In a more recent study, Dorman et al. ( 2000) dosed neonatal CD rats with 11 or 22 mg Mn ( in water)/ kg day for 21 days from birth to weaning. The high dose resulted in decreased body weight gain in the pups and affected brain neurochemistry. Manganese treatment induced a significant increase in the amplitude of response to an auditory stimulus but did not affect motor activity, performance in a passive avoidance task, or brain histopathology.

Colomina et al. ( 1995) conducted a study to determine which gestation day is most critical for developmental toxicity of manganese in mice. The investigators administered a 50 mg/ kg dose of manganese chloride by subcutaneous injection once during the period between gestation days 9 and 12. Late resorptions, post implantation loss, and skeletal anomalies increased in all treatment groups. Significant reductions in fetal body weight occurred following exposure on gestation day 9 or 10, indicating these days were most critical.

Treinen and Blazak ( 1995, abstract only) dosed female Sprague Dawley rats ( 15/ group) intravenously with 0, 5, 20, or 40 nmol/ kg MnCl 2 on days 6 to 17 of gestation. These doses correspond to approximately 0, 0.27, 1.1, or 2.2 : g Mn/ kg day. Treatment resulted in an 7 31 Manganese   February 2003 increased incidence of skeletal malformations ( doses which elicited effects were not reported). The observed malformations included angulated or irregularly shaped clavicle, femur, fibula, humerus, ilium, radius, scapula, tibia, and/ or ulna.

Grant et al. ( 1997) administered 6 or 30 : mol MnCl 2/ kg day to female mice ( 24/ group) by intravenous injection on gestation days 6 to 17. These doses correspond to approximately 0.3 and 1.6 mg Mn/ kg day. The experiment was terminated on gestation day 20. No significant differences were noted in manganese treated mice for number of corpora lutea, implantation sites, pre or post implantation losses, or number of viable fetuses per litter. Fetal weight was significantly increased ( p< 0.05) at the lower dose, and significantly decreased ( p< 0.05) at the 1.6 mg/ kg day dose. Skeletal abnormalities were noted in the fetuses of dams receiving the higher intravenous dose. In contrast, no increase in skeletal abnormalities was observed in the fetuses of mice administered 400 : mol of MnCl 2 ( approximately 22 mg Mn/ kg day) by oral gavage daily from days 6 to 17 of gestation.

One in vitro developmental study was located. Hanna et al. ( 1996, abstract only) cultured two stage mouse embryos in media containing varying concentrations of essential and nonessential minerals, including manganese. Embryos were incubated in culture media containing 0.05   200 : M manganese for 72 hours. Both essential and nonessential minerals were embryotoxic at relatively low doses.

Reproductive Studies Some inhalation data from occupational exposure studies suggest that male reproductive dysfunction is a primary endpoint of manganese toxicity. Toxicity is manifested in symptoms including loss of libido and impotence ( U. S. EPA, 1996a). Some evidence indicates that the hypothalamus and pituitary are sites of manganese accumulation ( see Section 6.2), suggesting disturbance of the hypothalamic pituitary gonadal axis hormones as a potential mechanism for reproductive effects. No human reproductive data for oral manganese exposure are available in the current literature. Reproductive studies in animals orally exposed to manganese are described below. Results of these studies are summarized in Table 7 6.

Chandra and colleagues consistently reported degenerative changes in the seminiferous tubules in the testes after parenteral exposure of rats and rabbits to manganese ( Chandra, 1971; Shukla and Chandra, 1977; Imam and Chandra, 1975; Chandra et al., 1973, 1975). However, similar changes were not observed in subchronic or chronic studies in mice or rats ( NTP, 1993).

Gray and Laskey ( 1980) exposed male mice to 1,100 mg Mn/ kg as Mn 3 O 4 in a casein diet from gestation day 15 to 90 days of age. Assuming a food consumption factor of 0.13 ( U. S. EPA, 1986d), the estimated daily dose at the termination of the study would be approximately 143 mg/ kg day. Sexual development was retarded, as indicated by decreased weight of testes, seminal vesicles and preputial glands. Reproductive performance was not evaluated. 7 32 Manganese   February 2003 Laskey et al. ( 1982) exposed Long Evans rats to 0, 400, 1,100 or 3,550 mg Mn/ kg ( as Mn 3 O 4) in the diet from day 2 of mother's gestation to 224 days of age. Assuming a food consumption factor of 0.05 ( U. S. EPA, 1986d), the average daily dose at the termination of the 7 33 Manganese   February 2003 Table 7 6. Reproductive Effects of Exposure to Manganese.

Compound Species Route Dose Effect Reference Mn 3 O 4 Mouse Oral ( diet) 143 mg Mn/ kgday Decreased weight of testes, seminal vesicles and preputial glands after 90 days. Gray and Laskey ( 1980)

Mn 3 O 4 Rat Oral ( diet) 20 mg Mn/ kgday 55 177 Dose related decrease in serum testosterone concentration. Reduced fertility at 3550 ppm after 224 days. Laskey et al. ( 1982)

Mn 3 O 4 Rat Oral ( gavage) 71 mg Mn/ kgday 214 Decreased body and testes weights. Reduction in serum testosterone. Laskey et al. ( 1985)

MnCl 2 Rat i. p. 8 mg/ kg day Degenerative changes in approx. 50% of seminiferous tubules after 150 and 180 days. Chandra ( 1971)

MnCl 2 ! 4H 2 0 Rat i. p. 15 mg/ kg day Increased Mn in testes; decreased nonprotein sulfhydryls and decreased activity of glucose 6 phosphate dehydrogenase and glutathione reductase after 15   45 days. Shukla and Chandra ( 1977)

MnCl 2 ! 4H 2 0 Rabbit i. v. 3.5 mg/ kg Inhibition of succinic dehydrogenase in seminiferous tubules after 5 days. Morphologic changes were not apparent. Imam and Chandra ( 1975)

MnSO 4 Rat i. p. 6 mg Mn/ kg Increased Mn in testes after 25   30 days. Degenerative changes in 10% of seminiferous tubules. Chandra et al. ( 1975)

MnO 2 Rabbit i. t. 250 mg/ kg single dose Destruction and calcification of the seminiferous tubules at 8 months. Infertile females. Chandra et al. ( 1973)

i. p. = intraperitoneal; i. v. = intravenous; i. t. = intratracheal 7 34 Manganese   February 2003 study was 0, 20, 55, or 177 mg Mn/ kg day. The investigators observed a dose related decrease in serum testosterone concentration ( without a concomitant increase in serum luteinizing hormone concentration), and reduced fertility at the highest dose. Testes weight, number of ovulations, resorption and preimplantation deaths, litter size, and fetal weights were unaffected by manganese exposure.

Laskey et al. ( 1985) conducted studies to assess the effect of manganese on hypothalamic, pituitary and testicular function. Long Evans rat pups ( 8/ litter) were dosed by gavage from day 1 to day 21 with a 50% sucrose solution containing particulate Mn 3 O 4. The average daily dose of manganese was calculated to be 0, 71 or 214 mg Mn/ kg day. Assessments of the hypothalamic, pituitary, or testicular functions were determined by measuring the endogenous or stimulated serum concentrations of follicle stimulating hormone, luteinizing hormone, and/ or testosterone at 21 or 28 days of age. Body, testes, and seminal vesicles weight and tissue concentrations of Mn were also evaluated. Effects attributed to manganese included slight decreases in body and testes weights, and a reduction in serum testosterone. There was no indication of hypothalamic or pituitary dysfunction. The authors suggested that the decrease in testosterone level resulted from manganese induced damage of Leydig cells.

Studies exist, however, that report no adverse reproductive effects in female rats following oral manganese exposure. Pappas et al. ( 1997) dosed pregnant rats with up to 620 mg Mn/ kg day ( as MnCl 2) throughout gestation. No treatment related effects were reported in dam health, litter size, or sex ratios of the pups. The study did not include more extensive analysis of female reproductive organs. Grant et al. ( 1997) administered 22 mg Mn/ kg day ( as MnCl 2) via gavage to pregnant dams on gestation days 6 17. No treatment related effects were reported in dams as measured by mortality, clinical signs, food and water intake, or body weights.

7.2.6 Chronic Toxicity NTP ( 1993) investigated the chronic toxicity of manganese in a 2 year oral exposure study. Concentrations of 0, 1,500, 5,000 or 15,000 mg/ kg manganese sulfate monohydrate were administered in the diet to male and female F344 rats ( 70/ sex). These dietary concentrations resulted in doses ranging from 30 to 331 mg Mn/ kg day for males, and 26 to 270 mg Mn/ kg day for females. Ten rats/ group were sacrificed at 9 and 15 months. Survival of males in the highdose group was significantly decreased starting at week 93 of the study, and death was attributed to advanced renal disease associated with manganese exposure. Food consumption was similar for all groups. However, by the end of the study, high dose males exhibited a mean body weight that was 10% lower than controls. No clinical findings or effects on hematologic or clinical chemistry parameters were attributed to manganese exposure in any group. Tissue concentrations of manganese were elevated in the livers of mid and high dose males, concurrent with a decrease in hepatic iron concentrations. Renal disease in high dose males was the only pathological effect noted. No increases in tumor incidence were attributed to manganese exposure.

The chronic oral toxicity of manganese was evaluated in mice in a concurrent study conducted by NTP ( 1993). Concentrations of 0, 1,500, 5,000, or 15,000 mg/ kg manganese 7 35 Manganese   February 2003 sulfate monohydrate were administered in the diet to B6C3F 1 mice ( 70/ sex) in a 2 year oral exposure study. These dietary concentrations were reported to be equivalent to doses ranging from 63 mg Mn/ kg day to 722 mg Mn/ kg day for male mice, and from 77 mg Mn/ kg day to 905 mg Mn/ kg day for female mice. Interim sacrifices of 11 mice/ group were made at 9 and 15 months. No clinical findings or effects on survival were observed in any group of mice. Mean body weights of males were not affected. Female mice had a dose related decrease in mean body weight after week 37. The final mean body weights for the low, mid and high dose females were 6%, 9% and 13% lower than controls, respectively. No differences were seen in food consumption for any group. No effects were reported on hematologic parameters. Tissue concentrations of manganese were significantly elevated in the livers of all exposed females, and in high dose males. Elevated manganese concentration was associated with decreased hepatic iron.

7.2.7 Carcinogenicity The carcinogenicity of ingested manganese was evaluated in concurrent 2 year oral exposure studies conducted in mice and rats by NTP ( 1993). An overview of these studies is provided below. No other studies of manganese carcinogenicity via the oral route were identified.

Groups of rats were exposed to dietary levels of manganese sulfate monohydrate that resulted in intakes ranging from 30 to 331 mg Mn/ kg day for males and 26 to 270 mg Mn/ kg day for females. At the termination of the study, no manganese related increase in any tumor type was observed ( NTP, 1993).

In a parallel study, NTP ( 1993) administered 0, 1,500, 5,000, and 15,000 mg/ kg manganese sulfate monohydrate in the diet to B6C3F 1 mice ( 70/ sex) for 2 years. These dietary concentrations resulted in intakes ranging from 63 to 722 mg Mn/ kg day for males and from 77 to 905 mg Mn/ kg day for females. The estimated manganese intake in the high dose mice was approximately 107 times greater than the recommended dietary allowance. Incidence of thyroid follicular cell hyperplasia was significantly greater in high dose male and female mice than in controls. The incidence of follicular cell adenomas is summarized in Table 7 7. In males, tumors were observed only at the highest dose ( 6% incidence). The highest incidence of tumors in females was also observed at the highest dose. No significant differences in tumor incidence relative to the controls were observed for either sex. The follicular cell tumors were seen only at the termination of the study ( 729 days), and their number was only slightly increased relative to the historical control range in female B3C6F 1 mice ( 0 to 9% historical range versus 10% tumor incidence in high dose females). Hence, the relevance of these findings to human carcinogenesis is questionable. The issues of concern are: 1) the large intake of manganese required to elicit a response seen only at the end of the study, and 2) tumor frequencies that are not significantly different from historical controls. While NTP ( 1993) has concluded that the marginal increase in thyroid adenomas of the mice was equivocal evidence of carcinogenicity, others have questioned the relevance of these data to human carcinogenicity ( U. S. EPA, 1993). 7 36 Manganese   February 2003 Table 7 7. Follicular Cell Tumor Incidence in B6C3F 1 Mice.

Sex Concentration of MnSO 4° H 2 O in Diet Control Low Medium High Males 0/ 50 0/ 49 0/ 51 3/ 50 Females 2/ 50 1/ 50 0/ 49 5/ 51 Source: NTP ( 1992)

Other studies reporting positive results for carcinogenicity are summarized in Table 7 8. Stoner et al. ( 1976) tested manganese sulfate in a mouse lung adenoma screening bioassay. These investigators exposed 6 to 8 week old Strain A/ Strong mice of both sexes ( 10/ sex) to 6, 15 or 30 mg MnSO 4/ kg via intraperitoneal injection. Doses were administered three times a week for a total of 21 injections. The cumulative doses were 132, 330 and 660 mg MnSO 4/ kg.

Table 7 8. Summary of Carcinogenicity Studies Reporting Positive Findings for Selected Manganese Compounds a.

Compound Species Route Dose Duration ( weeks intermittent Results Reference Manganese chloride Mouse Mouse i. p.

s. c. 0.1 mL of 1%

0.1 mL of 1%

0% ( control) 26 26 41%  Lymphosarcomas 67%  Lymphosarcomas 24%  Lymphosarcomas DiPaolo ( 1964)

Manganese sulfate Mouse i. p. 660 mg/ kg 0 mg/ kg 8 67%  Lung adenomas 31   37%  Lung ademomas Stoner et al. ( 1976)

Manganese acety lacetonate ( MAA) Rat i. m. 1,200 mg/ kgb 0 mg/ kg 26 40% ( males) Fibrosarcomas 24% ( females) Fibrosarcomas 4 % ( control males and females) Furst ( 1978)

i. p. = intraperitoneal; s. c. = subcutaneous; i. m. = intramuscular a Source: U. S. EPA ( 1984) 7 37 Manganese   February 2003 These doses corresponded to 42.9, 107.2 and 214.4 mg Mn/ kg. Observation continued for 22 weeks after the dosing period, and the mice were sacrificed at 30 weeks. Table 7 9 summarizes the results of this study. The percentage of mice with tumors was elevated at the highest dose level, but the difference was not significant ( Fisher Exact test) when compared with the vehicle controls. An apparent increase in the average number of pulmonary adenomas per mouse was noted both at the middle and high doses, but the increase was significant only at the high dose ( 660 mg MnSO 4/ kg) ( Student's t test, p < 0.5). Although these study results are suggestive of carcinogenic activity, they do not conclusively meet the positive response criteria ( increase in the mean number of tumors per mouse and an observable dose response relationship) for the interpretation of lung tumor data in this mouse strain ( Shimken and Stoner, 1975).

Table 7 9. Pulmonary Tumors in Strain A Mice Treated with Manganese Sulfate a.

Total Dose Group mg MnSO 4/ kg mg Mn/ kg Mortality Mice with Lung Tumors (%) Average Number Tumors/ Mouse b Value c Untreated control 0 0 1/ 20 6/ 19 ( 31) 0.28 ± 0.07 NA Solvent control ( 0.85% NaCl) 0 0 1/ 20 7/ 19 ( 37) 0.42 ± 0.10 NA Treated 132 42.9 1/ 20 7/ 19 ( 37) 0.47 ± 0.11 NS Treated 330 107.2 0/ 20 7/ 20 ( 35) 0.65 ± 0.15 NS Treated 660 214.4 2/ 20 12/ 18 ( 67) 1.20 ± 0.49 0.05d 20 mg urethane e 0 0 2/ 20 18/ 18 ( 100) 21.6 ± 2.81 NR a Source: Stoner et al. ( 1976) b X ± S. E. c Student t test d Fisher Exact Test p = 0.068 e Single intraperitoneal injection NA = Not applicable; NS = Not significant; NR = Not reported Furst ( 1978) injected F344 rats intramuscularly with manganese acetylacetonate and observed an increased incidence of fibrosarcomas at the injection site, but did not observe increased tumor incidence at other sites. 7 38 Manganese   February 2003 7.3 Other Key Data 7.3.1 Mutagenicity/ Genotoxicity In Vivo Studies No studies or reports were identified which describe mutagenic or genotoxic effects in humans following oral exposure to manganese. Table 7 10 summarizes the results of the most recent in vivo mutagenicity and genotoxicity studies in animals. Results from additional studies are noted in the text below.

Studies of genotoxicity in animals have shown mixed results. The bone marrow cells of rats receiving a 50 mg/ kg oral dose of manganese ( as manganese chloride) showed an increased incidence of chromosomal aberrations ( 30.9%) compared with those of control animals ( 8.5%) ( Mandzgaladze, 1966; Mandzgaladze and Vasakidze, 1966). However, Dikshith and Chandra ( 1978) administered repeated oral doses of manganese chloride ( 0.014 mg/ kg day) to male rats for 180 days and did not observe significant chromosomal damage in bone marrow or spermatogonial cells.

Table 7 10. Genotoxicity of Manganese In Vivo.

Species ( test system) Compound End Point Route Results Reference Nonmammalian systems:

Drosophila melanogaster MnSO 4 Sex linked recessive lethal Feeding Injection  Valencia et al. ( 1985)

Drosophila melanogaster MnSO 4 Sex linked recessive lethal Feeding Injection  NTP ( 1993)

Drosophila melanogaster MnCl 2 Somatic mutation Soaking larvae  Rasmuson ( 1985)

Mammalian systems:

Albino rat ( bone marrow cells) ( spermatogonial cells) MnCl 2 Chromosomal aberrations Oral  Dikshith and Chandra ( 1978)

Albino mouse MnSO 4 KMnO 4 Chromosomal aberrations Chromosomal aberrations Oral Oral + + Joardar and Sharma ( 1990) 7 39 Manganese   February 2003 Joardar and Sharma ( 1990) administered oral doses of manganese sulfate ( approximately 102, 202, and 610 mg/ kg) and potassium permanganate ( 65, 130, and 380 mg/ kg) to male Swiss albino mice for three weeks. Both compounds were clastogenic, with manganese sulfate being more potent. The frequencies of chromosomal aberrations in bone marrow cells and micronuclei were significantly increased by both salts. There was also a statistically significant, dosedependent enhancement of sperm head abnormalities. A LOAEL of 23 mg Mn/ kg day was identified for this effect by ATSDR ( 2000).

The divalent manganese ion ( Mn II) interacts with DNA and chromosomes ( Kennedy and Bryant, 1986; Yamaguchi et al., 1986). In cultured mammalian cells, both MnCl 2 and KMnO 4 produced chromosome aberrations, including breaks, exchanges and fragments ( Umeda and Nishimura, 1979). DNA strand breaks have also been induced by manganese in Chinese hamster ovary calls and human diploid fibroblasts ( Hamilton Koch et al., 1986; Snyder, 1988). Tests for induction of chromosomal aberrations and sister chromatid exchanges in cultured Chinese hamster ovary cells were positive for manganese sulfate monohydrate in the absence of S9 metabolic activation. In the presence of S9, only the sister chromatid exchange test was positive ( NTP, 1993).

Tests for mutagenicity in Drosophila melanogaster have given negative results. Manganese sulfate monohydrate did not induce sex linked recessive lethal mutations in germ cells of male Drosophila treated by feeding or injection ( Valencia et al., 1985, as reported in NTP, 1993). Treatment of D. melanogaster with manganese chloride by soaking did not induce somatic mutation ( Rasmuson, 1985).

In Vitro Assays Table 7 11 summarizes the results of the most recent in vitro mutagenicity and genotoxicity studies. Additional results from early studies are included in the text below.

Manganese chloride was mutagenic in Escherichia coli ( Demerec et al., 1951; Durham and Wyss, 1957; Zakour and Glickman, 1984), Photobacterium fischeri ( Ulitzer and Barak, 1988) and Serretia marcescens ( Kaplan, 1962). Both positive ( Nishioka, 1975) and negative ( Kanematsu et al., 1980) results have been reported for the Bacillus subtilis recombination assay. Positive ( Pagano and Zeiger, 1992; Wong, 1988) and negative results ( Wong, 1988) have also been reported for manganese chloride in the Salmonella typhimurium reversion assay. Assays in mammalian cell lines were positive for gene mutation in mouse lymphoma cells ( Oberley et al., 1982) and enhancement of transformation in Syrian hamster embryo cells ( Casto et al., 1979). An assay for DNA damage in human lymphocytes gave negative results with metabolic activation, and positive results without activation ( De Meo et al., 1991).

Manganese sulfate gave positive results in the T4 bacteriophage mutation test ( Orgel and Orgel, 1965), and the B. subtilis recombination assay with S9 activation ( Nishioka, 1975). Pagano and Zeiger ( 1992) obtained positive results for mutagenicity in S. typhimurium strain TA97. In contrast, manganese sulfate monohydrate was not mutagenic in S. typhimurium strains TA98, TA100, TA1535, or TA1537, either with or without exogenous metabolic ( S9) activation 7 40 Manganese   February 2003 Table 7 11. Genotoxicity of Manganese In Vitro.

Results Species ( test system) Compound End Point Strain With S9 Activation Without S9 Activation Reference Prokaryotic organisms:

Salmonella typhimurium MnCl 2 Gene mutation TA98 TA102 TA1535 TA1537        + Wong ( 1988)

Salmonella typhimurium MnSO 4° H 2 O Gene mutation TA97 TA98 TA100 TA1535 TA1537      /+     Mortelmans et al. ( 1986)

Salmonella typhimurium MnSO 4 Gene mutation TA97 ND + Pagano and Zeiger ( 1992)

Salmonella typhimurium MnCl 2 Gene mutation TA100 TA102 ND ND  + DeMeo et al. ( 1991)

Photobacterium fischeri ( bioluminescence test) MnCl 2 Gene mutation ( restored luminescence) Pf 13 ( dark mutant) ND + Ulitzur and Barak ( 1988)

Escherichia coli MnCl 2 Gene mutation KMBL 3835 ND + Zakour and Glickman ( 1984)

Bacteriophage ( E. coli lysis) MnSO 4 Gene mutation T4 ND + Orgel and Orgel ( 1965)

Bacillus subtilis ( recombination assay) MnCl 2 Mn( NO 3) 2 MnSO 4 Mn( CH 3 CO O) 2 KMnO 4 Inhibition of growth in recombination deficient mutant ( Rec) compared to wild type ( Rec+) M45 ( Rec) ND + + +

+  Nishioka ( 1975)

B. subtilis ( recombination assay) MnCl 2 Mn( NO 3) 2 Mn( CH 3 CO O) 2 Inhibition of growth in recombination deficient mutant ( Rec) compared to wild type ( Rec+) M45 ( Rec) ND    Kanematsu et al. ( 1980) Table 7 11 ( continued)

Results Species ( test system) Compound End Point Strain With S9 Activation Without S9 Activation Reference 7 41 Manganese   February 2003 Eukaryotic organisms:

Fungi: Saccharomyces cerevisiae MnSO 4 Gene conversion, reverse mutation D7 ND + Singh ( 1984)

Mammalian cells: Mouse lymphoma cells MnCl 2 Gene mutation L5178Y TK+/ ND + Oberley et al. ( 1982)

Mammalian cells: Syrian hamster embryo cells MnCl 2 Enhancement of SA7 transformation ND + Casto et al. ( 1979)

Mammalian cells: Human lymphocytes ( Single cell gel assay) MnCl 2 DNA damage  + DeMeo et al. ( 1991)

Mammalian cells: Chinese hamster ovary cells MnSO 4 Chromosomal aberrations Sister chromatid exchange

+ +

+ NTP ( 1993)

Notes:  = negative results + = positive results /+ = equivocal results ND = no data available DNA = deoxyribonucleic acid MnSO 4° H 2 O = manganese ( II) sulfate monohydrate Mn( CH 3 COO) 2 = manganous acetate MnCl 2 = manganous chloride Mn( NO 3) 2 = manganous nitrate MnSO 4 = manganous sulfate Rec = recombination Source: Modified from ATSDR ( 2000) 7 42 Manganese   February 2003 when assayed by Mortelmans et al. ( 1986). Results for strain TA97 were negative when assayed with S9 activation, and equivocal when assayed without metabolic activation. Assays in eukaryotic test systems were positive for mutagenicity in S. cerevisiae ( Singh, 1984) and chromosomal aberrations in Chinese hamster ovary ( CHO) cells ( NTP, 1993). Manganese sulfate gave negative results when assayed for induction of sister chromatid exchange in CHO cells ( NTP, 1993).

Comparatively little data are available that describes the genotoxic potential of other manganese compounds. Manganese oxide ( Mn 3 O 4) was not mutagenic in S. typhimurium or S. cerevisiae ( Simmon and Ligon, 1977). Data obtained for manganese nitrate ( Mn( NO 3) 2) in the B. subtilis recombination assay were inconsistent between studies ( Nishioka, 1975; Kanematsu et al., 1980). Manganese acetate ( Mn( CH 3 OO) 2) was mutagenic in the B. subtilis recombination assay without exogenous metabolic activation, and gave negative results with activation ( Nishioka, 1975; Kanematsu et al., 1980).

7.3.2 Immunotoxicity Immunotoxicity and lymphoreticular effects do not appear to be significant outcomes of oral exposure to manganese. A single report describes effects in this category following oral exposure. NTP ( 1993) administered diets containing 0, 1,600, 3,130, 6,250, 12,500, or 25,000 mg/ kg manganese sulfate monohydrate to F344 rats ( 10/ sex/ dose) in a 13 week study. Based on measured feed consumption, the study authors determined that the mean intake of manganese sulfate monohydrate ranged from 110 to 1,700 mg/ kg day ( equal to about 36 to 553 mg Mn/ kgday for males, and from 115 to 2,000 mg/ kg day ( equal to about 37 to 621 mg Mn/ kg day) for females. Increased neutrophil counts were noted at 32 mg Mn/ kg day in male rats. Decreased leukocyte counts were noted at 155 mg Mn/ kg day in female rats.

Studies in animals exposed to manganese chloride by intraperitoneal or intramuscular injection suggest that manganese can affect several immunological cell types ( ATSDR, 2000). Observed effects include stimulation of macrophage and natural killer cell activity in mice ( Rogers et al., 1983; Smialowicz et al., 1985, 1987). Other effects include alteration of the responsiveness of lymphoid cells to mitogens and inhibited antibody production in response to a T cell antigen ( Hart, 1978; Lawrence, 1981; Srisuchart et al., 1987). The significance of these findings for human immune function is presently unknown.

7.3.3 Hormonal Disruption No reports describing hormonal disruption associated with manganese exposure were located. 7 43 Manganese   February 2003 7.3.4 Physiological or Mechanistic Studies Biochemical and Physiological Role Manganese is a naturally occurring element that is required for normal physiological functioning in all animal species ( U. S. EPA, 1996a). It 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. Studies conducted to determine the biochemical and nutritional roles of manganese in human health are reviewed in greater detail by Wedler ( 1994) and Keen et al. ( 1999).

The frequency of occurrence and consequences of manganese deficiency are issues of some debate ( Keen et al., 1999). However, observations reported by Doisy ( 1973) and Friedman et al. ( 1987) suggest that manganese is an essential element for humans. Doisy ( 1973) reported decreased levels 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 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.

Manganese deficiency has been experimentally induced in multiple animal species. Outcomes associated with manganese deficiency in animals 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), ataxia ( Hurley et al., 1961), altered metabolism of carbohydrates ( Baly et al., 1988; Hurley et al., 1984) and lipids ( Abrams et al., 1976), and decreased cholesterol synthesis and excretion ( Davis et al., 1990; Kawano et al., 1987). The biochemical basis for these effects has not been established with certainty, but it may be related to the participation of manganese in numerous enzymatic reactions.

Low serum manganese levels are associated with several disease states, including epilepsy, exocrine pancreatic insufficiency, multiple sclerosis, cataracts, and osteoporosis ( Freeland Graves and Llanes, 1994). In addition, the metabolic disorders phenylketonuria and maple syrup urine disease, genetic disorders of amino acid metabolism, are associated with poor manganese status ( U. S. EPA, 1996a). 7 44 Manganese   February 2003 Mechanisms of Neurotoxicity The central nervous system ( CNS) has been identified as the major target of manganese toxicity ( U. S. EPA, 1993; ATSDR, 2000). The blood brain barrier ( BBB) is a major regulator of the ( CNS) milieu, and the rate and extent of manganese transfer across the BBB may be a determinant of manganese neurotoxicity ( Aschner and Aschner, 1991). The mechanism by which manganese crosses the BBB to gain access to neuronal tissue has not been fully elucidated, but may be a function of binding to transferrin ( Aschner and Aschner, 1991). In the portal circulation, manganese as Mn( II) initially binds to alpha 2 macroglobulin, and this complex cannot cross the BBB. The Mn( II) alpha 2 macroglobulin complex is transported by the bloodstream to the liver ( Tanaka, 1982), where a small fraction of the circulating Mn( II) may be oxidized to Mn( III). The iron transporting protein transferrin has been shown to also bind Mn( III), and may be responsible for its transport into the brain. The observation that some of the regions of the brain that accumulate manganese ( e. g., globus pallidus, striatum, and substantia nigra) receive neuronal input from the transferrin rich nucleus accumbens and the caudateputamen supports this argument. Both of these regions are rich in transferrin receptors.

Additional evidence for the transferrin transport hypothesis was provided by an experiment in which rats were given a 6 hour intravenous administration of ferric hydroxide dextran complex ( Aschner and Aschner, 1990). The uptake of radiolabeled manganese into the brain was significantly ( p < 0.05) inhibited following the administration of the iron complex as compared with rats administered iron free dextran. It was concluded that iron homeostasis may play an important role in the regulation of manganese transport across the BBB, since both metals are transported by transferrin and may be competing for binding sites.

Once manganese has crossed the BBB, several neurotransmitter systems in the brain appear to be potential targets for manganese toxicity. The primary targets appear to be the monoamines, including dopamine, noradrenaline and serotonin ( Neff et al., 1969; Mustafa and Chandra, 1971). The amino acid neurotransmitter ( amino butyric acid ( GABA) may also be affected ( Gianutsos and Murray, 1982). Effects on neurotransmitters may be both specific and highly localized. Manganese neurotoxicity, for example, is reportedly associated with a selective depletion of dopamine in the striatum, a site of manganese accumulation ( Neff et al., 1969; Bernheimer et al., 1973).

A resemblance exists between the symptoms of manganism and Parkinsonism, a condition characterized by loss of dopaminergic neurons in the substantia nigra and globus pallidus. In addition, several clinical features of manganism respond favorably to therapy with Ldopa in a manner similar to patients with Parkinson's disease ( Mena et al., 1970) although longterm response of manganism patients to L dopa has not been observed ( ATSDR, 2000; Calne et al., 1994). However, despite some similarities in symptoms, a comparative study of a 52 yearold worker exposed to manganese in an ore crushing plant and a patient with Parkinson's disease did not reveal any similarity in neuropathology ( Yamada et al., 1986). Barbeau ( 1984), Calne et al. ( 1994), and Pal et al. ( 1999) have summarized the similarities and differences between manganism and Parkinsonism. These researchers have noted that manganism characteristically occurs in phases of increasing severity and that sufferers exhibit dystonia ( disordered tonicity of 7 45 Manganese   February 2003 muscles), symptoms of extrapyramidal dysfunction such as bradykinesia ( extreme slowness of movements and reflexes), monotonic speech, and an expressionless or even grimacing face. Although the altered gait and fine tremor are common to both Parkinsonism and manganism, the syndromes are different in that manganism patients sometimes have psychiatric disturbances early in the onset of the syndrome, have a tendency to fall backwards, do not have the Lewy bodies in the substantia nigra that are commonly found in Parkinson's patients. Further, fluorodopa positron emission tomography ( PET) scans are normal in manganism patients but not in individuals with Parkinson's disease ( ATSDR, 2000).

Mapping studies by Yamada et al. ( 1986) indicate that most of the neuronal degeneration attributed to manganese exposure lies close to monoamine cell bodies and pathways. Histopathology in manganese exposed primates shows more widespread deposition of the metal, with intense signaling observed in both the globus pallidus and substantia nigra using MRI ( Newland and Weiss, 1992). Studies in humans indicate that excess manganese in the brain deposits primarily in the globus pallidus ( Fell et al. 1996; Kafritsa et al. 1998; Ono et al. 1995) and damage to the human brain from manganese deposition may be limited to that region. In a study that supports these findings, the globus pallidus exhibited atrophy in an autopsy performed on a worker with inhalation related manganese poisoning ( Yamada et al., 1986).

Although there is consensus that the monoaminergic systems, particularly the dopaminergic system, are affected by excess exposure to manganese, the precise mechanism of action remains obscure. One hypothesis proposes that oxidation of dopamine plays a key role in manganese neurotoxicity. Manganese ( III) has been shown to oxidize dopamine to its cyclized O quinone ( cDAoQ) ( Archibald and Tyree, 1987). This irreversible process ultimately results in decreased dopamine levels. The formation of cDAoQ may subsequently initiate the generation of reactive oxygen species, leading to oxidative stress and cell death ( Segura Aguilar and Lind, 1989). An alternative hypothesis for manganese toxicity proposes an effect on brain cytochrome P 450 activity. Liccione and Maines ( 1989) demonstrated a high sensitivity of rat striatal mitochondria to manganese induced increases in cytochrome P 450 activity. These authors speculated that the increase in mixed function oxidase activity may trigger an increase in the formation of active oxygen species ( e. g., superoxide anions) that exert a harmful effect on dopaminergic pathways.

Other mechanistic studies have identified tyrosine hydroxylase ( TOH), the rate limiting enzyme in dopamine synthesis, as a potential target in manganese induced neurochemical effects. Bonilla ( 1980) and Chandra and Shukla ( 1981) found that changes in TOH activity in the presence of manganese closely paralleled dopamine levels. Qato and Maines ( 1985) determined that alterations in the activity of TOH and other monooxygenases may be related to manganeseinduced alterations in brain heme metabolism.

Manganese toxicity may be selectively associated with adverse effects on mitochondria. Maynard and Cotzias ( 1955) originally proposed the mitochondrion as the target organelle for manganese cytotoxicity, with adverse effects expressed primarily as disruption of Ca( II) homeostasis. Mn( II) preferentially accumulates in the mitochondria in regions of the brain 7 46 Manganese   February 2003 associated with neurological symptoms and manganism. Once inside the mitochondria, Mn( II) disrupts oxidative phosphorylation. The fundamental role of mitochondrial energy metabolism in manganese neurotoxicity has been highlighted by the studies of Aschner and Aschner ( 1990) and Gavin et al. ( 1992), as cited in U. S. EPA ( 1996a) and ATSDR ( 2000).

The results of Brouillet et al. ( 1993) confirm that manganese impairs mitochondrial oxidative metabolism. In addition, their findings indicate that manganese neurotoxicity involves an N methyl D aspartate receptor mediated process similar to that observed for some other mitochondrial toxicants. Manganese may thus produce neuronal degeneration by an excitotoxic process secondary to its ability to disrupt oxidative energy metabolism.

7.3.5 Structure Activity Relationship Information on structure activity relationships is not available for manganese.

7.4 Hazard Characterization 7.4.1 Synthesis and Evaluation of Major Noncancer Effects Manganese is an ubiquitous element that is essential for normal physiological functioning in all animal species. The biochemical basis for this requirement is most likely the participation of manganese as a structural component or catalytic cofactor for many enzymes. The Adequate Intake levels for manganese range from 0.003 to 0.6 mg/ day for infants from birth to 6 months, 0.6 mg/ day for infants from 7 months to 1 year, 1.2 mg/ day for children aged 1 3 years, 1.5 to 1.9 mg/ day for children aged 4 13 years, and from 1.6 to 2.3 mg/ day for adolescents and adults ( IOM, 2002). Although outright manganese deficiency has not been observed in the general population, sub optimal intake may be of concern for some individuals.

In contrast to the beneficial effects of manganese as a nutrient, excess exposure to manganese may be associated with toxic effects. At present, the optimal level of oral exposure to manganese is not well defined ( Greger, 1999).

Ingested manganese appears to be primarily absorbed in the Mn( II) form, and may compete with iron and cobalt for common absorption sites. Absorption varies among individuals and is also influenced by dietary factors. Absorption of 3 to 10% of ingested dietary manganese is considered to be representative of the general population ( U. S. EPA, 1996a). Iron deficiency enhances the absorption of manganese in animals ( U. S. EPA, 1984). Uptake of dietary manganese may be reduced in the presence of other dietary components such as calcium and phytate.

Once absorbed, manganese has the potential to accumulate in mitochondria rich tissues, including liver, pancreas, and kidney. Lesser amounts accumulate in brain and bone. Manganese is efficiently removed from the blood by the liver and released into bile. Biliary secretion represents the major pathway for manganese transport to the intestine, and studies in humans indicate that manganese is primarily excreted in the feces. The rate of excretion responds 7 47 Manganese   February 2003 efficiently to increased manganese intake. The rate of biliary secretion acts in concert with absorptive processes to establish homeostatic control of manganese levels in the body. As long as physiological systems are not overwhelmed, humans appear to exert efficient homeostatic control over manganese levels, so that levels in the body are kept relatively constant despite moderate variations in intake. Manganese is also reabsorbed in the intestine through enterohepatic circulation ( Schroeder et al. 1966).

While it is apparent that exposure to excess manganese can result in increased tissue levels, the interrelationships between oral exposure levels, tissue accumulation, and health effects in humans are not completely understood. Epidemiological studies of workers exposed by inhalation to manganese dusts and fumes have identified the central nervous system ( CNS) as the primary target for chronic manganese toxicity by the inhalation route ( U. S. EPA, 1993). Both Mn( III) and Mn( II) have been associated with the neurotoxic effects of manganese. While some researchers note the similarities in CNS effects occurring following manganese exposure and in Parkinson's disease ( dystonia, rigidity, bradykinesia), there are significant differences in the two diseases. For example, manganism patients exhibit a less frequent resting tremor than do Parkinson's patients, extrapyramidal symptoms including fixed expression or a facial grimace, active tremor ( particularly in the upper body), a " cock walk" in which the patient walks on the toes with the back stiff and the elbows flexed, a propensity to fall backwards ( especially when pushed), and a failure to respond to dopaminomimetics ( Barbeau, 1984; Calne et al., 1994; Pal et al., 1999).

Several investigators have proposed a link between elevated oral manganese intake by humans and neurological symptoms resembling manganism ( Kawamura et al., 1941; Kilburn, 1987; Kondakis et al., 1989; Goldsmith et al., 1990). Results from these studies are described in detail in Section 7.1. In each case, the data from these studies were insufficient to establish that manganese was the causative factor ( ATSDR, 2000). The evidence for a similar pattern of neurotoxicity in humans following oral exposure is therefore considered equivocal.

Numerous studies have investigated manganese neurotoxicity in rodent models. However, the utility of rodent studies for evaluating the potential neurotoxic effects of manganese in humans has been questioned. Although biochemical and behavioral evidence of neurological effects has been observed, signs of impaired motor function resembling those seen in humans are usually not detected. In particular, studies of rodents exposed to manganese by drinking water or food have been unable to produce the characteristic signs of extrapyramidal neurologic disease seen in humans. In contrast, chronic administration of manganese to monkeys by oral ( one study) or parenteral routes ( two studies) has resulted in neurological signs consistent with chronic manganism. The failure to reproduce these signs in rodent studies may result from differences in manganese accumulation and distribution between rodents and primates. The dietary requirement for manganese in rodents, for example, is estimated to be 100 times higher than in humans. In addition, neurotoxic effects in humans are associated with manganese accumulation in neuromelanin rich regions of the brain, and the homologous regions in rats and mice lack this pigment. Although primates are likely to be better models of the neurological manifestations of manganese intoxication than rodent species, sufficient data from well designed oral studies are not currently available. 7 48 Manganese   February 2003 An additional drawback to animal studies of manganese neurotoxicity is the inability to identify certain psychological or neurobehavioral signs. Overt neurological impairment in humans is often preceded by psychological symptoms such as irritability and emotional lability. Since accurate dose response relationships based on neurobehavioral endpoints are generally not available from animal studies, neurochemical responses have been examined as alternative indicators of neurotoxicity. Such studies have been conducted on the assumption that since the toxic manifestations of chronic manganese exposure resemble Parkinsonism, altered biogenic amine metabolism in the CNS may be one of the underlying mechanisms. However the patterns of neurochemical response reported following manganese exposure are not consistent among studies. Although manganese exposure is generally thought to result in decreased dopamine concentrations, some studies report increased or fluctuating levels. The effect of manganese on dopamine levels, for example, appears to be age dependent. Neonatal rats and mice exposed to manganese from birth to 15 or 30 days of age have an increased levels of dopamine and norepinephrine in the brain ( Chandra et al., 1979; Cotzias et al., 1976; Shukla et al., 1980). Further, temporal changes in dopamine neurochemistry have been observed with prolonged or continuous manganese treatment and it is not established how these time related changes affect manganese induced neurotoxicity.

Route of administration is also an issue of concern in evaluating the results of animal studies. Scheuhammer ( 1983), for example, determined that intraperitoneal injection is not the route of choice for studies of manganese exposure that are longer than 30 days in duration, especially for investigations of neurotoxicity. Intraperitoneally administered manganese appears to have a selectively toxic effect on the pancreas. This effect may make it difficult to distinguish between subtle neurochemical changes resulting directly from manganese exposure, and changes that are secondary to cellular damage in the pancreas. In addition, U. S. EPA ( 1984) noted that results from parenteral studies are of limited value in predicting the reproductive hazards of ingested manganese. At least one study exists, however, that shows the differential uptake and distribution of manganese administered via injection compared to oral dosing. Roels et al. ( 1997) investigated the uptake and distribution of manganese ( as either MnO 2 or MnCl 2) in rats following intra peritoneal injection or gavage dosing. Manganese concentrations were not increased in the blood or brain following administration of 4 weekly doses of 1.22 mg Mn/ kg of the dioxide via gavage; following i. p. dosing, manganese concentrations were significantly increased in the blood, striatum, cerebellum and cortex. Steady state blood manganese concentrations were increased to similar levels by both gavage and i. p. dosing of MnCl 2. Gavage dosing of the dichloride significantly increased the cortex manganese concentrations, but not that of the other two regions. Intra peritoneal dosing of the compound increased the manganese levels in the striatum and cortex, but not the cerebellum. These data indicate that depending on the compound, injection administration of manganese results in higher blood and brain concentrations of the metal than does gavage administration.

Toxic effects of oral manganese exposure have also been reported in the hematopoietic, cardiovascular, reproductive, and digestive systems in animals. Hematological and biochemical outcomes vary depending on age and iron status, with young or iron deficient animals more likely to exhibit adverse effects. Other effects observed following manganese exposure include reduced body weight and reduced liver weight. Animal studies suggest that manganese is not a potent developmental toxicant. 7 49 Manganese   February 2003 Infants have been identified as a potentially sensitive subpopulation for excess manganese exposure. This determination reflects evidence for higher levels of manganese retention in the brains of neonates than in adults, although the relationship between manganese accumulation in the neonatal brain and toxicity remains unclear ( U. S. EPA, 1993). Additional concerns include evidence for greater extent of manganese transport across the blood brain barrier, the high concentration of manganese in some infant formulas, and evidence suggestive of a possible link between manganese exposure and learning disabilities. Although a causal relationship has not been established for elevated manganese intake and learning disabilities, a need for further research in this area has been noted ( U. S. EPA, 1993).

Other potentially sensitive subpopulations for manganese exposure have been identified. In general, these are groups who may have greater potential for increased body burdens due to increased absorption or altered clearance mechanisms. The list includes pregnant women, elderly persons, iron or calcium deficient individuals, and individuals with impaired liver function.

7.4.2 Synthesis and Evaluation of Carcinogenic Effects The carcinogenic potential of ingested manganese has not been systematically evaluated in epidemiological studies.

Data from animal studies are also limited. Currently, one of the few adequately designed investigations is the 2 year oral exposure study conducted by the National Toxicology Program ( NTP, 1993). Groups of F344 rats ( 70/ sex) were provided with diets containing 0, 1,500, 5,000, or 15,000 ppm manganese sulfate monohydrate. These dietary concentrations were reported to be equivalent to an intake ranging from 30 to 331 mg Mn/ kg day for males, and 26 to 270 mg Mn/ kg day for females. No increase in any tumor type could be attributed to manganese exposure.

In a concurrent study, B6C3F 1 mice were administered 0, 1,500, 5,000, or 15,000 mg/ kg manganese sulfate monohydrate ( NTP, 1993). These dietary concentrations were reported to be equivalent to an intake ranging from 63 to 722 mg Mn/ kg day for males and 77 to 905 mg Mn/ kg day for females. Compared to controls, the incidences of thyroid follicular cell hyperplasia were significantly greater in high dose males and in females at all dose levels. The incidence of follicular cell adenomas in high dose males ( 6%) was slightly greater than the range of historical incidence in NTP studies of follicular cell adenomas in male B6C3F 1 mice ( 0   4%). In high dose females, the incidence of follicular cell adenomas ( 10%) was also slightly above the historical control range ( 0   9%). Follicular cell tumors were seen only at the termination of the study ( 729 days). NTP ( 1993) reported that the manganese intakes in the high dose mice were 107 times greater than the recommended dietary level. While NTP ( 1993) concluded that these data provided " equivocal evidence of carcinogenic activity in mice," U. S. EPA ( 1993) questioned the relevance of these findings to human carcinogenesis. The basis for concern was 1) the large dose of manganese required to elicit a response observed only at the end of the study, and 2) tumor frequencies that were not statistically different from historical controls. 7 50 Manganese   February 2003 Three additional studies address the carcinogenicity of manganese. DiPaolo ( 1964) found that a larger percentage of DBA/ 1 mice exposed subcutaneously and intraperitoneally to manganese chloride developed lymphosarcomas when compared to controls. A comprehensive evaluation of these data was not possible, however, because they were published in an abstract form which lacked sufficient experimental detail. Stoner et al. ( 1976) found a higher frequency of lung tumors in strain A/ Strong mice administered manganese sulfate intraperitoneally as compared to controls. Although these results are suggestive of carcinogenic activity, they fail to meet the positive response criteria for the interpretation of lung tumor data in this strain of 1) an increase in the mean number of tumors per mouse, and 2) an observable dose response relationship ( Shimkin and Stoner, 1975). In the third study, Furst ( 1978) injected F344 rats intramuscularly with manganese acetylacetonate. An increased incidence of fibrosarcomas was observed at the injection site. Increased tumor incidence was not observed at other sites. When evaluated as a group, these studies do not provide convincing evidence for carcinogenicity of manganese.

Both negative and positive results have been obtained in assays for the genotoxic effects of manganese. Mutagenicity assays in multiple tester strains of Salmonella typhimurium gave predominately negative results for manganese sulfate monohydrate and manganese chloride when tested with or without exogenous metabolic activation by S9 fraction ( Wong, 1988; DeMeo et al., 1991; Pagano and Zeiger, 1992; NTP, 1993). Neither compound induced mutations in Drosophila melanogaster as evaluated by sex linked recessive lethal or somatic mutation assays ( Rasmuson, 1985; Valencia et al., 1985; NTP, 1993). Dikshith and Chandra ( 1978) did not observe increased incidence of chromosomal aberrations in rat bone marrow or spermatogonial cells following oral administration of manganese chloride.

In addition to the negative results described above, positive results for manganese compounds have been obtained in some assays for genotoxicity. Manganese sulfate induced sister chromatid exchange and chromosomal aberrations in vitro in Chinese hamster ovary cells, and induced chromosomal aberrations in vivo in albino mice following oral administration ( Joardar and Sharma, 1990). Manganese compounds also induced or enhanced mutation, transformation, chromosomal aberrations, and DNA damage in some assays conducted in mammalian cell lines ( Casto et al., 1979; Oberly et al., 1982; DeMeo et al., 1991; NTP, 1993), bacteria ( Orgel and Orgel, 1965; Nishioka, 1975; Zakour and Glickman, 1984), and yeast ( Singh, 1984). Although these results suggest that manganese may have genotoxic potential, there are presently no epidemiological or unequivocal animal data to suggest that manganese is carcinogenic.

7.4.3 Mode of Action and Implications in Cancer Assessment The molecular mechanisms responsible for the toxicity of manganese have not been identified with certainty. Most effort has focused on identification of mechanisms mediating the toxic effects observed in the central nervous system. Multiple researchers have proposed that elevated levels of Mn( II) and Mn( III) trigger the production of free radicals, reactive oxygen species, and other cytotoxic metabolites in brain tissue. Generation of these reactive species is hypothesized to occur via the oxidation or turnover of intracellular catecholamines, impacts on 7 51 Manganese   February 2003 mitochondrial metabolism, or stimulation of cytochrome P 450 activity. Manganese may also influence transport systems, enzyme activity and receptor function in the brain and other organs. At the present time, there is no evidence to link these proposed mechanisms of action to carcinogenic potential.

7.4.4 Weight of Evidence Evaluation for Carcinogenicity The U. S. EPA considers that there are " Inadequate Information to Assess Human Carcinogenic Potential" for manganese ( U. S. EPA, 1999b). In the past, the weight of evidence for manganese carcinogenicity resulted in Group D ( Not Classifiable) using the criteria of the U. S. EPA Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1986a). The classification of Group D was verified on May 25, 1988 by the cancer risk assessment verification endeavor ( CRAVE) Work Group of the U. S. EPA. The basis for this determination is the inadequacy of existing studies for assessment of manganese carcinogenicity ( U. S. EPA, 1996a).

7.4.5 Sensitive Populations Sensitive populations are defined as those which will exhibit an enhanced or altered response to a chemical when compared with most persons exposed to the same concentration of chemical in the environment. Factors that can contribute to this altered response include genetic composition, age, developmental stage, health status, substance use history, and nutritional status. These factors may alter absorption and excretory processes, or compromise the function of target organs. In general, the elderly with declining organ function and infants and children with developing organs and people with liver malfunction are expected to be more sensitive to toxic substances than healthy adults.

7.4.6 Potential Childhood Sensitivity Neonates have been identified as a potentially sensitive subpopulation for manganese exposure. This determination reflects observations in human ( Zlotkin and Buchanan, 1986) and animal ( Keen et al., 1986; Kostial et al., 1978; Rehnberg, et al., 1980) studies that suggest that neonates retain higher levels of administered manganese than adults.

In adults, manganese concentrations are retained within a narrow range by the ability of excretion systems to match the intake of this element ( Fechter, 1999). The process responsible for manganese excretion is generally believed to require a significant time period to mature into the adult pattern, with adult patterns of excretion developing at about the time of weaning ( Fechter, 1999). During this period of development, the young organism might be susceptible to manganese toxicity if exposed to high levels in the diet or via environmental contamination.

Data with respect to fetal accumulation are not numerous, but appear to consistently demonstrate that manganese is transported across the placenta to a limited extent ( Fechter, 1999). In 32 mother infant pairs, it was shown that the mean blood manganese level in the newborn was three times higher than in the maternal blood ( Chan et al., 1980). Up to the age of six weeks, infants' erythrocyte manganese concentrations are higher than adults by about 7 9% ( Hatano et 7 52 Manganese   February 2003 al., 1985). When all available data are examined, it appears that the fetus is relatively protected from manganese accumulation when maternal exposure occurs at relatively low doses. Under conditions of high maternal exposure, manganese accumulation also appears to be limited ( Fechter, 1999). The mechanism underlying this lack of accumulation is unknown, but may reflect increased maternal excretion, limited uptake across the placenta, or fetal elimination.

The greatest concern for developmental susceptibility has been generated by data which suggest the existence of a period prior to weaning when the neonate is unable to eliminate manganese. Fechter ( 1999) reassessed data in the published literature and concluded that the available literature does not support a toxicokinetic basis for accumulation in the fetal or neonatal organism [ relative to the adult organism], under conditions of excess exposure to manganese. While the available data indicate that manganese does reach brain tissue, currently available evidence does not support a clear regional distribution.

Kaur et al. ( 1980) found that younger neonates and 19 day fetuses were more susceptible to manganese toxicity than older rats. Studies with 54Mn indicated that manganese was localized to the liver and brain in younger animals, and there was more manganese per unit weight in younger animals when compared with older animals.

Collipp et al. ( 1983) found that hair manganese levels in newborn infants increased significantly from birth ( 0.19 : g/ g) to 6 weeks of age ( 0.885 : g/ g) and 4 months of age ( 0.685 : g/ g) when the infants were given formula. In contrast, there was no significant increase in babies who were breast fed ( 0.330 : g/ g at 4 months). These results were attributed to the difference in manganese content between infant formula and breast milk. Human breast milk is relatively low in manganese ( 7 to 15 : g/ L), while levels in infant formulas are 3 to 100 times higher. Collipp et al. ( 1983) further reported that the level of manganese in the hair of learning disabled children ( 0.434 : g/ g) was significantly increased in comparison to samples from normal children ( 0.268 : g/ g).

There is at least one study reporting different responses in manganese treated neonatal animals compared to treated adults ( Dorman et al., 2000). Pups were administered MnCl 2 in water at 11 or 22 mg Mn/ kg for 21 days by mouth and were dosed starting after birth, postnatal day 1 ( PND 1), until weaning, PND 21. At PND 21, the effect of manganese treatment on motor activity, learning and memory ( passive avoidance task), evoked sensory response ( acoustic startle reflex), brain neurochemistry, and brain pathology was evaluated. Manganese treatment at the highest dose was associated with decreased body weight gain in pups, although the authors indicated absolute brain weight was not significantly altered. There were no statistically significant effects on motor activity or performance in the passive avoidance task. However, manganese treatment induced a significant increase in amplitude of the acoustic startle reflex. Significant increases in striatal DA and DOPAC concentrations were also observed in the highdose treated neonates. No pathological lesions were observed in the treated pups. No effects on body weight or behavior were observed in treated adult animals in this study. The authors indicated that these results suggest that neonatal rats are at greater risk than adults for manganese induced neurotoxicity when compared under similar exposure conditions. This study, along with evidence for increased absorption and reduced elimination in the neonate, 7 53 Manganese   February 2003 suggests that the very young may be more susceptible to the harmful effects of manganese exposure due to differences in toxicokinetics.

Other investigators have reported an association between elevated hair levels of manganese and learning disabilities in children ( Barlow and Kapel, 1979; Pihl and Parkes, 1977). Although no causal relationship has been established for learning disabilities and manganese intake, further research in this area is warranted ( U. S. EPA, 1993). The studies by He et al. ( 1994) and Zhang et al. ( 1995) reported increased manganese levels in hair of school age children exposed to excess levels of manganese in drinking water and food stuffs. These studies conflict with the Kawamura et al. ( 1941) study which showed that children were not adversely affected by ingesting excess levels of manganese. The more recent studies differ in design, however, because they measured early preclinical neurological effects of manganese overexposure. The older studies did not have the sensitivity to measure such effects; this may explain why children were not previously identified as a sensitive population. None of the studies in children provide adequate exposure levels or properly control for confounding factors; therefore, they are not strong enough to indicate that children are more sensitive than adults. They do confirm the need for additional studies to investigate the possibility that children may be more susceptible than adults to the effects of manganese overexposure.

High levels of manganese in infant formulas may also be of concern since Lönnerdal et al. ( 1987) reported increased absorption and retention of manganese in neonatal animals. Manganese has also been shown to cross the blood brain barrier, with the rate of penetration in animal experiments being 4 times higher in neonates than in adults ( Mena, 1974). Dieter et al. ( 1992) stated that " if there were a toxicological limit to manganese according to the principles of preventive health care, then it would have to be set at 0.2 mg/ L of manganese in water for infants as a group at risk."

7.4.7 Other Potentially Sensitive Populations U. S. EPA ( 1996a) has identified additional sensitive subpopulations for manganese exposure. In general, these are groups who may have greater potential for increased body burdens due to increased absorption or altered clearance mechanisms. The list includes pregnant women, elderly persons, iron or calcium deficient individuals, and individuals with impaired liver function. 8 1 Manganese   February 2003 8.0 DOSE RESPONSE ASSESSMENT 8.1 Dose Response for Noncancer Effects 8.1.1 RfD Determination Choice of Principal Study and Critical Effect Manganese is an essential trace element that is required for normal physiologic function in humans and animals. Excess exposure to manganese, particularly via the inhalation route, is associated with neurotoxicological symptoms that resemble parkinsonism. Thus, derivation of the RfD must consider issues of both essentiality and toxicity.

The RfD is not based on rodent studies, because rodents do not exhibit the same neurologic deficits that humans do following exposure to manganese. For example, manganese at high doses induces parkinson like symptoms in humans and primates, but not in rodents. Because of the species difference in the response to manganese exposure, rodents are not good models for manganese toxicity studies. More details on this can be seen in IRIS ( U. S. EPA, 1996a).

The reference dose ( RfD) is based on the extensive information available for the dietary intake of manganese by human populations ( U. S. EPA, 1996a). Freeland Graves et al. ( 1987) reviewed human studies and proposed an estimated safe and adequate daily dietary intake of 3.5 to 7 mg for adults. WHO ( 1973) reviewed data on adult diets and concluded on the basis of manganese balance studies that 2 to 3 mg/ day is an adequate daily intake and 8 to 9 mg/ day is " perfectly safe."

Dose Response Assessment and Method of Analysis The current RfD for manganese was derived from information gathered in dietary surveys of manganese exposure. In various surveys, manganese intakes of adults eating western type and vegetarian diets ranged from 0.7 to 10.9 mg per day ( Freeland Graves, 1994; Gibson, 1994 as cited by IOM, 2002). Depending on individual diets, a normal intake may be well over 10 mg per day, especially from a vegetarian diet Based on this information, the U. S. EPA ( 1996a) considers a dietary intake of 10 mg/ day to be safe for a 70 kg adult. Thus, the resulting dose of 0.14 mg/ kg day represents a NOAEL for chronic human consumption of manganese in the diet ( U. S. EPA, 1996a).

Application of Uncertainty and Modifying Factors U. S. EPA ( 1996a) has recommended use of an uncertainty factor of 1 for derivation of the manganese RfD. This recommendation is based on the following considerations. Manganese is an essential trace element for human health. The information used to derive the RfD was collected from many large human populations consuming normal diets over an extended period of time. The available data suggest that as long as physiological systems are not overwhelmed, 8 2 Manganese   February 2003 humans exert effective homeostatic control over manganese so that body burden is kept relatively constant when concentration of manganese in the diet varies.

U. S. EPA ( 1996a) has recommended the use of a modifying factor of 3 when assessing exposure to manganese from drinking water. U. S. EPA ( 1996a) has outlined four reasons for this recommendation:

° While toxicokinetic data suggest that there is no significant difference in absorption of manganese from food versus water, uptake of manganese from water appears to be greater in fasted individuals.

° The study by Kondakis et al. ( 1989) raises concern for possible adverse health effects associated with a lifetime consumption of drinking water containing 2 mg/ L of manganese.

° Evidence exists that neonates absorb more manganese from the gastrointestinal tract, and excrete less of the absorbed manganese. Additional evidence suggests that absorbed manganese more easily crosses the blood brain barrier in neonates. However, this evidence comes from animal studies; similar absorption studies in human neonates have not been performed, although Collipp et al. ( 1983) observed increased hair manganese levels in infants fed prepared formula compared with infants fed breast milk.

° Infant formula typically contains a much higher concentration of manganese than human or cows' milk. Powdered formula reconstituted with drinking water represents an additional source of manganese intake for a potentially sensitive population.

These potential impacts on children, when considered in conjunction with the likelihood that the most adverse effects of manganese ( e. g., those seen in manganese miners or others with chronic overexposure to inhaled manganese) are likely to be irreversible and not manifested for many years after exposure, warrant caution until more definitive data are available ( U. S. EPA, 1996a). Recent data indicate, however, that in contrast to the symptoms of manganism, preclinical neurological effects of inhalation exposure of occupational workers to excess manganese are reversible ( Roels et al., 1999). Similarly, symptoms of oral exposure to excess manganese in compromised individuals ( e. g., individuals with liver disease who could not excrete manganese in the bile) were resolved when the exposure to excess manganese was decreased ( Devenyi et al., 1994; Fell et al., 1996). These data indicate that the human body can recover from certain adverse effects of overexposure to manganese if the exposure is stopped and the body can clear the excess. Significant uncertainty still exists, however, concerning at what level of manganese intake these preclinical neurological symptoms might occur. 8 3 Manganese   February 2003 The RfD for chronic exposure to manganese in drinking water is therefore calculated as follows:

RfD = 0.14 mg/ kg day = 0.047 mg/ kg day 1 × 3 where:

0.14 mg/ kg day = Chronic NOAEL for dietary manganese.

1 = Uncertainty factor.

3 = Recommended uncertainty factor for exposure in drinking water 8.1.2 RfC Determination The inorganic manganese compounds predominating in drinking water are non volatile. Inhalation of manganese during use of drinking water for residential activities is therefore not expected to be a significant pathway of exposure or toxicity.

U. S. EPA ( 1996a) has derived an inhalation Reference Concentration ( RfC) for manganese of 5 × 10 5 mg/ m3.

Choice of Principal Study and Critical Effect The RfC for manganese ( U. S. EPA, 1996a) was derived using data from two epidemiological studies of workers exposed to manganese dioxide dust in occupational studies ( Roels et al., 1987; Roels et al., 1992). The critical effect was impairment of neurobehavioral function, as assessed by medical questionnaire, audio verbal short term memory, visual simple reaction time, hand steadiness, and eye hand coordination.

Dose Response Characterization and Method of Analysis The toxicity data for manganese were evaluated using the conventional NOAEL/ LOAEL approach. Neither of the principal studies identified a NOAEL. The LOAEL from the Roels et al. ( 1992) is derived from an occupational lifetime integrated respirable dust ( IRD) concentration of manganese dioxide ( based on 8 hour time weighted average [ TWA] occupational exposures for various job classifications, multiplied by individual work histories in years). This LOAEL is expressed as mg Mn/ m3 year. The IRD concentrations ranged from 0.040 to 4.433 mg Mn/ m3 year, with a geometric mean of 0.793 mg Mn/ m3 year and a geometric standard deviation of 2.907. The geometric mean concentration ( 0.793 Mn/ m3 year) was divided by the average duration of manganese dioxide exposure ( 5.3 years) to obtain a LOAEL TWA of 0.15 mg Mn/ m3 year. The LOAEL ( Human Equivalent Concentration, HEC) is 0.05 mg/ m3.

The LOAEL identified in the Roels et al. ( 1987) study is based on an 8 hour TWA occupational exposure. The TWA of total airborne manganese dust ranged from 0.07 to 8.61 mg/ m3, and the median was 0.97 mg/ m3. The LOAEL ( HEC) is 0.34 mg/ m3. 8 4 Manganese   February 2003 Application of Uncertainty and Modifying Factors No modifying factor was used in derivation of the RfC. A composite uncertainty factor of 1,000 was used and reflects a factor of 10 for protection of sensitive individuals, a factor of 10 for use of a LOAEL, and a factor of 10 for database limitations. The factor of 10 for database limitations reflects an exposure period of less than chronic duration, lack of developmental data, and potential but unquantified differences in the toxicity of different forms of manganese.

8.2 Dose Response for Cancer Effects Manganese has in the past been classified as a Group D chemical   NOT CLASSIFIABLE as to HUMAN CARCINOGENICITY. This category is assigned to chemicals for which there is inadequate human and animal evidence of carcinogenicity, or for which no data are available. There are presently no human data to suggest an association of oral manganese exposure with increased cancer incidence. Data collected from a 2 year oral exposure study in rats did not reveal evidence for carcinogenic activity ( NTP, 1993). Data collected from a 2 year oral exposure study in mice revealed an apparent increase in tumor incidence at the highest dose administered, but only near the end of the study ( NTP, 1993). The observed increase was not significantly different from the historical control incidence. These results are considered to be equivocal. Based on the absence of any significant cancer response, a quantitative cancer dose response assessment for manganese will not be conducted.

Manganese has been evaluated using the criteria of the U. S. EPA Proposed Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1999b). Based on available data manganese is classified under the category of " Data are inadequate for assessment of human carcinogenic potential." This descriptor is appropriate when there is a paucity of data on carcinogenic effects, or when the data are conflicting. 9 1 Manganese   February 2003 9.0 RISK DETERMINATION AND CHARACTERIZATION OF RISK FROM DRINKING WATER 9.1 Regulatory Determination for Chemicals on the CCL The Safe Drinking Water Act ( SDWA), as amended in 1996, required the Environmental Protection Agency ( EPA) to establish a list of contaminants to aid the Agency in regulatory priority setting for the drinking water program. EPA published a draft of the first Contaminant Candidate List ( CCL) on October 6, 1997 ( 62 FR 52193, U. S. EPA, 1997). After review of and response to comments, the final CCL was published on March 2, 1998 ( 63 FR 10273, U. S. EPA, 1998). The CCL grouped contaminants into three major categories as follows:

Regulatory Determination Priorities  Chemicals or microbes with adequate data to support a regulatory determination,

Research Priorities  Chemicals or microbes requiring research for health effects, analytical methods, and/ or treatment technologies,

Occurrence Priorities  Chemicals or microbes requiring additional data on occurrence in drinking water.

The March 2, 1998 CCL included one microbe and 19 chemicals in the regulatory determination priority category. More detailed assessments of the completeness of the health, treatment, occurrence and analytical method data led to a subsequent reduction of the regulatory determination priority chemicals to a list of 12 ( one microbe and 11 chemicals) which was distributed to stakeholders in November 1999.

SDWA requires EPA to make regulatory determinations for no fewer than five contaminants in the regulatory determination priority category by August, 2001. In cases where the Agency determines that a regulation is necessary, the regulation should be proposed by August 2003 and promulgated by February 2005. The Agency is given the freedom to also determine that there is no need for a regulation if a chemical on the CCL fails to meet one of three criteria established by SDWA and described in Section 9.1.1.

9.1.1 Criteria for Regulatory Determination These are the three criteria used to determine whether or not to regulate a chemical on the CCL:

The contaminant may have an adverse effect on the health of persons,

The contaminant is known to occur or there is a substantial likelihood that the contaminant will occur in public water systems with a frequency and at levels of public health concern, 9 2 Manganese   February 2003 In the sole judgment of the administrator, regulation of such contaminant presents a meaningful opportunity for health risk reduction for persons served by public water systems.

The findings for all criteria are used in making a determination to regulate a contaminant. As required by SDWA, a decision to regulate commits the EPA to publication of a Maximum Contaminant Level Goal ( MCLG) and promulgation of a National Primary Drinking Water Regulation ( NPDWR) for that contaminant. The Agency may determine that there is no need for a regulation when a contaminant fails to meet one of the criteria. A decision not to regulate is considered a final Agency action and is subject to judicial review. The Agency can choose to publish a Health Advisory ( a nonregulatory action) or other guidance for any contaminant on the CCL independent of the regulatory determination.

9.1.2 National Drinking Water Advisory Council Recommendations In March 2000, the EPA convened a Working Group under the National Drinking Water Advisory Council ( NDWAC) to help develop an approach for making regulatory determinations. The Working Group developed a protocol for analyzing and presenting the available scientific data and recommended methods to identify and document the rationale supporting a regulatory determination decision. The NDWAC Working Group report was presented to and accepted by the entire NDWAC in July 2000.

Because of the intrinsic difference between microbial and chemical contaminants, the Working Group developed separate but similar protocols for microorganisms and chemicals. The approach for chemicals was based on an assessment of the impact of acute, chronic, and lifetime exposures, as well as a risk assessment that includes evaluation of occurrence, fate, and dose response. The NDWAC Protocol for chemicals is a semi quantitative tool for addressing each of the three CCL criteria. The NDWAC requested that the Agency use good judgement in balancing the many factors that need to be considered in making a regulatory determination.

The EPA modified the semi quantitative NDWAC suggestions for evaluating chemicals against the regulatory determination criteria and applied them in decision making. The quantitative and qualitative factors for manganese that were considered for each of the three criteria are presented in the sections that follow.

9.2 Health Effects The first criterion asks if the contaminant may have an adverse effect on the health of persons. Because all chemicals have adverse effects at some level of exposure, the challenge is to define the dose at which adverse health effects are likely to occur, and estimate a dose at which adverse health effects are either not likely to occur ( threshold toxicant), or have a low probability for occurrence ( non threshold toxicant). The key elements that must be considered in evaluating the first criterion are the mode of action, the critical effect( s), the dose response for critical effect( s), the RfD for threshold effects, and the slope factor for non threshold effects. 9 3 Manganese   February 2003 A full description of the health effects associated with exposure to manganese is presented in Chapter 7 of this document and summarized below in Section 9.2.2. Chapter 8 and Section 9.2.3 present dose response information.

9.2.1 Health Criterion Conclusion The available toxicological data indicate that manganese has the potential to cause adverse health effects in humans and animals at high doses. The primary route of exposure to toxic levels of manganese is through the inhalation of manganese dust. An increased potential exists for inhalation and ingestion exposure to manganese as a result of the use of MMT in fuels. Zayed et al. ( 1999a) measured airborne manganese concentrations ( as MMT, respirable, and total manganese) in five different microenvironments around Montreal, Canada. The authors determined that the average daily exposure to respirable manganese was 0.010 : g/ kg day and had a low contribution to air, food, and water. Oral exposure to levels of toxicological concern is rare. In humans, neurological effects are the most likely manifestation of manganese toxicity. There is no information available regarding the carcinogenicity of manganese in humans, and animal studies have reported mixed results. Manganese is classified as Group D, or Not classifiable as to human carcinogenicity. The Reference Concentration ( RfC) for manganese is 5 x 10 5 mg/ m3 ( U. S. EPA, 1998a) which is derived using data from two epidemiological studies of workers exposed to manganese dioxide dust in an occupational setting ( Roels et al., 1987; Roels et al., 1992). The critical effect was impairment of neurobehavioral function. The current RfD for manganese in food is 0.14 mg/ kg day; and for drinking water, 0.047 mg/ kg day. Despite the fact that it is possible for manganese to elicit some toxic effects at very high doses, the database is too uncertain, especially related to children and other sensitive populations. Based on the occurrence of adverse effects in humans and animals, the evaluation for Criterion # 1 is positive.

9.2.2 Hazard Characterization and Mode of Action Implications The primary health effect of manganese exposure is neurotoxicity, which is characterized at high doses by ataxia, increased anxiety, dementia, a " mask like" face, general extrapyrimidal syndrome, or manganism, a syndrome similar to Parkinson's disease. The precise mechanisms of manganese neurotoxicity are not known, although the observed effects of manganese on the globus pallidus region of the brain suggest that a likely mechanism involves impairment of dopaminergic function. Preclinical adverse neurological effects have been reported at much lower doses than those resulting in manganism, however. Therefore, the possibility exists that any potential neurological effects resulting from environmental exposures to manganese would likely be more comparable to these subtle, though potentially significant, changes in neurological function.

Studies in humans and animals are mixed, but most animal studies indicate that children are a potentially sensitive subpopulation based on decreased excretion in the neonate ( Lönnerdal, 1994). Additional potentially sensitive sub populations include the elderly, pregnant women, iron deficient individuals, and individuals with impaired liver function. 9 4 Manganese   February 2003 Because the primary route of elimination for manganese is biliary excretion, persons with impaired liver function may be especially susceptible to manganese toxicity ( Layrargues et al., 1998). Persons in a state of iron deficiency may also experience greater susceptibility to manganese absorption and toxicity ( Finley, 1999; Finley et al., 1994). In addition, infants and neonates, in which the capacity for excretion through the bile is not fully developed, may also be potentially susceptible to manganese toxicity ( Lönnerdal, 1994). Although animal studies have indicated an increased potential in neonates for gastrointestinal absorption of manganese, as well as decreased excretion potential, the degree to which these findings apply to human infants is unknown. Dorman et al. ( 2000) have shown, however, that there is increased sensitivity for neurotoxic effects following manganese exposure in neonatal rats compared to adult rats. Because manganese is an essential nutrient in developing infants, however, the potential adverse effects from manganese deficiency may be of greater concern than potential toxicity from overexposure

An added complication is the fact that many inhibitors of manganese absorption, such as phytates and plant fiber, are common in the diet and may thus lower the actual absorption of ingested manganese. Also, manganese absorption from foods that are potentially high sources may be inhibited by other factors such as the presence of co occurring plant proteins that bind manganese and decrease its bioavailability. Thus, although the manganese content in the soybased formula is higher than manganese content in human milk, the actual absorption of manganese in the formula may not be substantially greater since it is prepared with soy milk, which is high in phytate and vegetable protein. Data exist, however, that argue against this possibility. For example, Keen et al. ( 1986) demonstrated in rat pups that manganese uptake from human breast milk and cow's milk was higher (~ 80% and ~ 89 %, respectively) than that from soy formula (~ 60%), but the absolute amount of manganese retained from soy was 25 times the amount retained from human milk. Dorner et al. ( 1989) also reported increased retention of manganese in full term human infants fed cow's milk formulas compared to breast fed infants. Human milk and cow's milk contain different proteins that bind manganese. In some cases, the presence of these proteins may enhance manganese transport across the gut wall and hence increase absorption. If infant formula is prepared with contaminated water, then it is possible that the manganese will remain in a soluble form which may be more easily absorbed. More data are needed on the various factors affecting manganese absorption in infants before a confident determination can be made. Other instances in which high dietary levels of manganese may not necessarily correspond to high dose levels include vegetarian diets ( many vegetables contain high manganese levels but also high fiber and phytate levels) and possibly tea drinkers ( tea also contains high manganese levels accompanied by high levels of tannin, another inhibitor of manganese absorption).

Several studies have explored the level of manganese intake which may be considered safe in humans. The Food and Nutrition Board ( IOM, 2002) set an adequate intake level for manganese of 2.3 mg/ day for men and 1.8 mg/ day for women ( IOM, 2002; Trumbo et al., 2001). The Food and Nutrition Board also set a tolerable upper intake level of 11 mg Mn/ day for adults based on the Greger ( 1999) review, which suggested that people eating western type and vegetarian diets may have intakes as high as 10.9 mg/ day ( IOM, 2002). Further, for short term 9 5 Manganese   February 2003 duration, Davis and Greger ( 1992) found that daily intake of 15 mg/ day for 90 days resulted in no adverse effects in women; the only effect seen was an increase in superoxide dismutase activity.

No significant exposure related neurological effects were seen in a cohort in Germany exposed for up to 40 years to manganese in their well water at levels as high as 2.160 mg/ L ( 0.3 to 2.160 mg/ L; Vierrege et al., 1995). On the other hand, a study in Greece which examined older populations chronically exposed to well water containing up to around 2 mg/ L found effects on neurological function in the high exposure group ( Kondakis et al., 1989); however, this study did not adequately account for potential bias in subjective neurological test scores. Neither study reported the dietary or other sources of manganese intake.

9.2.3 Dose Response Characterization and Implications in Risk Assessment The dose response relationship for neurological effects of manganese by ingestion is not well characterized in animals or humans, but epidemiological data for humans indicate that intakes as high as 11 mg/ day ( 0.16 mg/ kg day) may not cause adverse effects in adult humans. Additional evidence suggests a safe level as high as 15 mg/ day ( 0.21 mg/ kg day for adult), based on a study in which women received daily supplements of 15 mg manganese for 90 days and exhibited only an increase in lymphocyte manganese dependent superoxide dismutase, but no measured adverse effects ( Davis and Greger, 1992). Characterizing dose response in humans is complicated by the fact that manganese is an essential nutrient, and therefore some minimal level of intake is necessary for good health. There are many reports of toxicity to humans exposed to manganese by inhalation; much less is known, however, about oral intakes resulting in toxicity. Rodents do not provide a good experimental model for manganese toxicity and only one limited study in primates by the oral route of exposure is available ( Gupta et al., 1980).

A review of acute animal toxicity studies of manganese indicates that the manganese has low to moderate oral toxicity. For example, the oral LD 50 values for manganese compounds in rats are in the range of 400 to 2,000 mg Mn/ kg. Some animal studies have also reported developmental and reproductive effects at high doses for some manganese compounds, but most data from oral exposure suggest that manganese has a low developmental toxicity.

EPA has calculated an RfD for manganese. The RfD for manganese in food is 0.14 mg/ kg day, based on dietary surveys that have reported that, for an average 70 kg adult, having a daily manganese intake of 10 mg presents no adverse effect. For drinking water, EPA recommends to apply a modifying factor ( MF) of 3 to yield a value of 0.047 mg/ kg day. This modifying factor is meant to address the concern raised by the epidemiology study ( Kondakis et al., 1989), and a potential higher absorption of manganese in water, especially when drinking fluids early in the morning, when the gut is empty. EPA has medium confidence in the RfD for manganese. 9 6 Manganese   February 2003 9.3 Occurrence in Public Water Systems The second criterion asks if the contaminant is known to occur or if there is a substantial likelihood that the contaminant will occur in public water systems with a frequency and at levels of public health concern. In order to address this question, the following information was considered:

° Monitoring data from public water systems ° Ambient water concentrations and releases to the environment ° Environmental fate Data on the occurrence of manganese in public drinking water systems were the most important determinants in evaluating the second criterion. EPA looked at the total number of systems that reported detections of manganese, as well those that reported concentrations of manganese above an estimated drinking water health reference level ( HRL). For noncarcinogens the estimated HRL risk level was calculated from the RfD assuming that 20% of the total exposure would come from drinking water. For carcinogens, the HRL was the 10 6 risk level. The HRLs are benchmark values that were used in evaluating the occurrence data while the risk assessments for the contaminants were being developed.

The available monitoring data, including indications of whether or not the contamination is a national or a regional problem, are included in Chapters 4 of this document and are summarized below. Additional information on production, use, and environmental fate are found in Chapters 2 and 3.

9.3.1 Occurrence Criterion Conclusion The available data for manganese production and use indicate a fairly stable trend for both. While release of manganese to surface water is variable within a wide range of values, release of manganese compounds to surface water is increasing. Releases of manganese and manganese compounds to land are generally decreasing, while releases of manganese to air are decreasing and air emissions of manganese compounds are stable ( Tables 3 4 and 3 5). MMT in gasolines provides a relatively new environmental source of manganese exposure. Recent testing suggests that when very low levels of MMT are combusted ( i. e., concentrations comparable to the currently allowed levels), manganese is emitted primarily as manganese phosphate and sulfate. Data on the occurrence of manganese in air resulting from combustion of MMT and other sources are presented in Section 4.2. Pfeifer et al. ( 1999) determined that two occupational groups, office workers and taxi drivers were exposed to comparable concentrations of manganese both before and after MMT was present in fuels. These data, however, are counter to other modeling data that indicate that taxi drivers are exposed to increased concentrations of manganese as a result of MMT use ( Lynam et al., 1994; Zayed et al., 1994; Riveros Rosas et al., 1997). Modeling data from five microenvironments in Canada indicate that with the currently acceptable levels of MMT allowed in fuel, little impact to air and surface water concentrations of 9 7 Manganese   February 2003 manganese is expected from the use of MMT in fuels ( Zayed et al., 1999a). Monitoring data indicate that manganese is infrequently detected in public water supplies. When manganese is detected, it rarely exceeds the HRL or a value of one half the HRL. Further, because manganese is an essential nutrient, the risks of over exposure must be weighed against the risks of manganese deficiency. Based on these data, it is unlikely that manganese will occur in public water systems at frequencies or concentration levels that are of public health concern. Therefore, the evaluation for Criterion # 2 is negative.

9.3.2 Monitoring Data Drinking Water Occurrence data for manganese in drinking water are presented and analyzed in Chapter 4 of this document. Estimates of exposed populations are derived in Section 4.3. The National Inorganic and Radionuclide Survey ( NIRS) data represent 49 States. Data were not available for the State of Hawaii. Since NIRS data lack occurrence information for surface water systems, occurrence data on manganese exposure from the States of Alabama, California, Illinois, New Jersey, and Oregon were used to obtain information on surface water.

At a health reference level ( HRL) of 0.3 mg/ L, approximately 6.1% of the NIRS PWSs had detections greater than one half the HRL ( about 3,600 ground water PWSs nationally), affecting approximately 4.6% of the population served ( estimated at 4.0 million people nationally).

The percentage of NIRS PWSs with detections greater than the HRL of 0.3 mg/ L was approximately 3.2% ( about 1,920 ground water PWSs nationally), affecting 2.6 % of the population served ( estimated at approximately 2.3 million people nationally).

The supplemental State data sets indicate that ground water PWS detections greater than the HRL of 0.3 mg/ L are between 0.6% and 12%. Again, the NIRS national average is within this range, with 3.2% of PWSs greater than the HRL. Notably, surface water PWSs showed fewer exceedances of the HRL than ground water PWSs at this higher concentration, ranging from 0% to 3%. Extrapolating national population exposures from these limited data sets is not possible because exposure to manganese through surface water is not quantified beyond the five States shown. However, exposure estimates incorporating surface water sources would certainly be larger than the estimates provided here for groundwater sources.

Ambient Water The National Ambient Water Quality Assessment ( NAWQA) program was begun in 1991 by the United States Geological Survey ( USGS) to monitor water quality in representative study basins located around the country. This program, which consists of 59 significant watersheds and aquifers, was described in Chapter 4 of this document in regard to its use for monitoring ambient levels of manganese in surface and ground waters. The Minimum Reporting Level 9 8 Manganese   February 2003 ( MRL) in water is 0.001 mg/ L, while the MRLs in sediments and aquatic biota tissue are 4 mg/ kg and 0.1 mg/ kg, respectively.

The data indicate that manganese is ubiquitous in surface and ground waters, presumably as a result of its natural occurrence in the earth's crust. The frequency of detection above the HRL is generally higher in ground water than in surface water, but the median concentration in sites reporting a detection is higher in surface water ( 0.016 mg/ L in surface water versus 0.005 mg/ L in ground water). Overall, the data indicate that, while manganese is nearly ubiquitous in surface and ground water, detections at levels of concern to public health are relatively few.

Manganese has been universally detected in stream sediments and aquatic biota tissues at low levels. Manganese is not thought to bioaccumulate in tissues to any significant degree, and desorption from sediments into the water column is also limited by the insolubility of most manganese compounds.

9.3.3 Use and Fate Data Manganese is a naturally occurring element and is commonly found in soil, water, air, and food, generally as a component of over 100 mineral compounds. Most manganese ore is imported to the United States, with the amount increasing from 308 thousand metric tons in 1984 to 535 thousand metric tons in 1999. Most of this ore is smelted to produce ferromanganese, which is used in steel production. Manganese compounds have a variety of other uses in industry and agriculture, as described in Table 3 3 of this document.

Examination of data from the Toxic Release Inventory ( TRI), shown in Tables 3 4 and 3 5 of this document, indicates that releases of manganese to water varied between 89 thousand and 2.4 million pounds for the period 1988 to 1998. Data for manganese compounds reveal an increasing trend in surface water discharges, from 681 thousand to 4.5 million pounds for the same period.

Once released to the environment, manganese is readily deposited in the soil and taken up by plants, whereupon it may enter the food chain. Significant bioaccumulation is not expected to occur. Manganese is an essential nutrient in the diet, so some minimal intake is necessary for good health. Manganese particles may also become airborne, and some manganese compounds are soluble in water. Manganese compounds may also adsorb to sediment surfaces and precipitate out of solution.

Manganese, in the form of potassium permanganate, may be used in drinking water treatment for oxidation and disinfection purposes ( ANSI/ NSF, 2000), in addition to its use in industrial wastewater purification and odor abatement ( ATSDR, 2000; U. S. EPA, 1984). The adsorption properties of some manganese compounds may cause them to be more prevalent in certain types of soils or sediments. 9 9 Manganese   February 2003 9.4 Risk Reduction The third criterion asks if, in the sole judgment of the Administrator, regulation presents a meaningful opportunity for health risk reduction for persons served by public water systems. In order to evaluate this criterion, EPA looked at the total exposed population, as well as the population exposed above the estimated HRL. Estimates of the populations exposed and the levels to which they are exposed were derived from the monitoring results. These estimates are included in Chapter 4 of this document and summarized in Section 9.4.2 below.

In order to evaluate risk from exposure through drinking water, EPA considered the net environmental exposure in comparison to the exposure through drinking water. For example, if exposure to a contaminant occurs primarily through ambient air, regulation of emissions to air provides a more meaningful opportunity for EPA to reduce risk than regulation of the contaminant in drinking water. In making the regulatory determination, the available information on exposure through drinking water ( Chapter 4) and information on exposure through other media ( Chapter 5) were used to estimate the fraction that drinking water contributes to the total exposure. The EPA findings are discussed in Section 9.4.3 below.

In making its regulatory determination, EPA also evaluates effects on potential sensitive populations, including the fetus, infants and children. The sensitive population considerations are included in Section 9.4.4.

9.4.1 Risk Reduction Criterion Conclusion Approximately 47.5 million people are served by ground water public water systems with detections greater than the MRL. More than 2.3 million of these individuals are served by systems with detections greater than the HRL. Manganese is an essential nutrient that is common and necessary in the diet. The estimated daily exposure to manganese from public water systems is far below the expected daily intake from the diet, and also far below the level determined to be safe and adequate. When average daily intakes from drinking water are compared with intakes from food, air and soil, drinking water accounts for a relatively small proportion of manganese intake. On the basis of these observations, the impact of regulating manganese concentrations in drinking water on health risk reduction is likely to be small. Therefore, the evaluation for Criterion # 3 is negative.

9.4.2 Exposed Population Estimates Estimates of exposed populations were derived in Chapter 4. National population estimates for manganese exposure were derived using summary statistics from the National Inorganic and Radionuclide Survey ( NIRS), which lacked surface water data, with supplemental surface water occurrence data that had been separately submitted to EPA from five States. An estimated 47.5 million people in the U. S. are served by public water systems supplied from ground water with detections of manganese above the minimum reporting level ( MRL). An estimated 4.0 million people ( 4.6% of the population) are served by ground water with levels above one half the health reference level ( HRL) of 0.3 mg/ L, and an estimated 2.3 million people 9 10 Manganese   February 2003 ( 2.6 % of the population) are served by ground water with levels above the HRL. It should be noted that these estimates are based on very limited and outdated data. The possibility exists that the number of people served by ground water with Mn levels that are above the HRL could be higher than these estimates; however, the data are lacking at this time to develop a more timely assessment.

9.4.3 Relative Source Contribution Relative source contribution analysis compared the magnitude of exposure expected via drinking water to the magnitude of exposure from intake of manganese from other media such as food, air, and soil. To perform this analysis, intake of manganese from drinking water must be estimated. Occurrence data for manganese are presented in Chapter 4 of this document. According to the NIRS data ( Table 4 1), the median and 99th percentile concentrations for manganese in ground water public water supplies were above the MRL of 0.001 mg/ L. This is not surprising considering the ubiquity with which manganese is present in the earth's crust.

Taking the median concentration of detections from the NIRS data ( 0.01 mg/ L), and assuming a daily intake of 2 L of drinking water by a 70 kg adult, the average daily dose would be 0.02 mg/ person day or 2.8 × 10 4 mg/ kg day. The corresponding dose for a 10 kg child consuming 1 L/ day of drinking water would be 0.01 mg/ child day or 1.0 × 10 3 mg/ kg day. These values are far below those expected from a normal diet ( 2.9   12.6 × 10 2 mg/ kg day for adults, 1.3 × 10 1 mg/ kg day for children, see Table 9 1 below), and are also less than the levels determined by the National Academy of Sciences to be safe and adequate. The NAS determined that a daily intake of 2.3 mg Mn is adequate for men and 1.8 mg is adequate for women, while the daily adult intake expected from drinking water containing 0.01 mg/ L Mn is 0.02 mg Mn. The NAS also determined that a daily intake of 1.9 mg Mn is adequate for boys and 1.6 mg is adequate for girls, while the daily intake expected from drinking water containing 0.01 mg/ L Mn is 0.01 mg for children. ( IOM, 2002).

Table 9 1. Comparison of Average Daily Intake from Drinking Water and Other Mediaa Medium Adult (: g/ kg day) Child (: g/ kg day)

Drinking Waterb 0.29 1.0 Food 28.6 126 128.0 ( 0.87 37.2 for infants)

Air 0.0087 0.034 Soil 0.0014  5.0 0.02  70 a See Chapter 5 for derivation of intakes from media other than water b based on median values 9 11 Manganese   February 2003 9.4.4 Sensitive Populations The sensitive populations identified for manganese include persons with impaired detoxification and excretory function, such as infants and the elderly. Individuals with damaged or impaired liver function may be particularly sensitive.

9.5 Regulatory Determination Decision As stated in Section 9.1.1, a positive finding for all three criteria is required in order to make a determination to regulate a contaminant. While there is evidence that manganese may have adverse health effects in humans at high doses through inhalation, the evidence for adverse effects through oral exposure at low or moderate levels is less compelling. Because manganese is an essential nutrient, concern over potential toxic effects from high oral exposure must be balanced against concern for adverse effects from manganese deficiency should intake be too low. Manganese has been found to occur in an estimated 1,920 ground water public water systems representing more than 2.3 million people exposed ( 2.6 % of the population) to levels at or above 0.3 mg/ L. The Agency believes that a meaningful opportunity for health risk reduction does not exist for persons served by public water systems because the average dietary intake of manganese exceeds the contribution normally found in public drinking water systems. Thus, based on the evaluation of available data using the criteria described above, the regulatory determination is " Do not regulate". 10 1 Manganese   February 2003 10.0 REFERENCES Abbott, P. 1987. Methylcyclopentadienyl manganese tricarbonyl ( MMT) in petrol: The toxicological issues. Sci. Total Environ. 67: 247 255.

Abrams, E., J. W. Lassiter, W. J. Miller, et al. 1976. Effect of dietary manganese as a factor affecting 54Mn absorbtion in rats. Nutr. Rep. Int. 14: 561 565 ( as cited in ATSDR, 2000).

Ali, M. M., G. S. Shukla, D. K. Saxena, et al. 1981. Behavioral dysfunctions and central neurotransmitters in manganese exposed rats. J. Environ. Biol. 2: 29 39 ( as cited in ATSDR, 2000).

Ali, M. M., R. C. Murthy, D. K. Saxena, et al. 1983. Effect of low protein diet on manganese neurotoxicity: I. Developmental and biochemical changes. Neurobehav Toxicol Teratol 5: 377 383.

Ali, M. M., R. C. Murthy, S. K. Mandal, et al. 1985. Effect of low protein diet on manganese neurotoxicity: III. Brain neurotransmitter levels. Neurobehav. Toxicol. Teratol. 7( 5): 427 431. Amdur, M. O., L. C. Norris and G. F. Heuser. 1944. The need for manganese in bone development by the rat. Proc. Soc. Exp. Biol. Med. 59: 254 255 ( as cited in ATSDR, 2000).

Andersen, M. E., J. M. Gearhart, H. J. Clewell, III. 1999. Pharmacokinetic data needs to support risk assessments for inhaled and ingested manganese. Neurotoxicology 20: 161 172.

ANSI/ NSF. 2000. American National Standards Institute, NSF International. Drinking water treatment chemicals  Health effects. ANSI/ NSF 60 2000. Ann Arbor, MI.

Archibald, F. S. and C. Tyree. 1987. Manganese poisoning and the attack of trivalent manganese upon catecholamines. Arch. Biochem. Biophys. 2: 638 650.

Ardeleanu, A., S. Loranger, G. Kennedy, et al. 1999. Emission rates and physico chemical characteristics of Mn particles emitted by vehicles using methylcyclopentadienyl manganese tricarbonyl ( MMT) as an octane improver. Water Air Soil Pollut. 115: 411 427.

Arnaud, J. And A. Favier. 1995. Copper, iron, manganese and zinc contents in human colostrum and transitory milk of French women. Sci Total Environ 159: 9 15.

Aschner, M. and J. L. Aschner. 1990. Manganese transport across the blood brain barrier: relationship to iron homeostasis. Brain. Res. Bull. 24: 857 860.

Aschner, M. and J. L. Aschner. 1991. Manganese neurotoxicity: Cellular effects and blood brain barrier transport. Neurosci. Biobehav. Rev. 15( 3): 333 340.

ASNS. 1999. Nutrient Information: Manganese. American Society for Nutritional Sciences. Available at http:// www. nutrition. org/ nutinfo. 10 2 Manganese   February 2003 ATSDR. 2000. Toxicological Profile for Manganese ( Final). U. S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

Autissier, N., L. Rochette, P. Dumas, et al. 1982. Dopamine and norepinephrine turnover in various regions of the rat brain after chronic manganese chloride administration. Toxicology 24( 2): 175.

Bales, C. W., J. H. Freeland Graves, P. H. Lin, et al. 1987. Plasma uptake of manganese: Influence of dietary factors. In: Kies, C., ed., Nutritional Bioavailability of Manganese. Washington D. C.: American Chemical Society, pp. 112 122.

Baly, D. L., B. Lönnerdal and C. L. Keen. 1985. Effects of high doses of manganese on carbohydrate homeostasis. Toxicol. Lett. 25: 95 102.

Baly, D. L., B. Lönnerdal and C. L. Keen. 1988. Mechanism of decreased insulinogenesis in manganese deficient rats. Decreased insulin mRNA. FEBS Lett. 239: 55 58 ( as cited in ATSDR, 2000).

Banta, R. G. and W. R. Markesbery. 1977. Elevated manganese levels associated with dementia and extrapyramidal signs. Neurology 27( 3): 213 216.

Barbeau, A. 1984. Manganese and extrapyramidal disorders ( a critical review and tribute to Dr. George C. Cotzias). Neurotoxicology 5( 1): 13 35 ( as cited in ATSDR, 2000).

Barlow, P. J. and M. Kapel. 1979. Hair metal analysis and its significance to certain disease conditions. 2nd Annual Trace Metals Health Seminar, Boston, MA.

Baxter, D. J., W. O. Smith and G. C. Klein. 1965. Some effects of acute manganese excess in rats. Proc. Soc. Exp. Biol. Med. 119: 966 970.

Bell, J. G, C. L. Keen, and B. Lönnerdal. 1989. Higher retention of manganese in suckling than in adult rats is not due to maturational differences in manganese uptake by rat small intestine. J. Toxicol. Environ. Health 26: 387 398. ( As cited in ATSDR, 2000).

Bernheimer, H., W. Birkmayer, O. Hornykiewicz, et al. 1973. Brain dopamine and the syndromes of Parkinson and Huntington: Clinical, morphological and neurochemical correlations. J. Neurol. Sci. 20: 415 455.

Bertinchamps, A. J. and G. C. Cotzias. 1958. Biliary excretion of manganese. Fed. Proc. 17: 428.

Bertinchamps, A. J., S. T. Miller and G. C. Cotzias. 1966. Interdependence of routes excreting manganese. Am. J. Physiol. 211( 1): 217 224. 10 3 Manganese   February 2003 Bienvenu, P., C. Noire and A. Cier. 1963. Comparative general toxicity of metallic ions. A relation with the periodic classification. Rech. Serv. Sante Armees, Lyons, France 256: 1043 1044.

Bleich S., D. Degner, R. Sprung, A. Riegel, W. Poser, and E. Ruther. 1999. Chronic manganism: Fourteen years follow up. J. Neuropsych. Clin. Neuro. 11: 117.

Bonilla, E. and M. Diez Ewald. 1974. Effect of L DOPA on brain concentration of dopamine and homovanillic acid in rats after chronic manganese chloride administration. J. Neurochem. 22( 2): 297 299.

Bonilla, E. 1978a. Flameless atomic absorption spectrophotometric determination of manganese in rat brain and other tissues. Clin. Chem. 24: 471 472.

Bonilla, E. 1978b. Increased GABA content in caudate nucleus of rats after chronic manganese chloride administration. J. Neurochem. 31( 2): 551 552.

Bonilla, E. 1980. L tyrosine hydroxylase activity in the rat brain after chronic oral administration of manganese chloride. Neurobehav. Toxicol. 2: 37 41.

Boyer, P. D., J. H. Shaw and P. H. Phillips. 1942. Studies on manganese deficiency in the rat. J. Biol. Chem. 143: 417 425 ( as cited in ATSDR, 2000).

Britton, A. A. and G. C. Cotzias. 1966. Dependence of manganese turnover on intake. Am. J. Physiol. 211( 1): 203 206.

Brouillet, E. P., L. Shinobu, U. McGarvey, et al. 1993. Manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism. Exp. Neurol. 120( 1): 89 94 ( as cited in ATSDR, 2000).

Burnett, W. T., Jr., R. R. Bigelow, A. W. Kimball, et al. 1952. Radio manganese studies on the mouse, rat and pancreatic fistula dog. Am. J. Physiol. 168: 620 625.

Calne, D. B., N. S. Chu, C. C. Huang, et al. 1994. Manganism and idiopathic parkinsonism: similarities and differences. Neurology 44: 1583 1586.

Carter, S. D., J. F. Hein, G. L. Rehnberg, et al. 1980. Chronic manganese oxide ingestion in rats: hematological effects. J. Toxicol. Environ. Health 6( 1): 207 216.

Casto, B. C., J. Meyers and J. A. DiPaolo. 1979. Enhancement of viral transformation for the evaluation of the carcinogenic or mutagenic potential of inorganic metal salts. Cancer Res. 39: 193 198.

Cawte, J. and M. T. Florence. 1989. A manganic milieu in North Australia: Ecological manganism: Ecology; diagnosis; individual susceptibility, synergism, therapy, prevention, advice for the community. Int. J. Biosocial Med. Res. 11: 43 56 ( as cited in U. S. EPA, 1993). 10 4 Manganese   February 2003 Chan W. Y., T. Z. Ramadan, M. Perlman, A. M. McCaffree and M. O. Rennert. 1980. Nutrition Rep. Int. 22: 939 948.

Chan, A. W. K., J. C. K. Lai, M. J. Minski, et al. 1981. Manganese concentration in rat organs: Effect after life long manganese treatment. Biochem. Soc. Trans. 9: 229.

Chan, W. Y., J. M. Bates, Jr. and O. M. Rennert. 1982. Comparative studies of manganese binding in human breast milk, bovine milk and infant formula. J. Nutr. 112( 4): 642 651.

Chan, W. Y., M. H. Raghib and O. M. Rennert. 1987. Absorption studies of manganese from milk diets in suckling rats. In: Kies, C., ed., Nutritional Bioavailability of Manganese. Washington, D. C.: American Chemical Society, pp. 80 89.

Chandra, S. V. and S. P. Srivastava. 1970. Experimental production of early brain lesions in rats by parenteral administration of manganese chloride. Acta Pharmacol. Toxicol. 28( 3): 177 183.

Chandra, S. V. 1971. Cellular changes induced by manganese in the rat testis  preliminary results. Acta Pharmacol. Toxicol. 29( 1): 75 80.

Chandra, S. V. 1972. Histological and histochemical changes in experimental manganese encephalopathy in rabbits. Arch. Toxikol. 29( 1): 29 38.

Chandra, S. V., R. Ara, N. Nagar, et al. 1973. Sterility in experimental manganese toxicity. Acta Biol. Med. Ger. 30( 6): 857 862.

Chandra, S. V., D. K. Saxena and M. Z. Hasan. 1975. Effect of zinc on manganese induced testicular injury in rats. Ind. Health 13: 51 56.

Chandra, S. V., and G. S. Shukla. 1978. Manganese encephalopathy in growing rats. Environ Res 15: 28 37 ( as cited in ATSDR, 2000).

Chandra, S. V., G. S. Shukla and D. K. Saxena. 1979. Manganese induced behavioral dysfunction and its neurochemical mechanism in growing mice. J. Neurochem. 33( 6): 1217 1221.

Chandra, S. V. and G. S. Shukla. 1981. Concentrations of striatal catecholamines in rats given manganese chloride through drinking water. J. Neurochem. 36( 2): 683 687.

Chang, L. W. 1996. Toxico neurology and neuropathology induced by metals. In: Chang, L. W., ed,. Toxicology of Metals. Boca Raton, FL: CRC Press, Inc., pp. 511 536.

ChemIDplus. 2000. Division of Specialized Information Services, National Library of Medicine ( NLM) http:// chem. sis. nlm. nih. gov/ chemidplus/

Cikrt, M. and J. Vostal. 1969. Study of manganese resorption in vitro through intestinal wall. Int. Z. Klin. Pharmakol. Ther. Toxikol . 2( 3): 280 285. 10 5 Manganese   February 2003 Cikrt, M. 1973. Enterohepatic circulation of 64Cu, 52Mn and 203Hg in rats. Arch. Toxikol. 31( 1): 51 59.

Clayton, C., E. Pellizzari, R. Rodes, et al. 1999. Estimating distribution of long term particulate matter and manganese exposures for residents of Toronto, Canada. Atmos. Environ. 33: 2515 2526.

Collipp, P. J., Chen, S. Y., and Maitinsky, S. 1983. Manganese in infant formulas and learning disability. Ann. Nutr. Metab. 27 ( 6): 488 494.

Colomina, M. T., J. L. Domingo, J. M. Llobet, et al. 1995. Embryotoxicity and fetotoxicity of manganese in mice: variability with the day of exposure. Toxicologist 15( 1): 160 [ abstract].

Cooper, W. 1984. The health implications of increased manganese in the environment resulting from the combustion of fuel additives: a review of the literature. J. Toxicol. Envir. Hlth. 14: 23 46.

Cotzias, G. C. 1958. Manganese in health and disease. Physiol. Rev. 38: 503 533.

Cotzias, G. C., K. Horiuchi, S. Fuenzalida, et al. 1968. Chronic manganese poisoning. Clearance of tissue manganese concentrations with persistence of the neurological picture. Neurology 18( 4): 376 382.

Cotzias, G. C., S. T. Miller, P. S. Papavasiliou, et al. 1976. Interactions between manganese and brain dopamine. Med. Clin. North Am. 60( 4): 729 738.

Dagli, A. J., D. Golden, M. Finkel, et al. 1973. Pyloric stenosis following ingestion of potassium permanganate. Digest Dis. 18: 1091 1094.

Dastur, D. K., D. K. Manghani, K. V. Raghavendran, et al. 1969. Distribution and fate of Mn54 in the rat, with special reference to the C. N. S. Q. J. Exp. Physiol. 54( 3): 322 331.

Dastur, D. K., D. K. Manghani and K. V. Raghavendran. 1971. Distribution and fate of 54Mn in the monkey: Studies of different parts of the central nervous system and other organs. J. Clin. Invest. 50: 9 20.

Davidsson, L., A. Cederblad, B. Lönnerdal, et al. 1989a. Manganese absorption from human milk, cow's milk, and infant formulas in humans. Am. J. Dis. Child 43( 7): 823 827.

Davidsson, L., A. Cederblad, B. Lönnerdal, et al. 1989b. Manganese retention in man: a method for estimating manganese absorption in man. Am. J. Clin. Nutr. 49( 1): 170 179.

Davies, N. T. and R. Nightingale. 1975. The effects of phytate on intestinal absorption and secretion of zinc, and whole body retention of Zn, copper, iron and manganese in rats. Br. J. Nutr. 34( 2): 243 258. 10 6 Manganese   February 2003 Davis, C. D., D. M. Ney and J. L. Greger. 1990. Manganese, iron and lipid interactions in rats. J. Nutr. 120( 5): 507 513 ( as cited in ATSDR, 2000).

Davis, C. D. and J. L. Greger. 1992. Longitudinal changes of manganese dependent superoxide dismutase and other indexes of manganese and iron status in women. Am. J. Clin. Nutr. 55( 3): 747 752 ( as cited in ATSDR, 2000).

Davis, C. D., E.. A . Malecki, and J. L. Greger. 1992. Interactions among dietary manganese, heme iron, and non heme iron in women. Am. J. Clin. Nutr. 56: 926 932.

Davis, C. D., L. Zech, J. L. Greger. 1993. Manganese metabolism in the rats: An improved methodology for assessing gut endogenous losses. Proc. Soc. Exp. Biol. Med. 202: 103 108.

Davis, J. M, A. M. Jarabek, D. T. Mage and J. A. Graham. 1998. The EPA health risk assessment of methylcyclopentadienyl manganese tricarbonyl ( MMT). Risk Anal. 18( 1): 57 70.

De Meo, M., M. Laget, M. Castegnaro, et al. 1991. Genotoxic activity of potassium permanganate in acidic solutions. Mutat. Res. 260( 3): 295 306 ( as cited in ATSDR, 2000).

Demerec, M., G. Bertani and J. Flint. 1951. A survey of chemicals for mutagenic action on E. coli. Am. Nat. 85: 119 136.

Deskin, R., S. J. Bursian and F. W. Edens. 1980. Neurochemical alterations induced by manganese chloride in neonatal rats. Neurotoxicology 2: 65 73.

Deskin, R., S. J. Bursian and F. W. Edens. 1981. The effect of chronic manganese administration on some neurochemical and physiological variables in neonatal rats. Gen. Pharmacol. 12( 4): 279 280.

Devenyi, A. G., T. F. Barron and A. C. Mamourian. 1994. Dystonia, hyperintense basal ganglia, and high whole blood manganese levels in Alagille's syndrome. Gastroenterology 106( 4): 1068 1071 ( as cited in U. S. EPA, 1999a).

Dieter, H. H., W. Rotard, J. Simon,. et al. 1992. Manganese in natural mineral waters from Germany. Die Nahrung 36: 477 484.

Dikshith, T. S. and S. V. Chandra. 1978. Cytological studies in albino rats after oral administration of manganese chloride. Bull. Environ. Contam. Toxicol. 19( 6): 741 746 ( as cited in ATSDR, 2000).

DiPaolo, J. A. 1964. The potentiation of lymphosarcomas in mice by manganese chloride [ Abstract]. Fed. Proc. 23: 393.

Doisy, E. A. 1973. Effects of deficiency in manganese upon plasma levels of clotting proteins and cholesterol in man. Trace Element Metabolism. In: Hoekstra, W. G., J. W. Suttie, A. E. 10 7 Manganese   February 2003 Ganther, et al., eds. Animals 2, 2nd eds. Baltimore: University Park Press, pp. 668 670 ( as cited in ATSDR, 2000).

Dorman, D. C., M. F. Struve, D. Vitarella, F. L. Byerly, J. Goetz, and R. Miller. 2000. Neurotoxicity of manganese chloride in neonatal and adult CD rats following subchronic ( 21 day) high dose oral exposure. J Appl Tox 20: 179 187.

Dörner, K., S. Dziadzka, A. Höhn, E. Sievers, H. D. Oldgix, G. Schulz Lell, and J. Schaub. 1989. Longitudinal manganese and copper balances in young infants and preterm infants fed on breastmilk and adapted cow's milk formulas. Br J Nutrition 61: 559 572.

Durham, N. N. and O. Wyss. 1957. Modified method of determining mutation rates in bacteria. J. Bacteriol. 74: 548 552.

Ejima, A., T. Imamura, S. Nakamura, et al. 1992. Manganese intoxication during total parenteral nutrition [ letter]. Lancet 339: 426.

Eriksson, H., S. Lenngren and E. Heilbronn. 1987. Effect of long term administration of manganese on biogenic amine levels in discrete striatal regions of rat brain. Arch. Toxicol. 59( 6): 426 431.

Eriksson, H., J. Tedroff, K. A. Thuomas, et al. 1992. Manganese induced brain lesions in Macaca fascicularis as revealed by positron emission tomography and magnetic resonance imaging. Arch. Toxicol. 66( 6): 403 710 ( as cited in ATSDR, 2000).

Ethyl Corporation. 1990. The case for an environmentally beneficial fuel additive. Baton Rouge, USA.

Ethyl Corporation. 1997. Characterization of manganese particulates from studies performed by Lawrence Livermore National Laboratory, Southwest Research Institute, Univ. of Minnesota, Research Triangle Institute, and Ethyl Research and Development. September 10, 1997. Submitted to U. S. EPA. ( as cited in Lynam et al., 1999).

Fechter, L. D. 1999. Distribution of manganese in development. Neurotoxicology 20: 197 201.

Fell, J. M. E., A. P. Reynolds, N. Meadows, et al. 1996. Manganese toxicity in children receiving long term parenteral nutrition. Lancet 347: 1218 1221.

Finley, J. W. 1999. Manganese absorption and retention by young women is associated with serum ferritin concentration. Am. J. Clin. Nutr. 70: 37 43.

Finley, J. and C. D. Davis. 1999. Manganese deficiency and toxicity: Are high or low dietary amounts of manganese cause for concern? Biofactors 10( 1): 15 24.

Finley, J. W., P. E. Johnson and L. K. Johnson. 1994. Sex affects manganese absorption and retention by humans from a diet adequate in manganese. Am. J. Clin. Nutr. 60( 6): 949 955. 10 8 Manganese   February 2003 Florence, T. M. and J. L. Stauber. 1989. Manganese catalysis of dopamine oxidation. Sci. Total. Environ. 78: 233 240.

Fore, H. and R. A. Morton. 1952. Manganese in rabbit tissues. Biochem. J. 51: 600 603 ( as cited in ATSDR, 2000).

Franz, R. D. 1962. Toxicities of some trace metals. Naunyn Schmiedebergs Arch. Exp. Path. Pharmakol. 244: 17 20.

Freeland Graves, J. 1994. Derivation of manganese estimated safe and adequate daily dietary intakes. In: Mertz, W., C. O. Abernathy and S. S. Olin, eds., Risk Assessment of Essential Elements. Washington, D. C.: ILSI Press, pp. 237 252.

Freeland Graves, J. H., C. W. Bales and F. Behmardi. 1987. Manganese requirements of humans. In: Kies, C., ed., Nutritional Bioavailability of Manganese. Washington, D. C.: American Chemical Society, pp. 90 104.

Freeland Graves, J. H. and C. Llanes. 1994. Models to study manganese deficiency. In: Klimis Tavantzis, D. J., ed., Manganese in Health and Disease. Boca Raton, FL: CRC Press, Inc., pp. 59 86.

Friedman, B. J., J. H. Freeland Graves, C. W. Bales, et al. 1987. Manganese balance and clinical observations in young men fed a manganese deficient diet. J. Nutr. 117: 133 143.

Furst, A. 1978. Tumorigenic effect of an organomanganese compound on F344 rats and Swiss albino mice. J. Natl. Cancer Inst. 60( 5): 1171 1173.

Garcia Aranda, J. A., R. A. Wapnir and F. Lifshitz. 1983. In vivo intestinal absorption of manganese in the rat. J. Nutr. 113( 12): 2601 2607.

Gavin, C. E., K. K. Gunter and T. E. Gunter. 1992. Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation. Toxicol. Appl. Pharmacol. 115( 1): 1 5 ( as cited in ATSDR, 2000).

Gianutsos, G. and M. T. Murray. 1982. Alterations in brain dopamine and GABA following inorganic or organic manganese administration. Neurotoxicology 3( 3): 75 81.

Gianutsos, G., M. D. Seltzer, R. Saymeh, et al. 1985. Brain manganese accumulation following systemic administration of different forms. Arch. Toxicol. 57( 4): 272 275.

Gibbons, R. A., S. N. Dixon, K. Hallis, et al. 1976. Manganese metabolism in cows and goats. Biochim. Biophys. Acta 444: 1 10.

Gibson, R. S. 1994. Content and bioavailability of trace elements in vegetarian diets. Am J Clin Nutr 59( 5 Suppl): 1223S 1232S. 10 9 Manganese   February 2003 Gilliom, R. J., D. K. Mueller, and L. H. Nowell. 1998. Methods for comparing water quality conditions among National Water Quality Assessment Study Units, 1992 95. U. S. Geological Survey Open File Report 97 589. Available on the Internet at: URL: http:// ca. water. usgs. gov/ pnsp/ rep/ ofr97589/. Last modified October 09, 1998.

Goldsmith, J., Y. Herishanu, J. Abarbanel, et al. 1990. Clustering of Parkinson's disease points to environmental etiology. Arch. Environ. Health 45: 88 94 ( as cited in ATSDR, 2000).

Gong, H. and T. Amemiya. 1996. Ultrastructure of retina of manganese deficient rats. Invest. Ophthalmol. Vis. Sci. 37: 1967 1974.

Gottschalk, L. A., T. Rebello, M. S. Buchsbaum, et al. 1991. Abnormalities in hair trace elements as indicators of aberrant behavior. Compr. Psychiatry 32( 3): 229 237.

Grant, D., W. F. Blazak and G. L. Brown. 1997. The reproductive toxicology of intravenously administered MnDPDP in the rat and rabbit. Acta Radiol. 38( 4 Pt 2): 759 769.

Gray, L. E., Jr. and J. W. Laskey. 1980. Mulitvariate analysis of the effects of manganese on the reproductive physiology and behavior of the male house mouse. J. Toxicol. Environ. Health 6: 861 867.

Greenberg, D. M. and W. W. Campbell. 1940. Studies in mineral metabolism with the aid of induced radioactive isotopes. IV. Manganese. Proc. Natl. Acad. Sci. 26: 448 452.

Greenberg, D. M., D. H. Copp and E. M. Cuthbertson. 1943. Studies in mineral metabolism with the aid of artificial radioactive isotopes. VII. The distribution and excretion, particularly by way of the bile, of iron, cobalt, and manganese. J. Biol. Chem. 147: 749.

Greger, J. L. 1998. Dietary standards for manganese: Overlap between nutritional and toxicological studies. J. Nutr. 128: 368S 371S.

Greger, J. L. 1999. Nutrition versus toxicology of manganese in humans: Evaluation of potential biomarkers. Neurotoxicology 20: 205 212.

Greger, J. L. and S. M. Snedeker. 1980. Effect of dietary protein and phosphorus levels on the utilization of zinc, copper, and manganese by adult males. J. Nutr. 110: 2243 2253.

Greger, J. L., C. D. Davis, J. W. Suttie, et al. 1990. Intake, serum concentrations, and urinary excretion of manganese by adult males. Am. J. Clin. Nutr. 51( 3): 457 461.

Gruden, N. 1984. The influence of iron on manganese metabolism in the first three weeks of rat's life. Nutr. Rep. Int. 30: 553 557.

Gupta, S. K., R. C. Murthy and S. V. Chandra. 1980. Neuromelanin in manganese exposed primates. Toxicol. Lett. 6: 17 20. 10 10 Manganese   February 2003 Hagenfeldt, K., L. O. Plantin and E. Diczfalusy. 1973. Trace elements in the human endometrium. 2. Zinc, copper and manganese levels in the endometrium, cervical mucus and plasma. Acta Endocrinol. ( Copenh.) 72( 1): 115 126.

Hamilton Koch, W., R. D. Snyder and J. M. Lavelle. 1986. Metal induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells. Chem. Biol. Interact. 59( 1): 17 28.