Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is conducted according to an appropriate method and in compliance with the U.S. Environmental Protection Agency’s Good Laboratory Practice Standards for Inhalation Exposure Health Effects Testing (40 CFR Part 79.60).

Data source

Reference
Reference Type:
publication
Title:
Tissue Manganese Concentrations in Young Male Rhesus Monkeys following Subchronic Manganese Sulfate Inhalation
Author:
Dorman DC, Struve MF, Marshall MW, Parkinsn CU, James A & Wong BA
Year:
2006
Bibliographic source:
TOXICOLOGICAL SCIENCES 92(1), 201–210

Materials and methods

Principles of method if other than guideline:
The objective of this study was to determine tissue manganese concentrations in rhesus monkeys following subchronic (6 h/day, 5 days/week) manganese sulfate (MnSO4) inhalation.
GLP compliance:
yes
Remarks:
(U.S. Environmental Protection Agency’s Good Laboratory Practice Standards for Inhalation Exposure Health Effects Testing (40 CFR Part 79.60).)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Manganese (II) sulphate monohydrate
IUPAC Name:
Manganese (II) sulphate monohydrate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Molecular formula (if other than submission substance): MnSO4.H2O
Manganese sulphate is a relatively water-soluble, pale pink, crystalline powder that contains approximately 32% manganese.

Test animals

Species:
monkey
Strain:
other: rhesus
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. (Alice, TX)
- Age at study initiation: 17–22 months old at the time of their arrival at CIIT and between 20 and 24 months of age at the start of the inhalation exposure.
- Individual metabolism cages: no
- Diet (e.g. ad libitum): A certified primate chow (# 5048) diet from Purina Mills (St Louis, MO) was fed twice a day (total daily amount fed was approximately 4% of the animal’s body weight). Mean (± SEM) manganese concentrations determined in the primate chow were 133 ± 14 ppm. Manganese intake occurring from the ingestion of the base diet was approximately 6.2 mg/kg/day. Dietary supplements including fruits (e.g., oranges, raisins, apples), vegetables (e.g., carrots), and treats (e.g., honey, candies, cereal, fruit juices) were also provided to the monkeys each day. These supplements provided an additional 0–200 µg Mn per serving.
- Water (e.g. ad libitum): Reverse osmosis purified water was available ad libitum. Manganese concentrations in the majority of water samples (36/50 samples) were below assay detection limits (< 0.24 µg Mn/l), while the highest manganese water concentration was 8.9 µg Mn/l.
- Acclimation period: Animals were acclimated to the facility for at least 43 days prior to the start of the first inhalation exposure.

ENVIRONMENTAL CONDITIONS
During non-exposure periods, domiciliary stainless steel cages (0.4 m2 3 0.8 m tall) suitable for housing macaque monkeys (Lab Products, Inc., Seaford, DE) were used to individually house monkeys. On each exposure day, animals were transferred to 0.2 m2 X 0.6 m tall stainless steel cages (Lab Products, Inc.) that were designed to fit within the 8-m3 inhalation chambers. Animals were moved back to their domiciliary cages after the end of each 6-h exposure. Additional details concerning these animals and their husbandry have been published (Dorman et al., 2005).

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: See above.
Details on inhalation exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
no data

CHARACTERISATION OF TEST ATMOSPHERE
No significant differences in the test aerosol characteristics were observed between the two exposure cohorts. Based upon optical particle sensor results, the overall average concentrations (± SD) for the MnSO4 atmospheres were 0.19 ± 0.01, 0.97 ± 0.06, 4.55 ± 0.33 (cohort 1), and 4.45 ± 0.35 (cohort 2) mg/m3 for the target concentrations of 0.18, 0.92, and 4.62 mg MnSO4/m3. The geometric mean diameters and geometric standard deviations (σg) of the MnSO4 atmospheres were determined to be 1.04 µm (σg = 1.51), 1.07 µm (σg = 1.54),
1.12 µm (σg = 1.58), and 1.04 µm (σg = 1.50) for the target concentrations of 0.06, 0.3, 1.5 (cohort 1), and 1.5 (cohort 2) mg Mn/m3, respectively. The calculated mass median aerodynamic diameters (MMAD) were 1.73, 1.89, 2.12, and 1.72 µm for the target concentrations of 0.06, 0.3, 1.5 (cohort 1), and 1.5 (cohort 2) mg Mn/m3, respectively. Particles of unknown composition (arising from animal dander and other background sources) were present in the control chamber at an overall average concentration (± SD) of 0.004 ± 0.002 mg/m3. The calculated MMAD for the particles in the control chamber was 3.88 µm.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Exposures were conducted for 6 h/day, 5 days/week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.18, 0.92, and 4.62 mg MnSO4/m3, corresponding to 0.06, 0.3, and 1.5 mg Mn/m3.
Basis:

No. of animals per sex per dose:
A group of monkeys (cohort 1) were exposed to air (n = 6) or MnSO4 at 0.06 (n = 6), 0.3 (n = 4), or 1.5 mg Mn/m3 (n = 4) for 65 exposure days. Another eight monkeys were exposed to 1.5 mg Mn/m3 for 65 exposure days and held for 45 or 90 days before evaluation (i.e., post-exposure recovery groups). A second group (n = 4 monkeys per time point) of monkeys (cohort 2) was exposed to MnSO4 at 1.5 mg Mn/m3 for either 15 or 33 exposure days and euthanized the day following their last exposure.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
EVALUATIONS PERFORMED:
- necropsy: The following brain structures were collected for determination of manganese concentrations: pituitary gland, olfactory bulb, olfactory tract, olfactory cortex, caudate, putamen, globus pallidus, cerebellum, trigeminal nerve, white matter, and frontal cortex. Samples of the following tissues were also collected for manganeseanalyses: olfactory epithelium, heart, femur, skullcap (parietal bone), liver, pancreas, kidney, skeletal muscle (quadriceps femoris m), testes, gall bladder contents (i.e., bile) and urine.

- Hematology, clinical chemistry, and serum electrolyte analyses.

- Tissue manganese concentrations: determined by graphite furnace atomic absorption spectrometry.
Statistics:
Data analysis and statistics.
The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene’s test), ANOVA, and Dunnett’s multiple comparison procedure for significant ANOVA. The ANOVA for the clinical chemistry and haematology data was performed on the parameter value obtained from the difference between an animal’s pre-exposure and post-exposure test values. In the event, the Levene’s test was significant, then the data were transformed using a natural log (ln) transformation. If the Levene’s test remained significant, then the data were analyzed by nonparametric statistics (Wilcoxon/Kruskal-Wallis). Statistical analyses were performed using SAS Statistical Software (Cary, NC). A probability value of < 0.01 was used for Levene’s test, while < 0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values ± SEM. Elimination half-lives for manganese in several tissues were estimated for animals exposed to 1.5 mg Mn/m3 for 65 exposure days using standard kinetic formulas (Shargel and Yu, 1999). Prior to analysis, mean tissue manganese concentrations were corrected for background manganese concentrations present in the air-exposed monkeys. This analysis was performed on tissues that were significantly increased following the 65th exposure and 90 days thereafter had a mean manganese concentration higher than that present in the air-exposed animals.

Results and discussion

Results of examinations

Details on results:
Details on distribution in tissues
Tissue manganese concentrations: Subchronic exposure to MnSO4 at the lowest exposure concentration (0.06 mg Mn/m3) resulted in increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, white matter, cerebellum, and heart. Monkeys exposed to MnSO4 at the mid-dose (0.3 mg Mn/m3) for 65 exposure days developed increased manganese concentrations in all the above tissues, as well as in the olfactory tract, caudate, pituitary gland, kidney, pancreas, lung, bile, blood, and urine. Monkeys exposed to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3) for 65 exposure days additionally had increased manganese concentrations in the frontal cortex, trigeminal nerve, liver, skeletal muscle, and parietal bone. Increased manganese concentrations were observed in the olfactory epithelium, olfactory bulb, olfactory tract, olfactory cortex, globus pallidus, putamen, caudate, frontal cortex, cerebellum, pituitary, femur, kidney, lung, pancreas, parietal bone, and bile of monkeys exposed to MnSO4 at the highest exposure concentration for 3 weeks (15 exposure days). Manganese concentrations were elevated in the olfactory epithelium, olfactory bulb, olfactory tract, olfactory cortex, globus pallidus, putamen, caudate, frontal cortex, cerebellum, pituitary, trigeminal nerve, heart, kidney, lung, pancreas, parietal bone, testes, blood, and bile of monkeys exposed to MnSO4 at the highest exposure concentration for 6 weeks (33 exposure days). Tissue manganese concentrations remained elevated (vs. airexposed controls) in the olfactory cortex, globus pallidus, putamen, pituitary gland, and blood 45 days after the end of the 13-week exposure (65 exposure days) to MnSO4 at 1.5 mg Mn/ m3 (Table 4). All tissue manganese concentrations had returned to levels observed in the air-exposed control animals by 90 days after the end of the exposure.

Details on excretion
Elimination of manganese from the monkey brain varied from region to region with the shortest halftime of elimination occurring in the olfactory bulb (4.9 days), intermediate in the globus pallidus, putamen, and caudate (15.7–16.7 days), with slower elimination occurring in the olfactory cortex (19.4 days), pituitary (23.6 days), and cerebellum (32.3 days). The apparent halftime of elimination in the olfactory epithelium, kidney, and heart were 12.9, 18.3, and 27.3 days, respectively.

Bodyweight: Subchronic inhalation exposure to MnSO4 did not affect body weight gain (data not shown) or terminal body weight.

Clinical signs: Clinical signs observed in the monkeys were of minimal veterinary concern (e.g., alopecia or pulling of hair on the arms and legs, intermittent abnormal stool) and were not related to MnSO4 inhalation (data not shown).

Organ weights: No statistically significant difference from control in absolute organ weights was observed with any organ in animals exposed to MnSO4 for 65 days and then assessed immediately thereafter. Because the animals continued to grow, evaluation of post-exposure organ weights was confounded by the animal’s increase in body weight. There was a statistically significant decrease (approximately 17%) in the relative heart weight (relative to body weight) in monkeys evaluated 90 days after the end of a 13-week exposure to MnSO4 at 1.5 mg Mn/m3. No other statistically significant differences in relative organ weight (relative to either body weight or brain weight) were observed in the MnSO4-exposed animals versus controls.

Haematology and Clinical Chemistry: statistically significant decrease in the difference between pre- and post-exposure total bilirubin concentrations was observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 65 exposure days when compared to air-exposed controls. However, end-of-exposure total bilirubin concentrations in the air- and MnSO4-exposed monkeys were 0.15 ± 0.02 and 0.15 ± 0.03 mg/dl, respectively. A twofold higher pre-exposure total bilirubin concentration was present in the monkeys assigned to the high-dose MnSO4 exposure group. Alkaline phosphatase activity was approximately 1.6-fold higher in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m3, when compared to controls (524 ± 53 IU/l). Mean corpuscular hemoglobin concentration (MCHC %)

was decreased in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 15 days (post-exposure value ¼ 33.5 ± 0.3%) and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m3 (post-exposure value ¼ 33.6 ± 0.3%) versus controls (post-exposure value ¼ 35.1 ± 0.1%). The preexposure and post-exposure total bilirubin, alkaline phosphatase, and MCHCs were in the normal reference range reported for male rhesus monkeys (Wolford et al., 1986). The most common post-exposure red blood cell morphological finding was slight anisocytosis. Anisocytosis was found in control and MnSO4-exposed monkeys. Observed differences in clinical chemistry or hematology parameters are unlikely to be toxicologically significant or related to MnSO4 exposure.

Effect levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
These results provide an improved understanding of MnSO4 exposure conditions that lead to increased concentrations of manganese within the nonhuman primate brain and other tissues.
Executive summary:

ABSTRACT

High-dose human exposure to manganese results in manganese accumulation in the basal ganglia and dopaminergic neuropathology.

Occupational manganese neurotoxicity is most frequently linked with manganese oxide inhalation; however, exposure to other forms of manganese may lead to higher body burdens. The objective of this study was to determine tissue manganese concentrations in rhesus monkeys following subchronic (6 h/day, 5 days/week) manganese sulfate (MnSO4) inhalation. A group of monkeys were exposed to either air or MnSO4 (0.06, 0.3, or 1.5 mg Mn/m3) for 65 exposure days before tissue analysis. Additional monkeys were exposed to MnSO4 at 1.5 mg Mn/m3 for 15 or 33 exposure days and evaluated immediately thereafter or for 65 exposure days followed by a 45- or 90-day delay before evaluation. Tissue manganese concentrations depended upon the aerosol concentration, exposure duration, and tissue. Monkeys exposed to MnSO4 at≥ 0.06 mg Mn/m3 for 65 exposure days or toMnSO4 at 1.5 mg Mn/m3 for ≥ 15 exposure days developed increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, and cerebellum. The olfactory epithelium, olfactory bulb, globus pallidus, caudate, putamen, pituitary gland, and bile developed the greatest relative increase in manganese concentration following MnSO4 exposure. Tissue manganese concentrations returned to levels observed in the air-exposed animals by 90 days after the end of the subchronic MnSO4 exposure. These results provide an improved understanding of MnSO4 exposure conditions that lead to increased concentrations of manganese within the nonhuman primate brain and other tissues.