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Toxicological information

Repeated dose toxicity: inhalation

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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:
other: Well conducted study focussing mainly on tissue concentrations with some other parameters investigated. However, a lack of histopathology significantly reduces the usefulness for this endpoint.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Principles of method if other than guideline:
Monkeys were exposed to an atmosphere containing manganese sulphate for a maximum period of 65 days, with exposure taking place for 5 days per week, 6 hrs per day. Tissue concentrations and body weights were assessed together with selected clinical and haematolgical parameters. Some gross pathology was undertaken, and organ weights assessed. Control animals were used.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- DESCRIPTION: Pale pink crystalline powder, approx 32% manganese.
- Name of test material (as cited in study report): Manganese (II) sulfate monohydrate
- Molecular formula (if other than submission substance): MnSO4.H2O

Test animals

Species:
monkey
Strain:
other: Rhesus
Sex:
male
Details on test animals and environmental conditions:
monkeys were 17 - 22 months old at time of arrival and were medically examined prior to start of the study. Animals were between 20 and 24 months of age at the start of the inhalation exposure. Animals were fed a certified primate chow diet twice a day. Mean manganese concentrations determined in the feed were 133 ± 14 ppm. Therefore manganese intake from the base diet was approximately 6.2 mg/ kg/ day. Mn levels in dietary supplements and drinking water were also determined. During non exposure periods monkeys were individually housed in domiciliary stainless steel cages . On each exposure day animals were transferred to stainless steel cages that were designed to fit within the 8m3 inhalation chambers. Animals were moved back to their domiciliary cages after each 6 hour exposure.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 1.73, 1.89, 2.12 and 1.72 µm for the target concentrations of 0.18, 0.92 and 4.62 (group 1) and 4.62 (group 2) mg MnSO4 / m3 respectively
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
65 days (group 1), 15 or 33 days (group 2)
Frequency of treatment:
5 days per week, 6 hours per day
Doses / concentrations
Remarks:
Doses / Concentrations:
0.18, 0.92 and 4.62 mg MnSO4 / m3
Basis:

No. of animals per sex per dose:
Group 1:
control : 6
0.18 mg MnSO4 / m3: 6
0.92 mg MnSO4 / m3: 4
4.62 mg MnSO4 / m3: 4

Post-exposure recovery groups:
4.62 mg MnSO4 / m3 (held for 45 or 90 days post-exposure): 8

Group 2:
4.62 mg MnSO4 / m3: 4

All animals were male.

Control animals:
other: Yes: filtered air

Examinations

Sacrifice and pathology:
NECROPSY: food was withheld overnight prior to necropsy. Monkeys were anesthetised with ketamine and blood was collected from a peripheral vein. A smalll volume was used to determined in the PCV and additional samples were collected for complete blood cell counts, clinical chemistry, evaluation of basal levels of lutenizing hormone (LH) and red blood cell glutathione (GSH) concentrations.

SACRIFICE: monkeys were euthanised with pentobarbital and exsanguinated.

The lungs and other thoraic organs were removed, weighed and inspected for gross lesions and manganese concentration was analysed. Brain was removed and dissected for the purpose of determining manganese concentrations. Mn concentration was also determined in the following tissues: olefactory epithelium, heart, femur, skullcap, liver, pancreas, kidney, skeletal muscle, testes, gall bladder contents and urine.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL OBSERVATIONS: Clinical signs observed were of minimal veterinary concern and were not related to MnSO4 exposure.
BODY WEIGHT AND WEIGHT GAIN: Subchronic inhalation exposure to MnSO4 did not effect bodyweight gain or terminal bodyweight.
HAEMATOLOGY: Mean corpuscular hemoglobin concentration (MCHC%) was decreased in monkeys exposed to MnSO4 at 4.62 mg MnSO4 /m3 for 15 days and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 4.62 mg MnSO4 /m3.
CLINICAL CHEMISTRY: A statistically significant decrease in the difference between pre- and post-exposure total bilirubin concentrations was observed in monkeys exposed to MnSO4 at 4.62 mg MnSO4/m3 for 65 exposure days. However, end-of-exposure total bilirubin concentrations in the air-and MnSO4-exposed monkeys were virtually identical. A twofold higher pre-exposure total bilirubin concentration was present in the monkeys assigned to the high-dose MnSO4 group. Alkaline phosphatase activity was approx 1.6 fold higher in monkeys exposed to MnSO4 at 4.62 mg MnSO4/m3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 4.62 mg MnSO4/m3. The pre-exposure and post-exposure total bilirubin, alkaline phosphatase and MCHCs were in the normal reference range reported for male rhesus monkeys (Wolford et al 1986).
Additional selected post-exposure clinical chemistry parameters were recorded. These were LH, iron, transferrin, total iron binding capacity, total GSH, reduced GSH and ratio GSSG:GSH.
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 continue to grow, evaluation of post-exposure organ weights was confounded by the animals increase in body weight. There was a statistically significant decrease (approx 17%) in relative heart weight in monkeys evaluated 90 days after the end of a 13 week exposure to MnSO4 at 4.62 mg MnSO4 / m3. No other statistically significant differences in relative organ weight
were observed in MnSO4 exposed animals versus controls.
GROSS PATHOLOGY
OTHER FINDINGS: issue manganese concentrations were recorded. Subchronic exposure at the lowest concentration resulted in increased Mn concentration in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, white matter, cerebellum and heart.
Monkeys at the mid-dose exposed for 65 exposure days developed increased Mn 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 the highest dose for 65 exposure days additionally had increased Mn concentrations in the frontal cortex, trigeminal nerve, liver, skeletal muscle and parietal bone.
The group of monkeys exposed to the top dose but assessed 45 or 90 days later showed tissue Mn concentrations remained elevated (vs controls) in the olfactory cortex, globus pallidus, putamen, pituitary gland and blood 45 days after the end of the 13-week exposure period. All tissue Mn concentrations had returned to levels observed in the air-exposed control animals by 90 days after the end of the exposure.
Elimination of Mn from the monkey brain varied from region to region with the shortest halftime occurring in the olfactory bulb (4.9 days) and longest in the cerebellum (32.3 days)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinical Observations:

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

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:

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/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 1.5mg Mn/m3 (post-exposure value ¼ 33.6 ± 0.3%) versus controls (post-exposure value ¼ 35.1 ± 0.1%).

Observed differences in clinical chemistry or haematology parameters are unlikely to be toxicologically significant or related to MnSO4 exposure.

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.

Applicant's summary and conclusion

Conclusions:
MnSO4 inhalation affected the haematology and resulted in increased Mn concentrations in the brain of the monkey. Due to the lack of histopathology however, the study is of limited value in fulfilling the sub-chronic inhalation endpoint.