Dimanganese trioxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Tissue Manganese Concentrations in Young Mlae Rhesus Monkeys following Subchronic Manganese Sulfate Inhalation

Author:

Dorman D.C. et al.

Year:

2006

Bibliographic source:

Toxicological Sciences 92 (1), 201 - 210

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: 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. Necropsies were performed the day following the last inhalation exposure (i.e., 18–23 h after termination of the final inhalation exposure). 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.
- Parameters analysed / observed: Detailed clinical observations, body weight, organ weight, some gross pathology, hematology and clinical chemistry examinations were made. Control animals were used.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

manganese(2+);sulfate;hydrate

EC Number:

600-072-9

Cas Number:

10034-96-5

Molecular formula:

MnSO4.H2O

IUPAC Name:

manganese(2+);sulfate;hydrate

Specific details on test material used for the study:

- Name Manganese (II) sulfate monohydrate
- Solubility: water soluble
- Appearance: pale pink, crystalline powder
- Manganese content: 32%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company, Inc., Milwaukee, WI

Test animals

Species:

monkey

Strain:

other: Rhesus

Details on species / strain selection:

Evidence suggests that monkeys are a appropriate animal model to study manganese neurotoxicity. Therefore, rhesus monkeys were used in this study.

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: 20-24 months
- Housing: Animals were housed in animal rooms or exposure chambers within the animal facility of the institute. During nonexposure periods, domiciliary stainless steel cages (0.4 m^2 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 m^2 x 0.6 m tall stainless steel cages (Lab Products, Inc.) that were designed to fit within the 8-m^3 inhalation chambers. Animals were moved back to their domiciliary cages after the end of each 6-h exposure.
- Diet (e.g. ad libitum): certified primate chow 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). 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
- Acclimation period: at least 43 days

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1.72 - < 2.12 µm

Geometric standard deviation (GSD):

1.04

Remarks on MMAD:

The calculated mass median aerodynamic diameters(MMAD) were 1.73, 1.89, 2.12, and 1.72 lm for the target concentrations of 0.06, 0.3, 1.5 (cohort 1), and 1.5 (cohort 2) mg Mn/m3, respectively

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
Methods describing chamber monitoring as well as generation and characterization of the MnSO4 aerosol have been previously described (Dorman, D. C. et al. (2004). Nasal toxicity of manganese sulfate and manganese phosphate in young male rats following subchronic (13-week) inhalation exposure. Inhal. Toxicol. 16, 1–8.; Dorman, D. C., Struve, M. F., Gross, E. A., Wong, B. A., and Howroyd, P. C. (2005). Sub-chronic inhalation of high concentrations of manganese sulfate induces lower airway inflammation in rhesus monkeys. Respir. Res. 21, 121)

- Exposure apparatus: Four 8 m^3 stainless steel and glass inhalation exposure chambers were used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

65 days

Frequency of treatment:

6 h per day, 5 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.18 other: MnSO4/m^3

Remarks:

corrsponding to 0.06 Mn/m^3; cohort 1

Doses / concentrations 2

Dose / conc.:

0.92 other: MnSO4/m^3

Remarks:

corresponding to 0.3 Mn/m^3; cohort 1

Doses / concentrations 3

Dose / conc.:

4.62 other: MnSO4/m^3

Remarks:

corresponding to 1.5 Mn/m^3; cohort 1 & 2

No. of animals per sex per dose:

Cohort 1: Air: 6 males. Test item: 6 males at 0.18 mg MnSO4/m^3, 4 at 0.92 mg MnSO4/m^3 and 4 at 4.62 mg MnSO4/m^3
Cohort 2. 4 males at 4.62 mg MnSO4 / m^3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The lowest exposure MnSO4 concentration (0.06 mg Mn/m^3) is below the current 8-h threshold limit value for inhaled manganese of 0.2 mg Mn/m^3 that has been established by the American Conference of Governmental Industrial Hygienists.
- Rationale for animal assignment: Randomization of animals to treatment groups occurred prior to the start of the inhalation exposure and was based upon a weight randomization procedure

Positive control:

No

Examinations

Observations and examinations performed and frequency:

EVALUATIONS PERFORMED:
- Observations conducted: Detailed clinical observations, body weight, organ weight, hematology (hematocrit), clinical chemistry and serum electrolyte analyses
- Tissue manganese concentrations: Determined by graphite furnace atomic absorption spectrometry
- Following organ weights were examined: brain, pituitary, liver, lung, kidney, heart, pancreas, testes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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 manganese analyses: olfactory epithelium, heart, femur, skullcap (parietal bone), liver, pancreas, kidney, skeletal muscle (quadriceps femoris m), testes, gall bladder contents (i.e., bile) and urine.

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 hematology 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/m^3 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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A 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/m^3 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.
Mean corpuscular hemoglobin concentration (MCHC %) was decreased in monkeys exposed to MnSO4 at 1.5 mg Mn/m^3 for 15 days (post-exposure value = 33.5 ± 0.3%) and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m^3 (post-exposure value = 33.6 ± 0.3%) versus controls (post-exposure value = 35.1 ± 0.1%). The pre-exposure and post-exposure total bilirubin were in the normal reference range reported for male rhesus monkeys. The most common post-exposure red blood cell morphological finding was slight anisocytosis. Anisocytosis was found in control and MnSO4 exposed monkeys.
Observed differences hematology parameters are unlikely to be toxicologically significant or related to MnSO4 exposure. Sub-chronic exposure to MnSO4 did not affect any other hematology parameters examined in this experiment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Alkaline phosphatase activity was approximately 1.6-fold higher in monkeys exposed to MnSO4 at 1.5 mg Mn/m^3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m^3, when compared to controls (524 ± 53 IU/l).
The pre-exposure and post-exposure total alkaline phosphatase were in the normal reference range reported for male rhesus monkeys. Observed differences in clinical chemistry parameters are unlikely to be toxicologically significant or related to MnSO4 exposure. Sub-chronic exposure to MnSO4 did not affect any other clinical chemistry parameters examined in this experiment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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. Because the animals continue to growth, evaluation of post-exposure organ weights was confounded by the animals increase in body weight.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Tissue manganese concentrations: Sub-chronic exposure to MnSO4 at the lowest exposure concentration (≥ 0.06 mg Mn/m^3) 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/m^3) 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/m^3) 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. air exposed 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/m^3. All tissue manganese concentrations had returned to levels observed in the air-exposed control animals by 90 days after the end of the exposure. 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.

Effect levels

Dose descriptor:

NOAEC

Effect level:

4.62 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this study, the NOAEC was determined to be 4.62 mg MnSO4/m³ body weight/day.

Executive summary:

In a sub-chronic toxicity study, the test item was administered to 4 – 6 male Rhesus monkeys at concentrations of 0.18, 0.92 and 4.62 mg MnSO4 / m³ via aerosol inhalation for 6 h a day, 5 days a week for 65 exposure days. Detailed clinical observations, body weight, organ weight, haematology and clinical chemistry examinations were conducted. No treatment related toxic effects for clinical observations, body weight, organ weight, hematology or clinical chemistry were observed. Based on these results, the NOAEC for the test item was determined to be 4.62 mg/m³.