, 10,000, or 20,000 mg MnCl 2° 4H 2 O/ L in drinking water to female Wistar rats. Exposure was initiated at conception by administration of manganese containing drinking water to the dams, and continued through age 60 days. The estimated doses were 38.9, 389, and 778 mg Mn/ kg day ( U. S. EPA, 1993). Treated rats exhibited liver necrosis and ultrastructural alterations that resembled human cholestasis. A LOAEL of 38.9 mg Mn/ kg day was identified in this study based on hepatic necrosis.

In a 13 week study, NTP ( 1993) administered diets containing 0, 1,600, 3,130, 6,250, 12,500, or 25,000 mg/ kg manganese sulfate monohydrate above basal levels to F344 rats ( 10/ sex/ dose). The concentration of manganese in the control diets was approximately 92 mg/ kg. Mean daily intake of manganese sulfate monohydrate ranged from 98 mg/ kg day ( 32 mg Mn/ kgday for the low dose to 1,669 mg/ kg day ( 542 mg Mn/ kg day) for the high dose males. For females, the range was 114 mg/ kg day ( 37 ) for the low dose group and 1,911 mg/ kg day ( 621 mg Mn/ kg day) for the high dose group. No rats died during the study, and no clinical or histopathology findings were attributed to manganese exposure. Females receiving diets with $ 6,250 mg/ kg manganese sulfate experienced decreased body weight gain. Absolute and relative liver weights were decreased in males receiving diets with $ 1,600 mg/ kg, and in 7 15 Manganese   February 2003 females in the highest dose group only. Hematological effects were also reported. All groups of exposed males exhibited a significantly increased neutrophil count. Lymphocyte counts were decreased in males receiving $ 6,250 mg/ kg in the diet and females in the three highest dose groups. The low dose of 1,600 mg/ kg ( about 32 mg Mn/ kg day) was identified as the LOAEL for this study, based on effects on liver weight and neutrophil counts in male rats.

In a concurrent 13 week study, NTP ( 1993) administered diets containing 0, 3,130, 6,250, 12,500, 25,000, or 50,000 mg/ kg manganese sulfate monohydrate above basal levels to B6C3F 1 mice ( 10/ sex/ dose). The concentration of manganese in the control diets was approximately 92 mg/ kg. Mean daily intake of manganese sulfate monohydrate ranged from 328 mg/ kg day ( 107 mg Mn/ kg day) for the low dose to 8,450 mg/ kg day ( 2,746 mg Mn/ kg day) for the high dose group. No deaths were attributed to manganese exposure. All groups of male mice and female mice in the highest dose group exhibited significantly decreased body weight gain. Relative and absolute liver weights were decreased in males in the highest dose group. Both sexes receiving the 50,000 mg Mn/ kg diet exhibited decreased hematocrit and hemoglobin concentration. The NTP report suggests that these findings may indicate microcytic anemia, which may have resulted from a sequestration or deficiency of iron. Males receiving $ 25,000 ppm also exhibited significantly lower leukocyte counts, although this finding was of questionable relevance to manganese exposure. No clinical findings were attributed to manganese exposure. The LOAEL for this study was 3,130 mg/ kg day ( 107 mg Mn/ kg day), based on significantly decreased body weight gain in male mice.

Komura and Sakamoto ( 1991) supplemented mouse diets with different chemical forms of manganese. Male mice ( 8/ group) were exposed either to a control diet containing 130 mg Mn/ kg, or a diet supplemented with an additional 2,000 mg Mn/ kg as MnCl 2° 4H 2 O, Mn( CH 3 COO) 2° 4H 2 O, MnCO 3, or MnO 2. Assuming an average food consumption of 13% of body weight, the average daily dose from the control diet was approximately 17 mg Mn/ kg day, while the average daily dose from the manganese enriched diet was 276 mg Mn/ kg day. The duration of treatment was 100 days. The mice were tested for spontaneous motor activity after 30 days. Blood and tissues were analyzed at the termination of the experiment. No significant difference in food intake among groups was seen. Body weight gain and red and white blood cell count was decreased in groups that received Mn( CH 3 COO) 2° 4H 2 O or MnCl 2. Motor activity was reduced in the MnCO 3 group. Tissue manganese concentrations in groups receiving supplemental manganese was 2 to 3 times that of controls. A LOAEL of 276 mg Mn/ kg day was identified in this study based on decreased weight gain and hematological effects.

Parenteral Exposure Suzuki et al. ( 1975) administered 250, 500, or 1,000 mg of MnO 2 in saline to monkeys ( Macaca mullata) by subcutaneous injection. Injections were given once a week for 9 weeks. The study authors reported a body weight of 4 kg for monkeys used in the study. Estimated timeaveraged doses correspond to 5.6, 11, and 23 mg Mn/ kg day. At autopsy, manganese treated monkeys had irregular arrangement of hepatic cords and lymphocytic infiltration.

7.2.4 Neurotoxicity 7 16 Manganese   February 2003 Occupational studies of miners, industrial workers, and agricultural workers have established injury to the central nervous system as the chief health effect associated with inhalation exposure to manganese. High level exposure by this pathway typically results in a suite of neurological effects collectively termed manganism. Chronic manganism associated with inhalation exposure is characterized by an extrapyramidal syndrome with symptoms that are somewhat similar to those observed in Parkinson's disease. One characteristic difference is the " cock walk" of the manganism patient, in which the patient walks on his toes with his spine erect and elbows flexed. Further, manganism patients do not often exhibit the " resting tremor" that Parkinson's patients do, and they have a propensity for losing their balance and falling backwards. The clinical course of manganism occurs in three phases: an initial phase of subjective and nonspecific symptoms; an intermediate phase of evolving neurological symptoms related to speech, dexterity, facial expression, and movement; and an established phase characterized by persistent, often irreversible neurological deficits ( Chang, 1996). While MRI scans of the brains of humans and non human primates exposed to excess manganese indicate that the metal deposits in the globus pallidus and to a lesser extent in the substantia nigra, degenerative lesions are limited to the globus pallidus ( Calne et al., 1994). An important question in the evaluation of health effects associated with manganese in drinking water is whether similar neurotoxicological effects occur following exposure by the oral route.

Oral Exposure studies of the neurotoxic effects of manganese exposure. A single study exists for evaluation of manganese exposure in primates by the oral route. Gupta et al. ( 1980) administered 25 mg MnCl 2 C4H 2 O/ kg orally to four male rhesus monkeys daily for 18 months. This level is equivalent to an average daily dose of 6.9 mg Mn/ kg day. Animals were maintained on monkey pellets, two bananas/ day, and tap water. The monkeys developed muscular weakness and rigidity of the lower limbs. Histological analysis revealed degenerated neurons in the substantia nigra and scanty neuromelanin granules in some of the pigmented cells.

Bonilla and Diez Ewald ( 1974) noted that chronic exposure of rats to manganese chloride produces a marked decrease in brain biogenic amines, particularly dopamine.

Singh et al. ( 1979) administered manganese ( 16 mg/ kg in a 10% sucrose solution) alone or in combination with ethanol to groups of 20 male albino rats for 30 days. Exposure to manganese alone led to a 72% increase in manganese concentration in the brain ( 3.13 : g/ g dry weight versus 1.82 : g/ g for controls). This outcome was not altered by ethanol exposure. There were no morphologic changes in the brain tissue of any group. Significant alterations in activity were reported for several brain enzymes. Manganese exposure resulted in significant increases in monoamine oxidase ( p < 0.001), adenosine triphosphatase ( p < 0.001), ribonuclease ( p < 0.001), and glutamate oxaloacetate transaminase (= aspartate aminotransferase, or AST; p < 0.001). Significant decreases were observed for succinic dehydrogenase ( p < 0.02 and deoxyribonuclease ( p < 0.001). Concurrent exposure to ethanol resulted in a synergistic effect with some 7 17 Manganese   February 2003 Table 7 4. Neurological Effects of Oral Exposure to Manganese.

Species Compound Route Dose Duration CNS Effects Reference Behavioral Histological Biochemical Mouse MnCl 2 drinking water 3 : g MnCl 2/ mL 6 months + NS + Chandra et al. ( 1979)

Mouse MnCl 2 diet 1% MnCl 2 ( 1 month),

4% MnCl 2 ( 5 months) 6 months NS NS + Gianutsos and Murray ( 1982)

Mouse MnO 2 diet 1 mg MnO 2/ g 7.5 months  NS NS Morganti et al. ( 1985)

Rat MnCl 2 drinking water 5 mg MnCl 2/ mL 7 months NS NS + Bonilla and Diez Ewald ( 1974)

Rat MnCl 2 gavage 1, 10, 20 mg Mn/ kg day Birth   24 days old NS NS + Deskin et al. ( 1980)

Rat MnCl 2 ! 4H 2 O gavage 10, 15, 20 mg Mn/ kg day Birth   24 days old NS NS + Deskin et al. ( 1981)

Rat MnCl 2 ! 4H 2 O drinking water 1 mg MnCl 2 ! 4H 2 O/ mL 12 months + NS + Chandra and Shukla ( 1981)

Rat MnCl 2 ! 4H 2 O drinking water 1 mg MnCl 2 ! 4H 2 O/ mL 28 months NS NS + Leung et al. ( 1981)

Rat MnCl 2 ! 4H 2 O drinking water 1 mg MnCl 2 ! 4H 2 O/ mL over 2 years NS NS + Lai et al. ( 1981)

Rat MnCl 2 ! 4H 2 O drinking water 1 mg MnCl 2 ! 4H 2 O/ mL 4 months NS  + Lai et al. ( 1982a)

Rat MnCl 2 gavage 150 mg Mn/ kg 42 days + NS + Kristensson et al. ( 1986) Table 7 4 ( continued)

Species Compound Route Dose Duration CNS Effects Reference Behavioral Histological Biochemical 7 18 Manganese   February 2003 Rat MnCl 2 ! 4H 2 O drinking water 1 mg MnCl 2 ! 4H 2 O/ mL 65 weeks + NS NS Nachtman et al. ( 1986)

Rat MnCl 2 ! 4H 2 O drinking water 4,360 mg Mn/ L 60   265 days NS NS + Eriksson et al. ( 1987)

Rat MnCl 2 gavage 25, 50 mg MnCl 2 ! 4H 2 O / kg day 14 or 21 days NS NS + Kontur and Fechter ( 1988)

Rat MnCl 2 ! 4H 2 O gavage 0.357 Mn mg/ kg day 30 days + NS NS Oner and Senturk ( 1995)

Rat Not specified 10% sucrose 16 Mn mg/ kg 30 days NS NS + Singh et al. ( 1979)

Monkey MnCl 2 ! 4H 2 O diet 25 mg MnCl 2 ! 4H 2 O/ kg 18 months + + NS Gupta et al. ( 1980)

Notes: NS = Not studied Source: U. S. EPA, 1993 7 19 Manganese   February 2003 enzymes and an antagonistic effect with others. No mechanism was proposed to explain the pattern observed in the presence of ethanol.

Chandra et al. ( 1979) evaluated the neurological effects of manganese in mice exposed from birth. Neonatal mice were initially exposed by nursing from dams given 5 mg/ mL MnCl 2 in their drinking water. After weaning at 25 days, the mice received manganese in their drinking water. Average exposures to manganese were determined to be 0.030 mg Mn/ day for 60 days, 0.036 mg Mn/ day through the 90th day, 0.075 mg Mn/ day through the 120th day and 0.090 mg Mn/ day for the interval between 150 and 180 days. Assuming a body weight of 0.03 kg at adulthood, the average daily dose at the termination of the experiment was approximately 3 mg Mn/ kg day. Elevated levels of striatal dopamine, norepinephrine, and homovanillic acid were observed at 60 and 90 days of age, with a concomitant increase in spontaneous locomotor activity. Exposure past 90 days did not influence motor activity. Chandra et al. ( 1979) proposed that the hyperactivity observed in these mice was an early behavioral effect of excess manganese exposure that resulted from elevated dopamine and norepinephrine levels. The study authors further suggested that the observed hyperactivity may be comparable to the psychomotor excitement observed in the early stages of human manganism.

Gray and Laskey ( 1980) found that dietary exposure to 1,100 mg/ kg manganese ( as Mn 3 O 4) in rats for 2 months produced only reduced reactive locomotor activity. Assuming a body weight of 0.35 kg, this level of exposure corresponds to an average daily dose of 55 mg Mn/ kg day. Deskin et al. ( 1980) studied neurochemical alteration induced by manganese chloride in neonatal CD rats. Rats were intubated with daily doses of 1, 10, or 20 mg Mn/ kg day from birth to 24 days old. Neurochemical components were subsequently analyzed in the hypothalamus and corpus striatum. Administration of 10 and 20 mg Mn/ kg day resulted in significantly elevated manganese concentrations in both regions, but neurochemical alterations were observed only in the hypothalamus. These alterations included a decrease in dopamine concentration and turnover. The highest dose of manganese also resulted in a significant decrease in hypothalamic tyrosine hydroxylase activity, and an increase in monoamine oxidase activity. Visible signs of toxicity were not observed in any group.

Deskin et al. ( 1981) intubated rats with daily doses of 10, 15 or 20 mg Mn/ kg day ( as MnCl 2 C4H 2 O) from birth to 24 days old. The authors reported a significant elevation of serotonin levels in the hypothalamus, but not the striatum, following exposure to 20 mg Mn/ kg.

Chandra and Shukla ( 1981) exposed male albino rats to 1,000 mg/ L MnCl 2° 4H 2 O ( 436 mg Mn/ L) in drinking water. Assuming water consumption of 0.049 L/ day and an average adult body weight of 0.35 kg, this level of exposure corresponds to an average daily dose of 61 mg Mn/ kg day. Levels of catecholamines, homovanillic acid, manganese, and the activity of monoamine oxidase were determined in the corpus striatum at time intervals up to 360 days. The investigators found initial increases in dopamine, norepinephrine, and homovanillic acid levels. This initial increase was followed by a period of normal levels. After 300 days, a decrease in all levels was observed. These changes were not correlated with the tissue concentration of manganese. The authors suggested that the decreased locomotor activity observed during later periods of manganese exposure may be related to lowered dopamine and norepinephrine levels in the brain, and that this stage of chronic toxicity may correspond to the later neurologic phase of 7 20 Manganese   February 2003 motor dyskinesia in humans. Ali et al. ( 1981) conducted concurrent behavioral studies, and found an initial increase in spontaneous locomotor activity followed by a decrease during later periods of manganese exposure.

Lai et al. ( 1981) exposed female Wistar rats to 1,000 mg/ L MnC1 2 C4H 2 0 ( 280 mg Mn/ L) in drinking water. Exposure was initiated at mating. Pups were exposed in utero by administration of manganese in drinking water to dams via maternal milk during nursing, and by inclusion in drinking water after weaning. Groups of rats were exposed to manganese for over 2 years and were either 2 months or 24 to 28 months of age at examination. Assuming a body weight of 0.35 kg and water consumption of 0.049 L/ day, the average daily dose for rats at adulthood was approximately 39 mg Mn/ kg day. The brains were dissected and analyzed for activity of glutamic acid decarboxylase ( GAD), choline acetyltransferase ( ChAT), and acetylcholinesterase ( AChE). GAD, ChAT, and AChE are neurochemical markers for the GABA and cholinergic systems, and had previously been implicated in manganese toxicity ( Sitaramayya et al., 1974; Bonilla, 1978a, b). Adverse effects of chronic manganese exposure on the activity of GAD, ChAT, or AChE were not apparent in 2 month old rats. The study authors reported that lifetime exposure to manganese produced effects that counteracted age related decreases in GAD, ChAT, and AChE.

Leung et al. ( 1981) analyzed the same groups of rats used by Lai et al. ( 1981) for monoamine oxidase ( MAO) activity. MAO is a key enzyme in oxidative degradation of neurotransmitter amines. The only effect observed following exposure of 2 month old rats to manganese was a small decrease in the neurotransmitter serotonin in the cerebellum. No significant differences were observed in manganese treated 24 to 28 month old rats.

Lai et al. ( 1982a) examined the effects of manganese exposure on male Wistar rats. The rats were initially exposed to manganese in utero. Following weaning, the rats were exposed to 1,000 mg MnCl 2 C4H 2 O/ L ( 280 mg Mn/ L) in drinking water for either 70 to 90 days or 100 to 120 days after birth. Assuming an adult weight of 0.35 kg, and water consumption of 0.049 L/ day, this level corresponds to a dose of approximately 39 mg Mn/ kg day. Levels of dopamine, noradrenaline, serotonin, and choline were determined. A significant decrease was seen in the uptake of dopamine by synaptosomes isolated from the hypothalamus, striatum and midbrain in 70 to 90 day old rats. No effect was observed in the 100 to 120 day old rats. Choline levels were higher in 70 to 90 day old exposed rats and lower in 100 to 120 day old exposed rats when compared with controls. The authors suggested that this finding may reflect involvement of both the dopaminergic and cholinergic systems in manganese toxicity. They concluded that, although the rat may not serve as an ideal model for understanding the neurotoxic effects of manganese, neurochemical effects are discernible when analyses are made at the appropriate period.

Lai et al. ( 1982b) investigated the effect of manganese exposure on the developmental profile of acetylcholinesterase activity in different regions of the brain. Female Wistar rats were exposed to manganese chloride tetrahydrate provided in drinking water at a concentration of 1,000 mg/ L. Exposure was initiated at conception. Male offspring were weaned onto drinking water containing 1,000 mg/ L manganese chloride tetrahydrate and exposed for up to 60 days. Enzyme activity in the cerebral cortex, striatum, midbrain, pons and medulla, hypothalamus, and 7 21 Manganese   February 2003 cerebellum was determined at 5, 12, 20, 30, and 60 days after birth. The developmental profile of the enzyme differed in the various regions. Activity was detected earlier in the more caudal regions, except in the cerebellum where there was no increase. Exposure to manganese from conception did not influence the developmental profile of acetylcholinesterase activity.

Gianutsos and Murray ( 1982) studied changes in the concentrations of dopamine and GABA in mice exposed to MnCl 2 in the diet. A 1% concentration of MnCl 2 was administered in the diet to an unspecified number of male CD 1 mice for 1 month. This level of exposure corresponds to 568 mg Mn/ kg day. The concentration was increased to 4% for an additional 5 months. During this period, the average daily dose was 2,272 mg Mn/ kg day. Dopamine content in the striatum and in the olfactory tubule at 6 months was reduced compared with controls ( p < 0.05). GABA content of the striatum was increased ( p < 0.05). Apparent increases in the substantia nigra area and a decrease in the cerebellum were not statistically significant. No changes in neurotransmitter levels were observed when assays were conducted after 1   2 months of exposure.

Morganti et al. ( 1985) conducted a behavioral study using male ICR strain Swiss mice. The mice were fed powdered Charles River's RMH 300 diet that contained 1,000 mg MnO 2/ kg. This dietary concentration corresponds to approximately 632 mg Mn/ kg. The mice consumed 5 g of food daily. Assuming a body weight of 0.03 kg ( U. S. EPA, 1986d), this level of exposure corresponds to an average daily dose of 105 mg/ kg day. Neurobehavioral evaluation began after 16 weeks of feeding and continued at 2 week intervals for 30 weeks. The endpoints evaluated were open field and exploratory behavior, passive avoidance learning, and rotarod performance ( a measurement of balance and coordination). Multivariate analysis of variance ( 2 treatments and 8 samples by week of exposure) was used to test for intergroup differences. No significant behavioral differences were apparent in any treatment group. In contrast, Morganti et al. ( 1985) observed significant effects in mice exposed to manganese by inhalation for 7 hours/ day, 5 days/ week, at levels greater than 50 mg Mn/ m3. The duration of exposure was 16 to 32 weeks. This level of inhalation exposure was considered by the authors to be comparable to the oral exposure.

Ali et al. ( 1985) studied the effect of dietary protein on manganese neurotoxicity. Rats received either a normal diet ( 21% casein) or a low protein diet ( 10% casein). Half of each dietary group served as a control while the other half received MnCl 2 C4H 2 O ( 3,000 mg Mn/ L) in the drinking water for 90 days. Assuming an adult body weight of 0.35 kg and water consumption of 0.049 L/ day, this corresponds to an average daily dose of 420 mg/ kg day. The low protein diet was associated with decreased levels of brain dopamine ( DA), norepinephrine ( NE), and serotonin. Manganese exposure resulted in a marked increase in DA and NE levels, which were more pronounced in the low protein group. A significant decrease in serotonin levels following manganese exposure occurred only in the low protein group. Weaned F 1 pups of treated rats exhibited the same pattern of effects. The study authors concluded that protein deficiency can increase vulnerability of rats to the neurotoxic effects of manganese.

Nachtman et al. ( 1986) studied the behavioral effects of chronic manganese exposure. Male Sprague Dawley rats were administered 0 or 1 mg MnCl 2 C4H 2 O/ mL in drinking water for 65 weeks. Assuming a body weight of 0.35 kg and water consumption of 0.049 L/ day, this 7 22 Manganese   February 2003 corresponds to an average daily dose of 39 mg Mn/ kg day. The treatment did not result in any change in body weight. The manganese exposed rats exhibited a significant increase in locomotor activity during weeks 5 to 7. However, the effects were transient, and by 8 weeks the activities had returned to control levels. Treated rats examined at 14 and 29 weeks were found to be more responsive to the effects of d amphetamine ( a locomotor stimulant that works primarily by releasing dopamine) than were controls. There was no difference between manganese treated rats and controls at 41 or 65 weeks. The investigators concluded that manganese exposure may result in a transient increase in dopaminergic function, as evidenced by increased spontaneous and d amphetamine stimulated locomotor activity.

Kristensson et al. ( 1986) studied the effect of manganese on the developing nervous system of young rats. Starting at 3 days of age, Sprague Dawley rats received a daily dose of 150 mg Mn/ kg day ( as MnCl 2) by gavage. The treatment continued until the rats reached 44 days of age. At days 15 to 22 there was a large but transient increase ( 7 to 40 fold) of manganese in the brain, and the rats displayed a rigid and unsteady gait. By 44 days, the rats appeared normal and brain manganese levels had declined to approximately 3 times the control level. Histological analysis revealed no abnormalities in the brains of manganese exposed rats. Axonal growth and the axon myelin relation were normal. A second group of rats was treated for 15 days. At this time point, half the rats were sacrificed and half were maintained untreated until sacrifice at 60 days of age. The rats were subsequently analyzed for brain content of dopamine and its metabolites, including 2,4 dihydroxyphenylacetic acid and homovanillic acid ( HVA), and serotonin and its major metabolite 5 hydroxyindolacetic acid. Only HVA levels in the hypothalamus and striatum were affected by manganese treatment. Significantly decreased HVA levels were seen at the 15 day sacrifice. Similar decreases in rats treated for 15 days and allowed to recover until 60 days of age were not observed. The investigators concluded that divalent manganese has a very low degree of toxicity for the developing nervous system in rats, but that longer term exposure to more active manganese compounds may cause severe damage to certain neurologic pathways. In addition, the investigators emphasized that rodents may not be appropriate for comparison with primates. Unpublished studies, where monkeys exposed to manganese oxide developed severe motor disturbances, were cited as the basis for this conclusion.

Eriksson et al. ( 1987) studied the effect of long term manganese exposure on biogenic amine levels in rat brain. Starting at 20 days of age, groups of male Sprague Dawley rats were provided with drinking water containing 10 g/ L manganese chloride ( MnC1 2 C4H 2 0) for 60, 100, 165, or 265 days. This concentration corresponds to 2,777 mg Mn/ L. Assuming an adult body weight of 0.35 kg and water consumption of 0.049 L/ day, this level of exposure results in an average daily dose of approximately 390 mg Mn/ kg day. There were no clinical signs of toxicity. Following 60 days of exposure, manganese concentration in the striatum was estimated to be 1.3 to 2.0 mg/ kg, in contrast to control levels of 0.4 to 0.5 mg/ kg. Levels of dopamine, 3,4 dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5 hydroxyindoleacetic acid were determined in discrete regions of the caudate putamen. Rats exposed for 60 and 165 days showed significantly increased levels of dopamine ( DA) and 3,4 dihydroxyphenylacetic acid, but these alterations were not seen in rats exposed for 100 or 265 days. This suggests an increased synthesis and turnover of dopamine that is reversible, even with continuous manganese exposure. 7 23 Manganese   February 2003 This study identifies a LOAEL of 390 mg Mn/ kg day based on increased levels of dopamine at 60 days.

Kontur and Fechter ( 1988) intubated neonatal Long Evans rats daily with 0, 25, or 50 mg/ kg day manganese chloride ( MnC1 2 C 4H 2 0) for 14 or 21 days. These doses correspond to 6.9 and 13.9 mg Mn/ kg day. The level of manganese in the brain was increased at both 14 and 21 days, but was greater at 14 days. Monoamine and metabolite levels were not altered in any brain region by manganese treatment. The study authors suggested that the different results reported by different laboratories may be attributable to species or strain differences, the dosing regimen or vehicle, the route of administration, or the time points chosen for testing. These data suggest a NOAEL of 6.9 mg Mn/ kg day for this study, based on the absence of effect on monoamine levels.

These collective studies suggest that preclinical neurological effects are possible in the human following oral exposure; however, there are dissimilarities in the spectrum of responses between rodent and primate models of toxicity that preclude a determination of the oral dose range that might be expected to induce these preclinical effects. Further, conflicting data concerning responses in humans and confounding factors in the limited human epidemiological studies prevent determination of any dose response effect in humans exposed to manganese excesses via ingestion.

Parenteral Exposure Although deficiencies exist in experimental design ( U. S. EPA, 1984), primate studies by parenteral routes of exposure have reported extrapyramidal signs and histologic lesions similar to those described in humans. Mella ( 1924) treated four rhesus monkeys with MnCl 2 for 18 months. Two monkeys served as controls. The treated monkeys received gradually increasing doses of MnCl 2 by intraperitoneal injection on alternate days. The doses started at 5 mg and reached a maximum of 25 mg per injection. The monkeys exhibited uncontrolled, involuntary movements ( chorea) followed by rigidity, disturbances of motility, fine hand tremors, and finally, contracture of the hands. Histological changes were reported in the putamen, the caudate, and the globus pallidus.

Suzuki et al. ( 1975) exposed monkeys subcutaneously to 39.5, 79.0 or 158.0 mg Mn/ kg as MnO 2 once a week for 9 weeks and found the latency of neurologic signs ( tremors, excitability, choreiform movement, loss of equilibrium, and contracture of hands) inversely related to cumulative dose. Signs appeared earlier when higher doses were administered, but the severity of symptoms was not completely dose related. The estimated daily doses in this experiment were 5.6, 11, and 23 mg Mn/ kg day.

Olanow et al. ( 1996) reported damage to the globus pallidus and substantia nigra in monkeys that were dosed intravenously with doses as low as 4.4 mg Mn/ kg/ week ( for 7 weeks). The brain damage was accompanied by neuromuscular toxicity including bradykinesia, rigidity, facial grimacing, and abnormal posturing of the limbs. Newland and Weiss ( 1992) administered repeated daily intravenous doses of manganese to Cebus monkeys so that the monkeys received cumulative doses of 5 or 10 mg/ kg for 450 days. The dosings were separated by at least one 7 24 Manganese   February 2003 week. The authors observed that single intravenous doses of 5 or 10 mg Mn/ kg day resulted in a significant increase in the number of incomplete responses of dosed monkeys to a spring loaded test device that measured physical exertion through a rowing motion. The increase in incomplete responses occurred within a few days after dosing began. Further, action tremor was observed in the monkeys who had received cumulative doses of 40 mg/ kg or higher; however, dystonia was never observed.

Eriksson et al. ( 1992) subcutaneously injected two monkeys with 0.4 g doses of MnO 2 ( 0.253 g Mn) in water. Eleven doses were administered over 4 months, followed by a final dose at 12 months. Both animals developed an unsteady gait and exhibited hypoactive behavior. PET scans indicated that degeneration of dopaminergic nerve endings occurred following Mn intoxication.

Additional studies have examined the neurotoxic effects of manganese administered by parenteral routes in non primate species. Mustafa and Chandra ( 1971) and Chandra ( 1972) reported paralysis of the hind limbs in rabbits administered 169 mg Mn/ kg ( as MnO 2) intratracheally. The paralysis developed after a period of 18 to 24 months. Examination of the of affected animals brains showed widespread neuronal loss and neuronal degeneration in the cerebral cortex, caudate nucleus, putamen, substantia nigra and cerebellar cortex. These findings are reminiscent of the characteristic histopathologic and neurologic consequences of manganism found in exposed workers ( U. S. EPA, 1993). A marked decrease in brain catecholamine levels and related enzyme activity was also noted.

Histopathologic evaluations of exposed rats by Chandra and Srivastava ( 1970), Chandra et al. ( 1979) and Shukla and Chandra ( 1976) found scattered neuronal degeneration in the cerebral and cerebellar cortex. Daily intraperitoneal administration of 2 to 4 mg Mn/ kg for # 120 days appeared to be the threshold for the appearance of microscopic lesions. These studies also demonstrated an association between the maximum number of degenerated neurons and maximum manganese concentration in the brain.

Scheuhammer ( 1983) treated male Sprague Dawley rats intraperitoneally for 30 days with either 3.0 mg Mn/ kg or an equal volume of 0.9% NaCl. Assuming an average adult body weight of 0.35 kg, this treatment corresponds to an average daily dose of 8.6 mg Mn/ kg day. Following sacrifice, the pancreas was removed, fixed in 10% buffered formalin, and subsequently processed for light microscopy. Significant pathological changes were observed in pancreatic tissue from manganese exposed rats. The changes were characterized by a pancreatitis like reaction consisting of expanded interacinar spaces, a thickened connective tissue capsule with invaginations of fibrotic connective tissue septa extending into the body of the gland, the presence of an inflammatory infiltrate of neutrophils, lymphocytes, macrophages, and the separation of groups of acini from the body of the pancreas with occasional destruction of acinar cells. Other peritoneal organs did not exhibit pathological changes. This study suggests that intraperitoneally injected Mn( II) exerts a selective toxicity on pancreatic tissue. Therefore, the study author cautioned against use of intraperitoneal injection as the route of administration for chronic Mn neurotoxicity studies. 7 25 Manganese   February 2003 Brouillet et al. ( 1993) administered 0, 0.5, 1, or 2 : mol of MnCl 2 in deionized water to male rats by a single intrastriatal injection. Assuming a body weight of 0.35 kg for an adult rat, these doses correspond to 0, 0.077, 0.171, and 0.314 mg Mn/ kg. Each treatment group contained 9 to 10 rats. The lowest dose produced a significant reduction in dopamine, but had no effect on the other neurochemical markers examined. Doses of 0.171 and 0.314 mg Mn/ kg produced a reduction in dopamine levels, changes in neurochemical markers, and indications of impaired oxidative metabolism.

7.2.5 Developmental/ Reproductive Toxicity Developmental Studies Studies are limited regarding developmental toxicity in humans following oral exposures to manganese. Kilburn ( 1987) reported an increased incidence in birth defects and stillbirths in a small population of indigenous peoples in Groote Eylandt, Australia. Although the area was rich in manganese deposits and ingestion of excess amounts of the metal was suspected, the study suffered from a lack of exposure data, small sample sizes, and no suitable control group. Further, inhalation exposure to manganese could not be ruled out. Studies by He et al. ( 1994) and Zhang et al. ( 1995) suggest that oral exposures to excess manganese can possibly result in increased neurological deficits measured as poorer performance in school and on standardized neurobehavioral exams. These studies also suffer from a lack of adequate exposure data and the potential presence of confounding factors, such as exposure to other potential neurotoxicants and possible inhalation exposure to manganese.

Developmental studies conducted in animals are summarized in Table 7 5. These studies suggest that manganese is a potential developmental toxicant, but additional studies that are better controlled are necessary in order to determine how potent it is.

Several studies have reported developmental effects in animal models following oral administration of manganese. Järvinen and Ahlström ( 1975) exposed female rats to 4, 24, 54, 154, 504, or 1,004 mg Mn/ kg ( as manganese sulfate heptahydrate) in the diet for 8 weeks after weaning and during pregnancy. No signs of embryotoxicity or fetotoxicity were observed. Increases in the whole body content of manganese in fetuses and in liver manganese content of the dams were reported at dietary levels above 154 mg Mn/ kg. No increase in liver manganese content was observed in non pregnant females. Chandra and Shukla ( 1978) administered bolus doses of 1 mg Mn/ kg day to neonatal rats for 60 days. Neuronal degeneration and increased monoamine oxidase were reported on days 15 and 30 of the study, but no clinical or behavioral signs of manganese neurotoxicity were reported.

Several studies have measured changes in brain chemistry in neonatal rats following oral exposure to manganese. Deskin et al. ( 1980, 1981) dosed rat pups via gavage with MnCl 2 in 5% 7 26 Manganese   February 2003 Table 7 5. Developmental Effects of Exposure to Manganese.

Compound Species Route Dose ( mg Mn/ kgday Effect Reference MnSO 4 ! 7H 2 0 Rat ( female) Oral ( diet) 0 4 24 54 154 504 1,004 Increased manganese concentration in fetus and maternal liver; no indications of embryo or fetotoxicity Järvinen and Ahlström ( 1975)

MnCl 2 Rat Bolus ( in water) 1 Neuronal degeneration; increased monoamine oxidase; no indications ( clinical or behavioral) of neurotoxicity Chandra and Shukla ( 1978)

MnCl 2 Rat Oral ( gavage) 0 1 10 20 Decreased dopamine; decreased tyrosine hydroxylase; increased monoamine oxidase activity ( all changes in hypothalamus only) Deskin et al. ( 1980)

Mn 3 O 4 Mouse ( Male) Oral ( diet) 1,050 Decreased preputial gland, seminal vesicle, and testes growth Gray and Laskey ( 1980)

MnCl 2 Rat Oral ( gavage) 0 10 15 20 Increased hypothalamic serotonin Deskin et al. ( 1981)

Mn 3 O 4 Rat Oral ( diet) 0 350 1,050 3,500 Decreased serum testosterone; decreased sperm count; decreased testes weight; prevented normal decrease in serum FSH Laskey et al. ( 1982)

MnCl 2 Rat Oral ( drinking water) 240 Delayed air righting reflex; delayed age of eye opening; delayed development of auditory startle Ali et al. ( 1983)

Mn 3 O 4 Rat Oral ( drinking water) 40 Decreased chloline acetyltransferase activity in cerebellum and midbrain Lai et al. ( 1984)

MnCl 2 Rat Oral ( drinking water) 0 68 136 232 Decreased water consumption and decrease in weight gain in two highest dose groups; no changes in catecholamine or startle response in the exposed pups Kontur and Fechter ( 1985)

Mn 3 O 4 Rat Oral ( gavage) 0 71 214 Decreased serum testosterone following 7 days of hCG induction Laskey et al. ( 1985) Compound Species Route Dose ( mg Mn/ kgday Effect Reference 7 27 Manganese   February 2003 MnCl 2 Rat ( male) Oral ( drinking water) 150 Transient ataxia; decreased striatal and hypothalamic homovanillic acid concentrations Kristensson et al. ( 1986)

MnCl 2 ! 4H 2 0 Mouse Subcutaneous injection 0 0.56 1.1 2.2 4.4 Decreased weight gain/ food consumption; increased late resorptions; reduced fetal body weight; increased incidence of morphological defects Sanchez et al. ( 1993)

MnCl 2 Rat ( female) Rabbit ( female) Oral ( gavage) 0 11 22 33 Delayed skeletal and internal organ development and increased external malformations in rat pups delivered by Caesarean section. No effects in rabbit Szakmáry et al. ( 1995)

MnCl 2 Mouse Subcutaneous injection 50 Late resorptions; postimplantation loss; skeletal anomalies; reduced fetal body weight Colomina et al. ( 1995)

MnCl 2 Rat Intravenous 0 0.27 × 10 3 1.1 × 10 3 2.2 × 10 3 Increased incidence of skeletal malformations including angulated or irregularly shaped clavicle, femur, fibula, humerus, ilium, radius, scapula, tibia, and/ or ulna Treinen and Blazak ( 1995)

MnCl 2 Mouse Intravenous 0.3 1.6 Increased fetal weight at low dose; decreased fetal weight at high dose; fetal skeletal abnormalities at high dose Grant et al. ( 1997)

MnCl 2 Rat Oral ( drinking water) 0 350 1,420 Thinning of cerebral cortex; absence of convincing brain histopathological or behavioral evidence from perinatal manganese exposure on the brain Pappas et al. ( 1997)

MnCl 2 Rat Oral ( drinking water) 11 22 Decreased body weight gain; increased response to auditory stimulus Dorman et al. ( 2000) 7 28 Manganese   February 2003 sucrose for 24 days starting on the first postnatal day. The administered doses in the earlier study were 0, 1, 10, and 20 mg Mn/ kg day. Decreased dopamine levels in the hypothalamus were reported at the two highest doses, and decreased tyrosine hydroxylase levels and increased monoamine oxidase activity ( perhaps due to increased levels of the enzyme) were reported in the hypothalamus at the highest dose. No other changes in brain chemistry were reported in the hypothalamus, and no other brain section was affected. In the latter study, doses of 0, 10, 15, and 20 mg Mn/ kg day were administered. Hypothalamic serotonin was observed to be increased at the highest dose; the level of this transmitter was unaffected in the striatum. Lai et al. ( 1984) reported small decreases in choline acetyltransferase activity in the cerebellum and midbrain of 2 month old rats that had been exposed to 40 mg Mn/ kg day from conception, throughout gestation, and throughout life. Other neuronal enzymes ( e. g., glutamic acid decarboxylase, acetylcholinesterase) were unaffected.

Kristensson et al. ( 1986) dosed 3 day old male rat pups with 150 mg Mn/ kg day ( in water) for 41 days. The authors reported a transient ataxia ( days 15 22), which was resolved by the end of the dosing period, in the pups. Manganese levels in the blood and brain ( brain levels were increased 7 40 fold) were elevated significantly over controls in 15 and 20 day old pups; brain levels had decreased to approximately 3 fold over control levels in 43 day old pups. Homovanillic acid ( metabolite of dopamine) concentrations were decreased in the striatum and hypothalamus, but not in other brain regions; no other monoamines or their metabolites were affected.

A few studies have measured reproductive endpoints in the developing rodent. Manganese administered to pre weanling male mice at a dose of 1,050 mg Mn/ kg day beginning on postnatal day 15 resulted in the decreased growth of reproductive organs ( preputial gland, seminal vesicle, and testes) measured on days 58, 73, and 90 but did not affect body growth or liver or kidney weights ( Gray and Laskey, 1980). Laskey et al. ( 1982) administered dietary manganese at doses of 0, 350, 1,050, and 3,500 mg Mn/ kg day to male and female rats fed a diet either adequate or deficient in iron. Males and females were mated during days 90 100 of the study; testes weights of male offspring fed the iron deficient diet were decreased as compared to controls at day 40 at the highest two doses and at day 100 at the intermediate dose. While 40 day old weanling rats did not exhibit any treatment related hormonal changes, exposed rats showed a dose related decrease in serum testosterone at 60 100 days of age ( when age related increases were expected), and no increase in serum luteinizing hormone was observed. The normal decrease in serum follicle stimulating hormone ( FSH) from 60 to 100 days was prevented by manganese exposure. Epididymal sperm count was decreased by the treatment only when given with the iron deficient diet.

In an additional study measuring the effects of manganese exposure on the developing reproductive system, Laskey et al. ( 1985) administered 0, 71, and 214 mg Mn ( as Mn 3 O 4)/ kg day via gavage to pre weanling rats on postnatal days 1 21. The study authors measured serum levels of FSH, LH, and testosterone in the pups at 21 or 28 days of age. Manganese exposure did not affect endogenous or stimulated serum levels of FSH or LH, nor did it affect endogenous or acute human chorionic gonadotropin ( hCG) induced serum testosterone at 2 hours. Serum testosterone was decreased following 7 days of hCG induction, however. The delayed decrease 7 29 Manganese   February 2003 in testosterone was hypothesized by the study authors to be a result of an unknown manganeseinduced effect on the Leydig cell.

Ali et al. ( 1983) evaluated potential changes in developmental endpoints in rat pups after administering excess manganese in drinking water to pregnant dams fed a normal or low protein diet. Manganese exposure was started 90 days prior to mating and continued throughout gestation and nursing. The offspring of dams who had ingested 240 mg Mn/ kg day exhibited delayed air righting reflexes. Significant delays in the age of eye opening and the development of auditory startle were reported in pups from dams ingesting protein deficient diets. No decreases in body weight or brain weight were reported in the offspring of rats fed normalprotein diets.

Kontur and Fechter ( 1985) exposed pregnant Long Evans rats to 0, 5,000, 10,000, or 20,000 mg/ L of manganese chloride in drinking water throughout the gestation period. Rats in the 10,000 and 20,000 mg/ L groups showed reduced water intake and a significant decrease in weight gain. A significant decrease in birth weight was observed in the 20,000 mg/ L group. At one day of age, pups from the 10,000 and 20,000 mg/ L groups had increased manganese levels in the forebrain, although there was no difference in the extent of accumulation between the two groups. The increased manganese levels were not associated with any changes in catecholamine function or startle response in the exposed pups. The authors concluded that manganese is not particularly toxic to developing rats, perhaps as a result of limited placental transfer.

The developmental effects of manganese have also been evaluated following parenteral administration. Sánchez et al. ( 1993) investigated the embryotoxic and teratogenic potential of manganese during organogenesis. Pregnant Swiss mice received daily subcutaneous injections of 0, 2, 4, 8 or 16 mg/ kg day of MnCl 2 A4H 2 O on days 6 to 15 of gestation. These doses correspond to 0, 0.56, 1.1, 2.2, or 4.4 mg Mn/ kg day, respectively. Dams were sacrificed on gestational day 18. Significant reductions in weight gain and food consumption were reported in dams receiving 8 mg/ kg day and above, and treatment related deaths were reported at 16 mg/ kgday A significant increase in the number of late resorptions was observed at doses of 4 mg/ kgday and higher, and reduced fetal body weight and an increased incidence of morphological defects were reported at doses of 2.2 mg Mn/ kg day and higher. No difference was seen in the incidence of individual or total malformations in treated groups when compared with controls. A NOAEL of 1.1 mg/ kg day was identified by the study authors for maternal toxicity. A NOAEL of 0.56 mg/ kg day was identified for embryo/ fetal toxicity Pappas et al. ( 1997) assessed behavioral, neurohistological, and neurochemical endpoints in rats exposed to manganese from conception to weaning. The investigators administered 0, 2,000, or 10,000 mg Mn/ L as manganese chloride in drinking water to female rats ( 10/ group) and their litters from conception until postnatal day ( PND) 30. The average daily consumption of manganese during gestation was 350 and 1,420 mg/ kg day, respectively, for the two manganese treatment groups. No effects were observed on pregnancy or birth parameters and no physical abnormalities were evident in the offspring of treated dams. The findings reflect a lack of effects on reproductive capability. Fifty male pups from each treatment group were subsampled for behavioral tests ( 10 to 22 per group), histopathology ( 6 to 8 per group) and neurochemical 7 30 Manganese   February 2003 analyses ( 6 to 8 per group). The rats exposed to 10,000 mg Mn/ L showed a 2.5 fold increase in brain cortical Mn levels. They also experienced reduced weight gain during PND 9 to 32, and were hyperactive at PND 17. Behavioral tests were conducted on pups from all groups at PND 17, 90 or 95. No significant differences in performance were noted for the radial arm maze, elevated plus apparatus, or Morris water maze behavioral tests. Both doses resulted in thinning of the cerebral cortex. The observed thinning may have been a consequence of either perinatal malnutrition or a direct effect on cortical development. Brain monoamine levels and choline acetyltransferase activity were unaffected by manganese exposure. Tyrosine hydroxylase immunohistochemistry indicated that dopamine neurons of the substantia nigra were intact. Glial fibrillary acidic protein immunoreactivity, an indicator of neuronal damage, was not increased in cortex, caudate nucleus or hippocampus. The authors emphasized that the most noteworthy result of this study was the absence of convincing histopathological and behavioral evidence for persistent effects of perinatal manganese exposure on the brain.

Grant et al. ( 1997) failed to observe any effects of manganese exposure on weight gain, gross malformations, or skeletal malformations in the offspring of pregnant rats dosed via gavage with 22 mg Mn/ kg day on gestational days 6 17. Another study indicates a lack of persistent developmental effects from oral manganese exposure during gestation. Szakmáry et al. ( 1995) reported the developmental effects of manganese administered via gavage to pregnant rats throughout gestation and to pregnant rabbits through organogenesis ( gestation day 6 20) at doses of 0, 11, 22, and 33 mg/ kg day. No developmental effects in the rabbit were observed. The highest dose resulted in retardation of development of the skeleton and internal organs of the rat, as well as a significant increase in external malformations ( e. g., clubfoot) in pups delivered by caesarean section. These effects, however, were not observed in 100 day old offspring of dams that had been similarly dosed, indicating that the developmental effects were self correcting. Manganese treatment did not affect the following endpoints in either the pup group that was surgically delivered or the group born live: ears, teeth, eyes, forward motion, clinging ability, body posture, correction reflex, or negative geotaxis reflex.