Abstract

Manganese (Mn) is an essential element that is neurotoxic under certain exposure conditions. Monkeys and humans exposed to Mn develop similar neurological effects; thus, an improved understanding of the dose-response relationship seen in nonhuman primates could inform the human health risk assessment for this essential metal. A previous analysis of this dose-response relationship in experimental animals (Gwiazda, R., Lucchini, R., and Smith, D., 2007, Adequacy and consistency of animal studies to evaluate the neurotoxicity of chronic low-level manganese exposure in humans, J. Toxicol. Environ. Health Part A70, 594–605.) relied on estimates of cumulative intake of Mn as the sole measure for comparison across studies with different doses, durations, and exposure routes. In this study, a physiologically based pharmacokinetic model that accurately accounts for the dose dependencies of Mn distribution was used to estimate increases in brain Mn concentrations in monkeys following Mn exposure. Experimental studies evaluated in the analysis included exposures by inhalation, oral, iv, ip, and sc dose routes, and spanned durations ranging from several weeks to over 2 years. This analysis confirms that the dose-response relationship for the neurotoxic effects of Mn in monkeys is independent of exposure route and supports the use of target tissue Mn concentration or cumulative target tissue Mn as the appropriate dose metric for these comparisons. These results also provide strong evidence of a dose-dependent transition in the mode of action for the neurological effects of Mn that needs to be considered in risk assessments for this essential metal.

Manganese (Mn) is an essential nutrient that is required for many physiological functions. Mn occurs naturally in soil, water, and air, leading to a widespread presence in the environment. The primary source of Mn exposure is through the diet. Adult dietary intake in people has been estimated to range from 1 to 10mg Mn/day with only a small fraction (1–5%) absorbed by the gastrointestinal (GI) tract (Aschner et al., 2005; ATSDR, 2000). Homeostatic controls regulate intestinal absorption and biliary excretion to ensure adequate and stable tissue Mn concentrations and to prevent Mn deficiency or toxicity despite mild fluctuations in daily exposure levels.

As with other essential elements, Mn may induce toxicity under certain high-dose exposure conditions. Adverse neurological, reproductive, and respiratory effects characterize Mn toxicity in humans. Mn-induced neurotoxicity is of particular concern and is considered one of the most sensitive endpoints (Aschner et al., 2005). Early manifestations of Mn overexposure include fatigue, headache, muscle cramps, loss of appetite, apathy, insomnia, and diminished libido. As overexposure continues and the disease progresses, patients may develop prolonged muscle contractions (dystonia), decreased muscle movement (hypokinesia), rigidity, and muscle tremors (Pal et al., 1999). Structural changes in the globus pallidus of Mn-exposed people indicate that this brain region is a target site for Mn accumulation and effects (Perl and Olanow, 2007). Individuals with chronic Mn neurotoxicity resemble patients with Parkinson’s disease; however, unlike in Parkinson’s disease, the substantia nigra is largely spared and dopamine levels remain generally unaffected during Mn neurotoxicity (Guilarte, 2010).

Mn-induced neurological effects have been reported from studies of occupational exposure to high inhaled (> 0.2mg/m3) Mn concentrations (Myers et al., 2003; Roels et al., 1987), environmental studies of residents near ferroalloy plants (Lucchini et al., 2007), individuals exposed to Mn-laden drinking water (Bouchard et al., 2011; Kawamura et al., 1941; Kondakis et al., 1989), and from people with prolonged use of total parenteral nutrition formulations that contain Mn (Alves et al., 1997; Fell et al., 1996). These observations suggest that brain Mn concentration is the critical determinant for Mn neurotoxicity, regardless of exposure route. This is an important distinction to consider when assessing long-term health risks from chronic, low-level environmental Mn exposure.

Studies on Mn neurotoxicity have been conducted in rodents and nonhuman primates using various exposure routes and a wide range of doses and durations. Gwiazda et al. (2007) conducted a review of subchronic to chronic rodent and nonhuman primate studies to determine whether a consistent dose-response relationship existed among different studies. One goal of Gwiazda et al.’s work was to determine whether animal studies could be used to evaluate the neurotoxicity of chronic low-level Mn exposures in humans. They estimated internal cumulative Mn dose as “the total amount of Mn that was taken up into the circulatory system by the time the endpoint was detected.” The range of exposures associated with adverse changes was profound (over two orders of magnitude) in the animal studies evaluated by Gwiazda et al., leading them to conclude that most existing animal studies might be of limited relevance for the risk assessment of chronic low-level Mn exposure to humans.

Since Gwiazda et al.’s study was published, several physiologically based pharmacokinetic (PBPK) models for Mn have been reported (Nong et al., 2009; Schroeter et al., 2011; Yoon et al., 2011). These PBPK models effectively simulate Mn tissue kinetics from inhaled, oral, and parenteral Mn intake (Andersen et al., 2010). This multi–dose route capability is achieved by incorporating homeostatic control processes, saturable tissue binding capacities, and preferential fluxes in various tissue regions (see Taylor et al., 2012 for review). PBPK models have been widely used in risk assessment of chemical compounds to support extrapolation across species, high to low doses, and across exposure routes and to allow for calculation of target tissue dose, which is more biologically relevant than administered dose, exposure concentration, or estimated systemic dose as a metric for predicting toxic outcomes.

The purpose of this study was to apply PBPK modeling approaches to a subset of the studies initially identified by Gwiazda et al. (2007). We focused our attention on work performed in nonhuman primates because unlike rodents, Mn-exposed monkeys develop regionally selective increases in brain Mn concentrations (Aschner et al., 2005; Dorman et al., 2006b; Eriksson et al., 1992b; Newland et al., 1989) and behavioral effects similar to those seen in Mn-affected humans (Olanow et al., 1996). To this end, we used a PBPK model developed specifically for rhesus monkeys (Schroeter et al., 2011) to simulate the exposure scenarios from each study and to predict the corresponding increases in brain Mn concentrations for various dose routes, exposure concentrations, and durations.

Materials and Methods

Study Selection

The 15 nonhuman primate studies identified by Gwiazda et al. (2007) were used as a starting point for this analysis. Three of the studies identified by Gwiazda used cebus monkeys (Cebus spp.) or squirrel monkeys (Saimiri sciureus) (Neff et al., 1969; Newland and Weiss, 1992; Ulrich et al., 1979). These studies were excluded from our analysis because the PBPK models were developed and calibrated using pharmacokinetic data obtained in rhesus monkeys (Macaca mulatta) or phylogenetically similar macaque relatives. A fourth excluded study (Van Bogaert and Dallemagne, 1946) used macaques but included an inhalation coexposure to sodium perchlorate, thus confounding our interpretation of the reported clinical signs. In addition to the studies identified by Gwiazda, we also included two recent studies (Dorman et al., 2006a; Guilarte et al., 2006) and the report by Coulston and Griffin (1976). The study by Chandra et al. (1979) was also included for model calibration although clinical effects were not reported. In the end, 15 studies were identified in our analysis (summarized in Table 1). Exposure routes for these studies included inhalation, oral delivery, and iv, ip, sc, and im injection. Different forms of Mn were used in the exposures and included both soluble (e.g., MnCl2 and MnSO4) and relatively insoluble (e.g., MnO2 and Mn3O4) forms of Mn. These studies also included a wide range of Mn dose levels, dose frequencies, and exposure durations.

TABLE 1

Summary of Mn Exposure Studies in Rhesus Monkeys

Study Mn form Dose route Body weight (kg) Dosing regimen 
Chandra et al. (1979) MnCl2 oral 5.6mg/kg Mn in MnCl2 solution given once daily for 18 months 
Gupta et al. (1980) MnCl2 oral 5–6 7.0mg/kg Mn in MnCl2 solution given once daily for 18 months 
Van Bogaert and Dallemagne (1946) MnSO4 oral 2.85 1.3–6.3mg/kg Mn in MnSO4 solution given once daily for 292 days 
Mella (1924) MnCl2 ip 5a 0.5–2.2mg/kg Mn given every other day for up to 14 months 
Olanow et al. (1996) MnCl2 iv 12–18 2.8–4.0mg/kg Mn given by iv injection 7 times at ~1-week intervals 
Guilarte et al. (2006)/Schneider et al. (2006) MnSO4 iv 6.5 3.6–5.4mg/kg Mn injected weekly for approximately 40 weeks 
Suzuki et al. (1975) MnO2 sc 3.5–4.5 Up to 158mg/kg Mn injected once a week for 9 weeks 
Eriksson et al. (1987) MnO2 sc 3.5–4.5 Approximately 112mg/kg Mn injected on 18 occasions over 5 months 
Eriksson et al. (1992a) MnO2 sc 4.5–6.0 38mg/kg Mn injected on 13 occasions over 26 months 
Eriksson et al. (1992b) MnO2 sc 4–5 89mg/kg Mn injected on 11 occasions over 4 months 
Pentschew et al. (1963) MnO2 im 5a Single doses of 252 and 441mg/kg Mn injected 2 months apart 
Bird et al. (1984) MnO2 inhalation 2–3 Exposure to 30mg Mn/m3 for 6h/day, 5 days/week for 2 years 
Nishiyama et al. (1977) MnO2 inhalation 3–4 Exposure to 0.7 or 3.0mg Mn/m3 for 22h/day, 7 days/week for 10 months 
Coulston and Griffin (1976) Mn3O4 inhalation 2.75–6.5 Exposure to 0.1mg Mn/m3 for 23h/day, 7 days/week for up to 15 months or to 5.0mg Mn/m3 for 23h/day, 7 days/week for 23 weeks 
Dorman et al. (2006) MnSO4 inhalation 2.5 Exposure to 0.06, 0.3, or 1.5mg Mn/m3 for 6h/day, 5 days/week for 90 days 
Study Mn form Dose route Body weight (kg) Dosing regimen 
Chandra et al. (1979) MnCl2 oral 5.6mg/kg Mn in MnCl2 solution given once daily for 18 months 
Gupta et al. (1980) MnCl2 oral 5–6 7.0mg/kg Mn in MnCl2 solution given once daily for 18 months 
Van Bogaert and Dallemagne (1946) MnSO4 oral 2.85 1.3–6.3mg/kg Mn in MnSO4 solution given once daily for 292 days 
Mella (1924) MnCl2 ip 5a 0.5–2.2mg/kg Mn given every other day for up to 14 months 
Olanow et al. (1996) MnCl2 iv 12–18 2.8–4.0mg/kg Mn given by iv injection 7 times at ~1-week intervals 
Guilarte et al. (2006)/Schneider et al. (2006) MnSO4 iv 6.5 3.6–5.4mg/kg Mn injected weekly for approximately 40 weeks 
Suzuki et al. (1975) MnO2 sc 3.5–4.5 Up to 158mg/kg Mn injected once a week for 9 weeks 
Eriksson et al. (1987) MnO2 sc 3.5–4.5 Approximately 112mg/kg Mn injected on 18 occasions over 5 months 
Eriksson et al. (1992a) MnO2 sc 4.5–6.0 38mg/kg Mn injected on 13 occasions over 26 months 
Eriksson et al. (1992b) MnO2 sc 4–5 89mg/kg Mn injected on 11 occasions over 4 months 
Pentschew et al. (1963) MnO2 im 5a Single doses of 252 and 441mg/kg Mn injected 2 months apart 
Bird et al. (1984) MnO2 inhalation 2–3 Exposure to 30mg Mn/m3 for 6h/day, 5 days/week for 2 years 
Nishiyama et al. (1977) MnO2 inhalation 3–4 Exposure to 0.7 or 3.0mg Mn/m3 for 22h/day, 7 days/week for 10 months 
Coulston and Griffin (1976) Mn3O4 inhalation 2.75–6.5 Exposure to 0.1mg Mn/m3 for 23h/day, 7 days/week for up to 15 months or to 5.0mg Mn/m3 for 23h/day, 7 days/week for 23 weeks 
Dorman et al. (2006) MnSO4 inhalation 2.5 Exposure to 0.06, 0.3, or 1.5mg Mn/m3 for 6h/day, 5 days/week for 90 days 

a Body weights were not reported in the study, so a 5kg body weight for an adult rhesus monkey was assumed for model simulations.

Assessment of Clinical Signs

Clinical signs were evaluated using modifications of clinical sign scales developed for monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–induced parkinsonism (Imbert et al., 2000; Kurlan et al., 1991). Evaluations were based upon the original data and descriptions as published in the studies included in this review (Table 1). Scoring of clinical signs was performed by a veterinarian with expertise in neurotoxicology (David D. Dorman). The following effect categories and severity scoring system were used to assess the monkey studies:

  • Coordination

  • 0—normal

  • 1—very slight to no loss of balance

  • 2—moderate lapses in balance

  • 3—frequent and major lapses in balance

  • Bradykinesia

  • 0—normal speed and facility of movement

  • 1—mild (25–50%) reduction

  • 2—moderate (51–75%) reduction

  • 3—severe (> 75%) reduction

  • Rigidity/Gait disorders

  • 0—normal

  • 1—walks slowly and with difficulty

  • 2—markedly impaired; ambulate slowly +/− effort

  • 3—severe decrease in ability to ambulate

  • Postural instability

  • 0—absent

  • 1—minor

  • 2—marked

  • Muscle tremors

  • 0—absent

  • 1—mild

  • 2—moderate

  • 3—severe

  • Other neurological clinical signs (included CNS excitation and hyperactivity)

  • 0—none

  • 1—mild

  • 2—moderate

  • 3—severe

The severity scores for the categories listed above were summed to obtain a final severity score for each dose group in the studies listed in Table 1. The minimum possible score was 0 (no effect) and the maximum possible score was 17. If a sufficient description of a change in clinical signs was reported during the course of the exposure, then severity scores at multiple time points were used in the analysis.

Mn PBPK Model

The Mn PBPK model for rhesus monkeys was previously described in detail (Nong et al., 2009; Schroeter et al., 2011), so only a brief description is given of some of the important aspects of the model. The PBPK model contained compartments for the liver, lung, nasal cavity, bone, blood, and brain regions (Fig. 1). All other body tissues were combined into a single compartment. The brain was divided into compartments representing blood, globus pallidus, cerebellum, olfactory bulb, and pituitary gland. The globus pallidus, cerebellum, and pituitary gland were included because they accumulate Mn to varying degrees during inhalation exposure. The olfactory bulb was included because it receives direct transport from olfactory epithelium during Mn inhalation (Aschner et al., 2005; Dorman et al., 2006a,b).

Fig. 1.

Schematic of the Mn PBPK model for nonhuman primates. The PBPK model can accommodate the following Mn exposure routes: inhalation, diet/oral, ip, iv, and sc. Tissue-specific binding processes were controlled by association and dissociation parameters (ka and kd) and the binding capacity (Btissue). Diffusion rate constants (kin and kout) control increases in free Mn in brain regions. Qtissue parameters refer to tissue blood flow rates.

Fig. 1.

Schematic of the Mn PBPK model for nonhuman primates. The PBPK model can accommodate the following Mn exposure routes: inhalation, diet/oral, ip, iv, and sc. Tissue-specific binding processes were controlled by association and dissociation parameters (ka and kd) and the binding capacity (Btissue). Diffusion rate constants (kin and kout) control increases in free Mn in brain regions. Qtissue parameters refer to tissue blood flow rates.

Tissue Mn concentrations represent the sum of free Mn and bound forms within the tissues. Free Mn circulates in the blood throughout the body; the bound form is stored in tissue constituents. Tissue binding is a reversible, saturable process that likely represents incorporation of specific macromolecular Mn stores. The incorporation of Mn into tissue constituents is governed by tissue-specific association and dissociation rate constants (ka and kd). The distribution between bound and free Mn is determined by the dissociation binding ratio (kd/ka) and the tissue-specific maximum binding capacity (Bmax), which regulates the storage capacity of bound Mn in each tissue. Saturation of the binding capacity with increased exposure leads to increases in free Mn and subsequent increases in total tissue Mn. Following cessation of exposure, free Mn is rapidly cleared from the body and tissue Mn concentrations return to their basal levels.

The differential rise in free Mn concentration in brain regions was described with tissue-specific diffusion rate constants (kin and kout) to describe diffusion-limited flux between brain blood and brain tissues. The influx rates for the globus pallidus and pituitary regions were dose dependent in order to accurately describe changes in Mn concentration in these brain regions with increased Mn exposure. Increases in free Mn concentration in brain regions depend on asymmetric diffusional clearance rates, binding rate constants, and the binding capacity of each tissue. Tissue Mn binding capacity and diffusion parameters were calibrated by Schroeter et al. (2011) to the pharmacokinetic data from Mn-exposed monkeys in Dorman et al. (2006a) to simulate the rise in tissue Mn levels following the onset of inhalation exposure, followed by a return to basal levels after the exposure period. These parameters were unchanged for all simulations in this study.

A series of gut compartments were included in the PBPK model to track Mn absorption by the GI tract (Fig. 1). For dietary Mn, a small fraction (Fdietup) was absorbed by the GI tract and directed to the liver for systemic circulation. The remaining fraction of Mn in the gut lumen (1−Fdietup) was either transiently stored in gut epithelial cells (Fent) or transferred to the lower GI tract lumen (1−Fent) and excreted through the feces. Mn transfer from the gut lumen to the portal blood was reflected by the rate constant kGI. Mn transfer from the gut epithelium to the lower GI tract occurred by sloughing of enterocytes from the epithelial layer (kent). The stored Mn in enterocytes was not available to the systemic circulation due to rapid enterocyte turnover and was excreted into feces.

The elimination of Mn from the body occurs via biliary excretion in the liver. Biliary excretion was governed by the rate constant, kbile, which was induced by blood concentration to reflect the dose-dependent behavior of Mn elimination (Nong et al., 2009):

 
formula

where kbile0 is the basal biliary excretion rate constant, kbmax is the maximal increase in the excretion rate constant, kb50 is the arterial concentration at half the induced level of Mn in the arterial blood (Cart), and n is the slope factor. Arterial blood concentration (Cart) was used as a surrogate for free liver Mn concentration because Mn blood levels were directly measured in exposed monkeys (Dorman et al., 2006a).

As described in Schroeter et al. (2011), fractional dietary absorption (Fdietup = 0.0021) and the biliary excretion rate constant (kbilec = 0.051) were calibrated by fitting basal Mn tissue concentrations to tissue levels from the control monkeys in Dorman et al. (2006a). Parameters governing GI absorption of Mn were calibrated using clearance data of oral doses of 54Mn (Furchner et al., 1966). Biliary induction parameters were originally estimated by Nong et al. (2009) for low-dose Mn inhalation exposures (Dorman et al., 2006a), where a maximum induction factor of kbmax = 2.5 was sufficient to describe the increase in biliary excretion following Mn exposure. However, most of the studies that were considered in this analysis used much higher Mn doses, so a larger maximal induction factor was needed to simulate increased biliary excretion rates following increased Mn intake. For all of the simulations in this study, the parameter kbmax was increased to 20.0 and the other parameters in the biliary induction equation were modified accordingly (kb50 = 0.105, n = 2.1) to remain consistent with the low-dose behavior described by Nong et al. (2009). Studies in rats have shown that the biliary excretion of Mn is saturable, indicating a carrier-mediated transport mechanism (Ballatori et al., 1987; Klaassen, 1974). To date, the transport protein(s) responsible for transporting Mn from hepatocytes into bile remain unknown. Because of known interaction between Mn and iron, it is plausible that Mn may share iron efflux transporters including ferroportin (Fpn1/Slc40a1), divalent metal transporter-1 (DMT-1), or copper ATPase (Atp7a or Atp7b) (Abboud and Haile, 2000; Goss et al., 2008). The eightfold increase in kbmax is consistent with studies evaluating Atp7a and DMT-1 hepatic expression in a rodent model of iron deficiency (which mimics Mn toxicity) (Jiang et al., 2011).

Simulation of Mn Exposure Routes

The Mn PBPK model included the exposure routes that have been used in experimental studies (Table 1). For all exposures, the nominal dose used in modeling simulations was calculated from the Mn atomic weight fraction in the administered solution (or exposure concentration). Typical commercial diets provided to research monkeys contain 70 to 100 ppm Mn (Knapka et al., 1995). All simulations were, therefore, conducted with monkeys maintained on an 80 ppm diet, simulated as a continuous dietary exposure. The one exception was the study by Dorman et al. (2006a), in which a 133 ppm diet was specified. Simulated exposures followed the dosing schedules precisely as described in the studies (e.g., dosing every day, every other day, once a week, etc.). All model parameters governing internal Mn kinetics, such as diffusion constants, biliary induction parameters, and GI absorption constants, remained unchanged for all of the simulations in this study, regardless of exposure route.

Oral exposure. Oral Mn exposures were given once daily in either MnCl2 or MnSO4 solutions (Chandra et al., 1979; Gupta et al., 1980; Van Bogaert and Dallemagne, 1946). For PBPK model simulations, these exposures were treated as a daily bolus dose and intestinal absorption was modeled in the same way as dietary Mn exposure. Even though GI absorption of Mn may decrease with time in reaction to increased oral dosage levels, uncertainties surrounding the timing and magnitude of this change led us to keep Fdietup constant. This decision also reflected the desire to maintain the most conservative scenario by assuming that Fdietup did not decrease with increased Mn oral doses.

Intraperitoneal injection. Following the dosing schedule given by Mella (1924), which was the only study in this analysis to use the ip route, injections of MnCl2 solution were given every other day with gradually increased dosage levels. Injection of Mn solution by the ip route was simulated by assuming that the majority of the ip dose was directly absorbed in the liver and a small fraction (Fip) was absorbed into the peritoneal cavity for slow release into the liver (Fig. 1). The rate constant kip governs the movement of Mn from the peritoneal cavity to the liver. The parameters Fip =0.03 and kip = 1.0×10−7 h−1 were previously calibrated to the whole-body elimination data of 54Mn given by ip injection (Dastur et al., 1971), as described by Schroeter et al. (2011). These parameters were unchanged for simulations of the Mella study.

Intravenous injection. Dosing for iv studies was typically once per week (Guilarte et al., 2006; Olanow et al., 1996) although longer durations between injections were sometimes needed depending on the state of the animal. For model simulations, the iv dose was input directly into venous blood (Fig. 1).

sc injection. Studies on sc administration used MnO2 powder mixed with saline, olive oil, or water for injection into sc tissue (Eriksson et al., 1987, 1992a,b; Suzuki et al., 1975). For model simulations, the entire Mn dose from the sc injection went into a compartment representing sc tissue (Fig. 1). Mn delivery by im injection (Pentschew et al., 1963) was treated the same as a sc injection. The rate constant ksc = 0.0007h−1, which governs the release of Mn from the sc tissue into the venous blood, and the bioavailability of MnO2 at the injection site (42%) were calibrated by fitting model predictions of globus pallidus Mn concentrations to the data from Suzuki et al. (1975) and Eriksson et al. (1987), where globus pallidus Mn concentrations were measured at multiple time points following sc Mn injections. These model parameters were kept constant for all sc exposure simulations.

Inhalation exposure. Inhalation exposures were simulated using the exposure concentrations and schedules (e.g., days/week, hours/day) used in the experimental procedures (Bird et al., 1984; Coulston and Griffin, 1976; Dorman et al., 2006a; Nishiyama et al., 1977). Respiratory tract deposition was estimated for inhalation of MnSO4 particles with a mass median aerodynamic diameter of 2.0 µm, a geometric SD of 1.5 and a particle density of 2.95g/cm3 (Dorman et al., 2006a). Lung and nasal deposition efficiencies for these aerosol particles were estimated to be 40 and 27%, respectively (Schroeter et al., 2011). No distinction was made between deposition on tracheobronchial or pulmonary airways due to uncertainties regarding clearance and absorption rates from these lung regions. Nasal deposition was further partitioned onto respiratory and olfactory epithelium according to an airflow allocation of 91 and 9%, respectively (Kepler et al., 1998). For model simulations of the Dorman study, deposited Mn from MnSO4 particles was rapidly absorbed from lung tissues and nasal respiratory epithelium into the systemic circulation or transported from nasal olfactory epithelium to the olfactory bulb. For simulations of other studies that used MnO2 and Mn3O4 (Bird et al., 1984; Coulston and Griffin, 1976; Nishiyama et al., 1977), lung deposition was decreased to account for different particle size fractions and reduced bioavailability of less soluble Mn forms in lung tissues (Table 3).

TABLE 3

Summary of Aerosol Parameters and Lung Deposition Factors Used for the Simulation of Mn Inhalation Exposures

Study Mn form Particle size Density (g/cm3Exposure concentration (mg Mn/m3Deposition adjustment factor Reported increase in globus pallidus Mn concentration (%) Predicted increase in globus pallidus Mn concentration (%) 
Dorman et al. (2006a) MnSO4 MMAD = 2.0 µm, GSD = 1.5 2.95 1.5 1.0a 513 471 
Bird et al. (1984) MnO2 < 5 µm 5.0 30 0.0028b 79 80 
Nishiyama et al. (1977) MnO2 < 2 µm 5.0 0.7
3.0 
0.03b 133c
183c 
83
250 
Coulston and Griffin (1976) Mn3O4 < 6 µm 4.9 0.1
5.0 
0.4b 122c
Not reported 
107
990 
Study Mn form Particle size Density (g/cm3Exposure concentration (mg Mn/m3Deposition adjustment factor Reported increase in globus pallidus Mn concentration (%) Predicted increase in globus pallidus Mn concentration (%) 
Dorman et al. (2006a) MnSO4 MMAD = 2.0 µm, GSD = 1.5 2.95 1.5 1.0a 513 471 
Bird et al. (1984) MnO2 < 5 µm 5.0 30 0.0028b 79 80 
Nishiyama et al. (1977) MnO2 < 2 µm 5.0 0.7
3.0 
0.03b 133c
183c 
83
250 
Coulston and Griffin (1976) Mn3O4 < 6 µm 4.9 0.1
5.0 
0.4b 122c
Not reported 
107
990 

aThe Dorman et al. (2006a) study was used to estimate model parameters, so there was no need to further adjust lung deposition.

bDeposition adjustment factors were calibrated to reported globus pallidus Mn concentrations from the respective study.

cMn concentrations were reported in the basal ganglia. These values were used as surrogates for globus pallidus Mn concentrations.

Dose Metrics

PBPK model simulations were run from the beginning of the exposure period (time = 0) until exposures stopped or clinical outcomes were observed (time = T), as reported in each study. Simulations were run using AcslX version 3.0 (Aegis Technologies Group, Inc., Huntsville, AL). For each simulation, time-course profiles of tissue Mn concentrations following Mn exposure were evaluated. Mn concentrations in the globus pallidus were considered for the dose-response analysis because it is the likely target tissue for Mn-induced motor effects and is a conservative surrogate for other potential target brain regions that do not accumulate Mn as readily. The following three dose metrics for globus pallidus Mn concentration (Cgp(t)) were used to correlate with observed clinical effects in monkeys:

Peak concentration = graphic

Area under the curve (AUC) = graphic

Average concentration = graphic.

Categorical Regression

A regression analysis was performed using the U.S. EPA CatReg software (available for download at www.epa.gov/ncea). CatReg fits a cumulative probability distribution using the method of maximum likelihood estimation to estimate the probability of a specific severity level (i.e., a category) at any specified dose. The dose can be defined using any relevant dose metric. The dose-response behavior was evaluated by plotting the severity scores for each dose group from the Mn exposure studies versus the corresponding dose metrics of peak concentration, average concentration, and AUC in the globus pallidus from the PBPK model simulations.

RESULTS

Clinical Observations

In general, studies performed prior to the 1990s had limited histological, neurochemical, or neuropathological assessments. These studies generally relied on very high-dose Mn exposures, and monkeys often developed postural instability, increased excitability, hypoactivity, falling, muscular rigidity, intention and/or action tremors, and ataxia. Studies using lower Mn doses were often associated with hypoactivity, loss of fine motor control, action tremor, or deficits in working memory. The severity scores applied to each dose group in each of the studies are shown in Table 2. Using this scoring system, the maximum summed severity score in this analysis was 9. With the exception of the inhalation exposures, most studies either used one monkey in each dose group or reported effects for individual monkeys.

TABLE 2

Severity Scores Applied to Clinical Effects Observed in Mn Exposure Studies in Monkeys

Study Dose route Monkey ID Time until effect (days) Observation Sum 
Coordination Bradykinesia/ Gross motor skills Rigidity/Gait disorders Postural instability Muscle tremors Other clinical signs 
Gupta et al. (1980) oral Group I 540         
Van Bogaert and Dallemagne (1946) oral 292             
Mella (1924) ip 253         
255       
275       
370         
429       
444       
473     
196       
204       
110           
112         
144         
Olanow et al. (1996) iv 10     
12     
49             
Guilarte et al. (2006) iv Group 1 140           
196         
Suzuki et al. (1975) sc 14           
42         
21           
28       
42     
49           
56       
35         
56       
49           
42           
56         
63       
Eriksson et al. (1987) sc 90       
150       
210   
90     
150     
3 and 4 150       
Eriksson et al. (1992a) sc 189             
189           
Eriksson et al. (1992b) sc 1 and 2 120       
Pentschew et al. (1963) im 270         
435     
Bird et al. (1984) inhalation Group 1 730             
Nishiyama et al. (1977) inhalation 90       
300             
Coulston and Griffin (1976) inhalation Low dose 450             
High dose 161             
Dorman et al. (2006) inhalation Low, mid, and high exposure groups 90             
Study Dose route Monkey ID Time until effect (days) Observation Sum 
Coordination Bradykinesia/ Gross motor skills Rigidity/Gait disorders Postural instability Muscle tremors Other clinical signs 
Gupta et al. (1980) oral Group I 540         
Van Bogaert and Dallemagne (1946) oral 292             
Mella (1924) ip 253         
255       
275       
370         
429       
444       
473     
196       
204       
110           
112         
144         
Olanow et al. (1996) iv 10     
12     
49             
Guilarte et al. (2006) iv Group 1 140           
196         
Suzuki et al. (1975) sc 14           
42         
21           
28       
42     
49           
56       
35         
56       
49           
42           
56         
63       
Eriksson et al. (1987) sc 90       
150       
210   
90     
150     
3 and 4 150       
Eriksson et al. (1992a) sc 189             
189           
Eriksson et al. (1992b) sc 1 and 2 120       
Pentschew et al. (1963) im 270         
435     
Bird et al. (1984) inhalation Group 1 730             
Nishiyama et al. (1977) inhalation 90       
300             
Coulston and Griffin (1976) inhalation Low dose 450             
High dose 161             
Dorman et al. (2006) inhalation Low, mid, and high exposure groups 90             

PBPK Model Simulations

Three studies used the oral route to deliver a Mn solution given as a bolus dose once daily. In the study by Chandra et al. (1979), brain Mn concentrations were reported in monkeys following an 18-month exposure period. Increases of 173 and 24% above control levels were reported in the midbrain region and cerebellum, respectively. Model predictions for this scenario yielded maximum tissue Mn concentrations of 138 and 57% for the globus pallidus and cerebellum, respectively. The study by Gupta et al. (1980) used an identical exposure scenario except that slightly higher Mn doses were used (7.0 vs. 5.6mg/kg in Chandra et al. (1979)) and clinical observations were reported at the end of the exposure period. In the study by Van Bogaert and Dallemagne (1946), oral doses were also administered daily but were varied during the exposure period. The PBPK model simulation of this exposure is shown in Figure 2. Globus pallidus Mn concentrations were observed to fluctuate with the daily dosing regimen and also increased with higher Mn doses. A maximum globus pallidus concentration of 1.5 µg/g was predicted at the highest oral dose of 6.3mg/kg. Although the oral doses were high in these studies, only a small fraction was predicted to be absorbed into the systemic circulation from the GI tract, resulting in moderate increases in brain Mn concentrations.

Fig. 2.

PBPK model simulation of the globus pallidus Mn concentration with the dosing schedule from Van Bogaert and Dallemagne (1946). Oral Mn doses were given once daily: day 1–20: 1.3mg/kg/day; day 21–89: 2.5mg/kg/day; day 89–123: 3.8mg/kg/day; day 124–183: 0mg/kg/day; day 184–291: 6.3mg/kg/day.

Fig. 2.

PBPK model simulation of the globus pallidus Mn concentration with the dosing schedule from Van Bogaert and Dallemagne (1946). Oral Mn doses were given once daily: day 1–20: 1.3mg/kg/day; day 21–89: 2.5mg/kg/day; day 89–123: 3.8mg/kg/day; day 124–183: 0mg/kg/day; day 184–291: 6.3mg/kg/day.

In the study by Mella (1924), monkeys were injected by the ip route every other day with progressively higher Mn doses. The predicted globus pallidus Mn concentration for monkey no. 6 from Mella (1924) is shown in Figure 3. The initial ip dose of Mn was 0.9mg/kg and continued for 96 days, followed by doses of 1.8mg/kg for 48 days and 2.2mg/kg for 22 days. The maximum predicted globus pallidus Mn concentration of 31.5 µg/g occurred during the highest dosing period. Predicted brain Mn concentrations exhibited a wide fluctuation between doses due to rapid systemic delivery from ip injection. This was followed by rapid clearance of Mn from the body after the ip injection.

Fig. 3.

Predicted Mn globus pallidus concentrations in monkey no. 6 from Mella (1924) subject to ip injection of Mn every other day. Dosing schedule: day 1–96: 0.9mg/kg; day 97–144: 1.8mg/kg; day 145–166: 2.2mg/kg.

Fig. 3.

Predicted Mn globus pallidus concentrations in monkey no. 6 from Mella (1924) subject to ip injection of Mn every other day. Dosing schedule: day 1–96: 0.9mg/kg; day 97–144: 1.8mg/kg; day 145–166: 2.2mg/kg.

Simulation of Mn globus pallidus concentration following exposure by iv injection is shown in Figure 4. In this exposure (monkey no. 2 in Olanow et al., 1996), the monkey was injected once per week with variable Mn doses of 2.8–3.6mg/kg. Extremely high brain Mn concentrations (> 50 µg/g) were predicted due to the high Mn doses and rapid delivery to tissues following direct systemic exposure from iv injection. These rapid increases in tissue Mn concentrations were followed by returns to near basal levels after several days. Similar behavior was observed in the other exposed monkeys from the Olanow study and in the iv exposures from Guilarte et al. (2006).

Fig. 4.

Predicted Mn globus pallidus concentration in a monkey exposed by iv injection according to the dosing schedule from Olanow et al. (1996). The monkey was injected once a week with 2.8–3.6mg/kg Mn.

Fig. 4.

Predicted Mn globus pallidus concentration in a monkey exposed by iv injection according to the dosing schedule from Olanow et al. (1996). The monkey was injected once a week with 2.8–3.6mg/kg Mn.

Studies that used sc injection for Mn delivery typically used much higher Mn doses (Table 1) to overcome the poor bioavailability and absorption of the insoluble MnO2 form. Simulations of the two dose levels in Suzuki et al. (1975) and the single dose level in Eriksson et al. (1987) are shown in Figure 5. Globus pallidus Mn concentrations from the other sc studies (Eriksson et al., 1992a,b; Pentschew et al., 1963) displayed similar kinetic behavior, including a rapid rise in brain Mn concentrations immediately following injection followed by a slow decline to near basal levels that in some instances took over one year. Compared with ip and iv exposures, brain Mn concentrations from sc exposures remained elevated for much longer periods of time, which may be due to slow absorption of relatively insoluble Mn particles at the injection sites, as was reported by Eriksson et al. (1987). The model did underpredict globus pallidus concentration 1 year following the last sc exposure in Eriksson et al. (1987); however, because the focus of this analysis was on a comparison of brain Mn concentrations with clinical effects observed during the exposure period or shortly thereafter, parameters governing transfer from the sc compartment were selected to calibrate simulation results closer to the end of the exposure period. This underprediction could also be due to liver impairment following high-dose Mn exposure, which would reduce biliary excretion and lead to higher tissue Mn concentrations. This effect was not included in the model due to a lack of quantitative information regarding changes in hepatobiliary excretion rates following Mn exposure.

Fig. 5.

Simulated globus pallidus concentrations in monkeys exposed by sc injection: (A) Monkeys were injected once a week for 9 weeks with 315 or 630mg Mn/injection (Suzuki et al., 1975); (B) Monkeys were injected on 18 occasions over 5 months with 444mg Mn/injection (Eriksson et al., 1987). The curves are model simulations, and the data points are measured globus pallidus concentrations from individual animals from Suzuki et al. (1975) and Eriksson et al. (1987).

Fig. 5.

Simulated globus pallidus concentrations in monkeys exposed by sc injection: (A) Monkeys were injected once a week for 9 weeks with 315 or 630mg Mn/injection (Suzuki et al., 1975); (B) Monkeys were injected on 18 occasions over 5 months with 444mg Mn/injection (Eriksson et al., 1987). The curves are model simulations, and the data points are measured globus pallidus concentrations from individual animals from Suzuki et al. (1975) and Eriksson et al. (1987).

Inhalation exposure studies used several different forms of Mn (MnO2, Mn3O4, and MnSO4) of varying degrees of solubility (Bird et al., 1984; Coulston and Griffin, 1976; Dorman et al., 2006a; Nishiyama et al., 1977). Model parameters were calibrated by Schroeter et al. (2011) to the time-course data from Dorman et al. (2006a) for a 90-day exposure to a MnSO4 atmosphere containing 1.5mg Mn/m3 to simulate a rapid rise in brain Mn concentrations during the 90-day exposure followed by a return to near basal levels during the 90 days following cessation of exposure (Fig. 6). To account for differences in lung deposition and absorption due to the different particle densities, particle sizes, and solubilities of the Mn aerosols used in the other studies, a Deposition Adjustment Factor (DAF) was applied to the lung deposition fraction of Mn oxide particles to account for their reduced bioavailability (Table 3). The DAF was calibrated based on measured end-of-exposure globus pallidus Mn concentrations. Simulations of the exposures from the Nishiyama et al. (1977) study are shown in Figure 6B. As with the Dorman simulations, brain Mn concentrations were predicted to rise during the exposure event and decrease once exposures stopped, although the predicted daily fluctuations from the Nishiyama simulations were less than those from the Dorman simulations due to the reduced bioavailability of the deposited Mn oxide particles in the lung. Even though the exposure concentration of 3.0mg/m3 was higher in the Nishiyama study, brain Mn concentrations were lower than in the Dorman study due to the lower solubility of MnO2. Model simulations of the Bird et al. (1984) and Coulston and Griffin (1976) studies showed similar behavior.

Fig. 6.

Predicted globus pallidus concentrations in monkeys exposed by inhalation: (A) the exposure scenario from Dorman et al. (2006a): exposure to MnSO4 at 1.5mg Mn/m3 for 6h/day, 5 days/week for 90 days; (B) the exposure scenario from Nishiyama et al. (1977): exposure to MnO2 at 0.7 or 3.0mg Mn/m3 for 22h/day, 7 days/week for 10 months.

Fig. 6.

Predicted globus pallidus concentrations in monkeys exposed by inhalation: (A) the exposure scenario from Dorman et al. (2006a): exposure to MnSO4 at 1.5mg Mn/m3 for 6h/day, 5 days/week for 90 days; (B) the exposure scenario from Nishiyama et al. (1977): exposure to MnO2 at 0.7 or 3.0mg Mn/m3 for 22h/day, 7 days/week for 10 months.

Dose-Response Analysis

The peak concentration, AUC (which represents cumulative internal dose), and average concentration for globus pallidus Mn concentrations during the exposure periods were computed from the PBPK model simulations for all of the dose groups from the Mn exposure studies included in this review. Severity scores applied to each dose group were plotted against all three dose metrics (Fig. 7). A steep dose-response behavior was observed when severity scores were plotted against peak globus pallidus Mn concentration (Fig. 7A). A visible pattern of increased severity was observed for peak concentrations greater than ~7 µg/g. When plotted against AUC, the progression of severity scores with exposure duration can be clearly observed (Fig. 7B). With the exception of a few dose groups (Nishiyama et al., 1977; Olanow et al., 1996), the severity scores started to increase for AUC values > 6000 (µg/g)-h. Although the dose-response behavior for all dose metrics exhibited significant scatter, the dose-response behavior for average concentration was not as evident, with increased severity scores for globus pallidus Mn concentrations between 4 and 30 µg/g (Fig. 7C).

Fig. 7.

Dose-response relationship for the Mn exposure studies using various dose metrics in the globus pallidus: (A) peak concentration, (B) area under the concentration curve (AUC), and (C) average concentration.

Fig. 7.

Dose-response relationship for the Mn exposure studies using various dose metrics in the globus pallidus: (A) peak concentration, (B) area under the concentration curve (AUC), and (C) average concentration.

A CatReg analysis was performed using the severity scores for the dose metrics of Mn peak concentration and AUC in the globus pallidus following Mn exposure. The dose metric of average globus pallidus Mn concentration was not considered for the regression analysis because the severity scores did not display a clear dose-dependent increase with concentration. Because the CatReg software limits severity scores to four categories, the severity scores summarized in Table 2 were combined to generate new severity scores for the regression analysis: 0:0, 1:1–3, 2:4–6, 3:7–9. These data were log transformed prior to the regression analysis. For the CatReg analysis, the p value was < 0.05 for both cases of peak concentration and AUC, indicating that the model fits were acceptable. R2 values were 0.20 and 0.16 for the dose metrics of peak concentration and AUC, respectively, indicating that a small amount (20 and 16%) of the variability in the data was accounted for with the regression curve. The low R2 values are likely due to the fact that most studies reported incidence fractions of 0 or 1 because only one animal was typically used in each dose group.

CatReg was also used to provide a maximum likelihood estimate of the peak globus pallidus Mn concentrations and AUC values that would result in 10, 20, and 30% extra risk (denoted as ERD10, ERD20, and ERD30, respectively) of attaining severity grades 1, 2, and 3 (Table 4). For example, the estimated 10% extra risk of attaining a severity score of 1, as informed by the severity scores applied to each dose group, was achieved with a peak globus pallidus Mn concentration of 0.8 µg/g or an AUC value of 813.1 µg/g-h.

TABLE 4

Results of the CatReg Analysis of Severity Scores for the Dose Metrics of Peak Concentration and AUC from the PBPK Model Simulations. ERD10, ERD20, and ERD30 Correspond to 10, 20, and 30% Extra Risk, Respectively, of the Corresponding Severity Score

Severity score ERD10 ERD20 ERD30 
Peak concentration (µg/g) 
0.8 1.4 2.0 
5.5 9.6 13.9 
18.1 31.6 45.9 
AUC ((µg/g)-h) 
813.1 1477.7 2198.2 
4860.7 8834.5 13141.7 
20283.9 36866.5 54840.2 
Severity score ERD10 ERD20 ERD30 
Peak concentration (µg/g) 
0.8 1.4 2.0 
5.5 9.6 13.9 
18.1 31.6 45.9 
AUC ((µg/g)-h) 
813.1 1477.7 2198.2 
4860.7 8834.5 13141.7 
20283.9 36866.5 54840.2 

DISCUSSION

An improved understanding of the dose-response relationships between Mn exposure, abnormally increased tissue Mn concentrations, and the development of neurotoxicity and other adverse clinical effects is a critical issue for Mn risk assessment. Many studies have been conducted to examine Mn-induced neurotoxicity in nonhuman primates. Most of these studies used elevated Mn doses, parenteral exposures, and variable dosing scenarios, making it difficult to extrapolate high-dose effects to potential responses from chronic low-dose inhalation exposure. There is also significant variability across studies regarding the onset and severity of neurotoxic effects. This variability is primarily due to the use of different forms of Mn and different exposure routes, leading to differences in Mn absorption, elimination, and tissue accumulation. Although it is difficult to extrapolate results from an individual study that may have only relied on several animals each acutely exposed to high Mn doses, a meta-analysis that utilizes a consistent measure of internal dose can reveal a clearer understanding of the dose-response behavior of Mn.

The analysis by Gwiazda et al. (2007) considered multiple studies on Mn neurotoxicity in Mn-exposed nonhuman primates to see whether there was a consistent dose-response relationship. In their analysis, internal cumulative Mn dose was estimated from the nominal dose provided in the reviewed study (corrected for the weight fraction of Mn in the compound administered), the measured or estimated daily consumption of the exposure media, treatment duration, estimated fraction of Mn absorbed into the bloodstream, and the body weight of the animal. For the nonhuman primate studies, Gwiazda assumed Mn uptake rates of 3% for oral exposures and 100% for all other exposure routes. They reported that the cumulative Mn dose at which adverse effects were detected in monkeys was dependent on the chemical species of Mn administered. Adverse effects also demonstrated a dose dependency. For example, in monkeys, motor deficits and/or effects on the globus pallidus were seen at relatively low cumulative doses (between 20 and 70mg Mn/kg). More profound effects and involvement of additional brain regions (e.g., caudate and putamen) were noted at higher cumulative exposure doses (> 260mg Mn/kg). The range of exposures associated with adverse changes was over two orders of magnitude in the animal studies, which led Gwiazda et al. (2007) to conclude that most existing animal model studies might be of limited relevance for the risk assessment of chronic low-level Mn exposure to humans. Newland (1999) also reviewed the progressive effects of Mn exposure with increased dose from several studies, where dose was defined as the “cumulative dose of Mn in mg of Mn/kg body mass that had been administered when the sign appeared.” He concluded that there is considerable variability across studies in the latency to onset of neurotoxic effects associated with Mn.

Although these earlier reviews of Mn neurotoxicity estimated cumulative Mn dose, they did not take into account internal Mn kinetics, solubility differences among the sulfate, phosphate, and oxide forms of Mn used in different studies, dose-dependent biliary excretion, or increases in Mn concentrations within brain regions (e.g., the globus pallidus) that are target tissues for Mn neurotoxicity. In the present analysis, PBPK model simulations were used to estimate toxicologically relevant internal measures of Mn accumulation within the globus pallidus: peak concentration, average concentration, and cumulative internal dose (AUC) during the Mn exposure period. Rodent studies were not described in this evaluation because, unlike nonhuman primates, they lack behavioral similarities to humans and are less sensitive to Mn than are humans and nonhuman primates (Guilarte, 2010).

The PBPK model for Mn dosimetry was derived from a rich pharmacokinetic data set in rhesus monkeys exposed to Mn by inhalation (Dorman et al., 2006a). Additional exposure routes in the model (oral, ip, iv, and sc) were validated using 54Mn clearance data, which showed differences in elimination rates depending on exposure route, but with all tracer studies displaying a biphasic elimination (Schroeter et al., 2011). Because tracer studies reflect the overall kinetics of Mn in the body, this gives us significant confidence that the model captured the main dose-dependent characteristics of Mn uptake, elimination, and distribution in monkeys. By accounting for dose-route differences in Mn absorption and elimination, we were able to estimate common dose metrics among the many Mn exposure studies that have been conducted in nonhuman primates, regardless of the exposure route, solubility, or dosing schedule used in the animal studies.

The PBPK model structure used in this study was originally developed to describe Mn tissue dosimetry in rats subject to dietary and inhalation exposure (Nong et al., 2008). This model structure was subsequently extended to monkeys and humans with additional exposure routes added as needed to accommodate various experimental methods used to administer Mn and 54Mn (Nong et al., 2009; Schroeter et al., 2011). These modeling efforts helped inform key processes involved in Mn kinetics, including saturable tissue binding, asymmetric flux into brain regions, and dose-dependent biliary excretion that allow tissue Mn concentrations to remain fairly constant during low levels of exposure and to rise rapidly during high-dose episodes, as was observed in animals. The models have also been recently used to guide a tissue dose–based risk assessment approach and to study Mn tissue accumulation in sensitive subpopulations (Andersen et al., 2010; Taylor et al., 2012). The ability of the PBPK models to consistently describe Mn kinetics across multiple species and exposure routes indicates that the models have accurately captured the dose-dependent characteristics of Mn disposition. For this study, several parameters governing portal-of-entry effects were estimated to accurately simulate the delivery of Mn to the systemic circulation from multiple exposure routes. However, the parameters governing internal Mn kinetics, such as diffusion rate constants and tissue binding parameters, were unchanged for all simulations regardless of exposure route. Once further biochemical details for tissue binding, membrane transport, and Mn retention in enterocytes (to name a few examples) become available, then these parameters can be appropriately refined in the model. Slight reparameterization of other rate constants may then be necessary to maintain the same quality as the current model-fit with pharmacokinetic data in animals because, as pointed out by Schroeter et al. (2011), model simulations are sensitive to some parameters such as the biliary excretion and diffusion rate constants.

This analysis depended not only on an accurate estimation of tissue Mn concentrations following Mn exposure but also on a consistent application of severity scores to multiple studies from numerous investigators over a wide period of time. Our clinical understanding of neurochemical changes associated with Mn neurotoxicity has undergone significant revisions during the past decade as single photon emission computed tomography and positron emission tomography studies have been used. In addition, some studies performed in monkeys have assessed dopamine, γ-aminobutyric acid, and other neurotransmitters in Mn-exposed monkeys. Guilarte (2010) performed a thorough review of the available human and nonhuman primate Mn neurotoxicity data and concluded that there is overwhelming evidence showing that Mn-induced neurological signs do not involve degeneration of midbrain dopamine neurons. Moreover, handling of brain tissue samples in some early neurochemistry studies (e.g., Neff et al., 1969) was of concern because tissue samples from control and Mn-treated animals were subjected to different storage times and conditions (Guilarte, 2010). Thus, unlike Gwiazda et al. (2007), we did not focus on brain dopamine changes as an endpoint of interest in this re-evaluation because too few contemporary studies would support this analysis.

Clinical evaluations, including an assessment of animal behavior, are a key component in neurotoxicity testing (Tilson and Moser, 1992). One challenge associated with this study is the confidence we have in the ability of the original study investigators to assess clinical signs in Mn-exposed monkeys. The a priori use of a scoring system is known to improve the ability to detect subclinical disease in animals. Among the studies we reviewed, only the one performed by Guilarte et al. (2006) fully described their observational methods and provided a scoring scale. Guilarte rated a variety of behaviors using a scale developed for a monkey model of parkinsonism (Schneider and Kovelowski, 1990). Each item was rated as 0 (normal), 1 (mild), 2 (moderate), or 3 (severe) with disability (up to a maximum score) and dystonia/dyskinesia (maximum score of 21) being assessed individually. Guilarte also used an automated system to assess gross motor activity and a separate test apparatus to assess fine motor skills in their Mn-exposed animals. Guilarte et al. (2006) reported that behavioral rating scores in Mn-exposed monkeys increased slightly as the exposure duration progressed. Throughout the course of the study, animals appeared grossly normal and detected changes were considered very subtle (we rated these effects as mild with affected animals receiving a score of 2/17 in our scoring system).

Our data analysis is further complicated by the lack of detailed reporting of when clinical signs emerged (often given in weeks or months rather than days). For example, on the one hand, Mella (1924) provided a detailed account of clinical signs associated with Mn exposure every other day during the exposure period, whereas the study by Gupta et al. (1980) merely reported that clinical signs were evident after 18 months of exposure. In the latter case, because no other information was given regarding the onset of effects, we had to assume that they began at 18 months. Another confounder that should be recognized is that many of the studies fail to describe whether or not animal selection was randomized (unlikely given the extremely small numbers of animals used) or whether the person performing the animal observations were blinded to treatment groups. As in human studies, failure to control for these confounders often leads to an overestimate of the effect seen in animals (Bebarta et al., 2003).

Our data analysis method also warrants examination. The clinical scoring system we used develops categorical data. The analysis of such categorical data has been less robustly explored in neurotoxicology (Markgraf et al., 2010). We utilized a descriptive approach that relied on the experience of the evaluator (David D. Dorman) to assess whether the reported clinical signs were abnormal and consistent with Mn neurotoxicity. Markgraf et al. (2010) compared the use of an experienced behavioral toxicologist to more routine statistical approaches in interpreting rodent functional observational battery data. Similar to our methods, the behavioral toxicologist evaluating the data sets in the Markgraf study was not blinded prior to evaluation. Markgraf found that professional judgments and statistical approaches yielded similar estimates of an individual study no-effect level.

Despite the use of different Mn forms, multiple exposure routes, a wide range of dose levels, and possible inconsistencies among studies regarding the reporting of Mn-induced effects, a clear dose-response behavior was observed when severity scores were analyzed versus the PBPK model–derived dose metrics of peak concentration and cumulative dose (AUC) in the globus pallidus region. This analysis can help inform threshold levels for dose-dependent transitions in effects from Mn exposure. The fact that peak concentration demonstrated a sharp transition in effects indicates the existence of a threshold brain Mn concentration for Mn-induced effects. For example, from the CatReg analysis, a 10% extra risk for a mild response (severity level 1) occurred at a peak concentration of 0.8 µg/g (Table 4). This value is 81% higher than the average basal Mn concentration in the globus pallidus from these studies. A rapid increase in severity score was observed for peak Mn concentrations > 7 µg/g. Likewise, when plotted against AUC, an increase in severity with cumulative dose was also observed, indicating a progression of effects with increasing dose and exposure duration.

Although most of the data points followed a sigmoidal dose-response curve, there were several exceptions, which were expected given the wide range of experimental conditions used in the studies. For example, for peak globus pallidus concentration (Fig. 7A), the results from the Gupta et al. (1980) study (oral dosing) and from the high-dose group (3.0mg/m3 inhalation exposure concentration) of the Nishiyama et al. (1977) study displayed mild clinical effects at lower peak brain concentrations (~2 µg/g). The Nishiyama study was the only inhalation exposure study in this analysis to observe Mn-induced clinical effects, despite other inhalation studies using higher exposure concentrations or having higher estimated brain Mn concentrations. Some possible reasons for these discrepancies are that brain Mn concentrations were reported by Nishiyama as µg Mn per g dry tissue weight in the basal ganglia region. To be consistent with model predictions, these values were reduced by 80% (Molokhia and Smith, 1967) to convert to wet weight. It is also possible that basal ganglia Mn concentrations are not a suitable surrogate for globus pallidus Mn concentrations, as the basal ganglia includes other brain regions in addition to the globus pallidus. The Gupta study observed mild effects after oral Mn dosing, whereas the study by Van Bogaert and Dallemagne (1946) did not report any effects after similar oral dosing schedules. The peak concentration predicted for the Gupta study was slightly greater than that for the Van Bogaert and Dallemagne (1946) study, but when the data points were plotted versus AUC, both points fell in line with other studies, indicating the importance of accounting for exposure duration as a predictor of toxicity. These discrepancies may also be due to differences in Mn absorption among individuals or may reflect a dose-dependent transition region where some animals begin to show effects whereas others do not.

Another aberration in the dose-response curve for peak concentration occurred from one of the animals in the Olanow et al. (1996) study, where globus pallidus Mn concentrations were predicted to be almost 90 µg/g, yet no clinical effects were observed. This contrasts with two other animals from the same study that received slightly lower Mn doses yet demonstrated severe clinical effects. Olanow attributed this to individual variability to Mn intoxication. Simulations of the Guilarte et al. (2006) study also predicted high brain Mn concentrations following iv injection of Mn, yet only mild effects were observed. Model predictions for high-dose Mn iv injections demonstrated rapid increases in tissue Mn concentrations immediately following the injection, followed by rapid decreases in the days following injection. This behavior led to lower AUC values compared with sc injections, where much slower decreases in tissue Mn concentrations were predicted. There was no data available to validate the rise in tissue Mn concentrations following iv injection. Model predictions could possibly be improved if data existed for brain Mn concentrations at several time points following iv injection. Although Guilarte et al. (2006) reported globus pallidus Mn concentrations in iv-injected monkeys, measurements were taken 33 days after the last Mn injection and therefore were of limited use for validating peak tissue concentrations that occur soon after injection.

Finally, we offer one additional comment regarding the interpretation of the dose-response data. Cumulative dose (AUC) correlated with toxicity at the high-exposure situations considered in this analysis. This conclusion does not imply that equivalent cumulative doses experienced over a much longer duration (e.g., 70 years) at lower daily doses would generate neurological effects. This current analysis clearly showed that the tissue concentration correlated with neurological responses. In the case of inhalation, the PBPK models demonstrate that at or below an identifiable dose-dependent transition point for tissue accumulation (exposures of approximately 0.01mg/m3), tissue Mn does not increase significantly above background concentrations because of natural homeostatic mechanisms (Andersen et al., 2010; Schroeter et al., 2011). Our analysis, then, indicates that neurological responses in rhesus monkeys correlate well with integrated tissue exposure (i.e., AUC) above a limiting globus pallidus concentration of about 7 µg/g (see Figure 7A).

An approach to risk assessment for inhaled Mn has been suggested that considers increases in target tissue dose above background tissue levels (Andersen et al., 2010). The dose-response behavior presented here lends validity to this approach by evaluating internal measures of dose in the central nervous system that relate to Mn toxicity. Homeostatic controls maintain relatively stable Mn tissue concentrations by regulating intestinal absorption and biliary excretion. Toxicity results when these homeostatic controls are overwhelmed under conditions of excessive exposure, which was common in many of the high-dose studies examined in this study. This analysis clearly demonstrates that the dose response for the neurotoxic effects of Mn is independent of exposure route and supports the use of peak or cumulative tissue Mn concentrations as appropriate dose metrics. This analysis also provides evidence of a dose-dependent transition in the mode of action for the neurological effects of Mn that should be considered in risk assessments for this essential metal.

Funding

This work was sponsored in part by the University of Ottawa. The development of the PBPK models used in this publication was sponsored by Afton Chemical Corporation in satisfaction of registration requirements arising under Section 211(a) and (b) of the Clean Air Act and corresponding regulations at 40 C. F. R. Subsections 79.50 et seq.

Acknowledgments

The authors gratefully acknowledge Drs Harry Roels and Daniel Krewski for helpful conversations regarding this analysis.

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