Manganese oxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results. The focus of the study was on biological changes in lung; not a traditional repeated dose study as required observations not made. The study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds, including the registration substance, manganese carbonate.

Data source

Reference

Reference Type:

publication

Title:

Rabbit lung after inhalation of manganese chloride: a comparison with the effects of chlorides of nickel, cadmium, cobalt and copper.

Author:

Camner P, Curstedt T, Jarstrand C, Johannsson A, Robertson B and Wiernik A

Year:

1985

Bibliographic source:

Environmental Research, 38: 301 - 309

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rabbits were exposed to MnCl2 via aerosol for 6 hours a day, 5 days a week, for a period of 4 to 6 weeks. Within 3 days after the end of the exposure period the rabbits were sacrificed and the lungs excised. The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Burker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophage were air dried, fixed in methanol and stained. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis. The functionality, phagocytic activity and bacteriocidal capacity of the macrophages was investigated. Lipid analysis was also performed.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Manganese dichloride

EC Number:

231-869-6

EC Name:

Manganese dichloride

Cas Number:

7773-01-5

Molecular formula:

Cl2Mn

IUPAC Name:

manganese(2+) dichloride

Test material form:

solid: particulate/powder

Details on test material:

- Molecular formula: MnCl2

Test animals

Species:

rabbit

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.1 -2.2 ± 0.3 kg
- Housing: During the exposure period animals were kept in 0.6 m exposure chambers (4 rabbits per chamber)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 1 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: MnCl2 aerosols were produced using an ultrasonic nebulizer
- Method of particle size determination: Mass median aerodynamic diameter of both aerosols was estimated with an impactor

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Manganese concentration was measured daily for 3 hours by air suction through a membrane filter (Gelman GN-4, 0.8 µm) and the amount of metal deposited on the filter was measured by atomic absorption spectroscopy (Varian AA6).

Duration of treatment / exposure:

6 hours/day, 5 days/week for 4 to 6 weeks

Frequency of treatment:

daily (5 days a week)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.01, 3.9 mg/m³
Basis:
other: concentration of manganese (as MnCl2)

No. of animals per sex per dose:

8 males per group

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

No in-life examinations were reported.

Sacrifice and pathology:

SACRIFICE
Within 3 days after the end of the exposure period the rabbits were sacrificed by an overdose of sodium pentobarbital and the lungs excised.
The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Bürker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophages were air dried, fixed in methanol and stained with Giemsa solution. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe, about 1 mm³ each, were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis.

LIGHT MICROSCOPY
The left upper lobe was fixed in 10% formalin and routine paraffin sections were stained with hematoxylin and eosin.

ELECTRON MICROSCOPY
Morphometric measurements were performed on 21 randomly selected fields from each rabbit in the control group and in the group exposed to the high Mn(II) concentrations, at a primary magnification of 1000. The area occupied by type II cell profiles divided by the area occupied by alveolar tissue profiles, was determined for each rabbit. the size of the type II cells was estimated on toluidine blue-stained Epon sections by means of a Lanameter.

FUNCTIONAL TESTS
The oxidative metabolic activity of the macrophages was estimated by measuring their ability to reduce nitroblue tetrazolium to formazan at rest and in the presence of Escherichia coli. The phagocytic activity of the macrophages was measured. A suspension of cells in Eagle's medium was incubated with yeast cells (Saccharomyces cerevisiae) labelled with fluorescein isothiocyanate and opsonized with pooled rabbit serum. After 30 and 60 minutes the phagocytosis was interrupted and the preparation stained with crystal violet. Ingested particles were recognised by their fluorescence and the attached ones being stained with the dye.
Bacterial capacity was tested by incubating the macrophages with Staphylococcus aureus "Oxford" in a suspension in Eagle's medium containing 0.1% gelatin and diluted pooled rabbit serum. After 90 minutes, colony forming units in this and in the original suspension were determined.

LIPID ANALYSIS
The left lower lobe was homogenised at 4°C and extracted with chloroform:methanol 2:1 (v/v). After filtration, 0.58% sodium chloride in water was added. The lower phase was dried and the lipids were separated by reverse-phase chromatography. The quantities of phospholipids were estimated by phosphorus determinations.

Results and discussion

Results of examinations

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

LUNG MORHPHOLOGY
The gross appearance of the lungs was normal both in MnCl2- exposed rabbits and in controls. The weight of the left lower lung weight was similar in all three groups.
Light microscopy showed focal infiltration of eosinophils (indicative of inflammation) in 4 controls, 3 in the low-dose group and 4 in the high-dose experimental animals. A few alveoli with increased accumulation of macrophages were found in 2 high-dose animals, 1 low-dose animal and 1 control. The majority of both control and experimental animals showed scattered areas of atelectasis. Slight inflammatory changes were non-significant and therefore were concluded to be unrelated to the experimental protocol.
Electron microscopy showed apparently normal alveolar septa, with the exception of 1 control and 1 exposed rabbit which showed focal oedema of alveolar type I cells. Values for volume density of type II cells were similar in all groups.

MACROPHAGE DATA
The cell diameter was significantly larger in the high dose group animals compared to the controls. The cell viability was above 90% in all animals.
By electron microscopy, macrophages from both exposed rabbits and controls had an undulating surface with some protrusions and their cytoplasm was rich in lysosomes. Some macrophages from exposed and control rabbits contained one or a few laminated inclusions. The oxidative metabolic activity of the macrophages was similar in the three groups both at rest and after stimulation with E. coli, as were the number of yeast particles ingested or attached to the macrophage surface. The bacteriacidal capacity was similar in exposed animals and controls.

PHOSPHOLIPID DATA
Phospholipids did not differ between controls and experimental animals.

Effect levels

Dose descriptor:

LOAEC

Effect level:

3.9 mg/m³ air

Based on:

other: concentration of manganese in test material

Sex:

male

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No abnormalities were found in Mn(II) exposed animals, except for an increase in the size of alveolar macrophages in the high-dose group.

Executive summary:

Rabbits were exposed to MnCl2 via aerosol for 6 hours a day, 5 days a week, for a period of 4 to 6 weeks. Within 3 days after the end of the exposure period the rabbits were sacrificed and the lungs excised. The right lung was lavaged and the alveolar macrophages collected. The macrophage concentration was measured in a Burker chamber and the cell viability tested by staining with eosin-y. Smears of lung macrophage were air dried, fixed in methanol and stained. Size distribution was determined by measuring the diameters of 100 -200 cells from each rabbit in a Lanameter. The upper left lobe was studied using light microscopy. Three tissue pieces from the middle part of the left lower lobe were sampled for electron microscopy and the remainder of the lobe was used for lipid analysis. The functionality, phagocytic activity and bacteriocidal capacity of the macrophages was investigated. Lipid analysis was also performed.

Under the conditions of the study, no abnormalities were found in Mn(II) exposed animals, except for an increase in the size of alveolar macrophages in the high-dose group.