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Diss Factsheets

Administrative data

Description of key information

There is a paucity of animal test data for MnO. However, information from mice studies on MnCO3  have been used to read across oral results from a sub-chronic (Komura et al 1991) and chronic (Komura et al 1992)  time periods and these show relatively mild effects given the high dietary dose.  Given that mice tend to be generally more sensitive than rats, these oral results do not cause concern for that route of exposure. The basis for the read across is justified by similarities between MnO and MnCO3; same oxidation state (+II), both are very poorly soluble in water (IUCLID section 4.8) and have similar leaching values in simulated gastric juice (IUCLID section 8.6.4 ). Both are not acutely toxic by the oral route (IUCLID section 7.2.1)

No relevant  inhalation studies are available. No relevant dermal studies are available.

Repeated Dose Oral Toxicity: Read-Across From NTP (1993)

Under the conditions of the study the NOAEL was 1 700 mg/kg bw for males and 2 000 mg/kg bw for females.

 

Repeated Dose Inhalation Toxicity: Read-Across From Camner (1985)

Under the conditions of the study the LOAEC was 3.9 mg/m3 based on an increase in the size of alveolar macrophages in the high-dose group.

 

Repeated Dose Dermal Toxicity

A short-term toxicity study via the dermal route does not need to be conducted as the physiological properties of the substance do not suggest a significant rate of absorption through the skin and no systemic effects or other evidence of absorption were seen in the skin or eye irritation studies (IUCLID section 7.3) and furthermore the water solubility of the substance is very poor (IUCLID section 4.8), and therefore a  limited amount of potential substance is available for systemic absorption via the dermal route. Therefore in accordance with Annex XI, section 1.1, this test is not considered necessary.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO ( IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. In addition there was no indication of any significant toxicity in the 100 day mouse study on MnCO3 (Komura et al, 1991), which can be read across to MnO, so a further study would appear scientifically unjustified. Therefore in accordance with Annex XI section 1.1, for the above reasons and animal welfare, this test is not considered necessary.
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not conducted to GLP, non-standard method using non-standard test subjects. Follows basic scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Sheep were maintained on a diet containing varying ppm levels of MnO for 84 days. Feed consumption, bodyweight, Haematological parameters and Mn organ concentrations were recorded.
GLP compliance:
not specified
Species:
sheep
Strain:
other: Florida native wether
Sex:
male/female
Details on test animals or test system and environmental conditions:
Sheep were housed individually in raised, slatted pens. Food and tap water was available ad libitum.
Route of administration:
oral: feed
Duration of treatment / exposure:
84 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 or 4000 ppm
Basis:

No. of animals per sex per dose:
3 animals per dose group
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
At termination of the study, sheep weights and total feed consumption were determined. Blood samples were obtained via jugular puncture for haemaglobin, haematocrit and serum mineral determination.
Sacrifice and pathology:
Sheep were euthanised via exsanguination and liver, kidney, spleen, heart and right metacarpus were excised and frozen for subsequent mineral determination.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
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
Dose descriptor:
LOAEL
Effect level:
4 000 ppm
Sex:
male/female
Critical effects observed:
not specified

Mortality: No mortalities were observed, no adverse clinical signs were recorded.

Food consumption: average daily food intake was reduced by high levels of dietary Mn.

Haematology: Haemoglobin and haematocrit were not affected by dietary Mn.

Mn levels in the liver, spleen and kidney were increased at the high dose level. Manganese in the heart was increased in all treatment groups with the exception of the 500 ppm dose group. Mn concentrations in bone increased with dose level.

Executive summary:

The subchronic repeated dose toxicity of the test material was assessed in sheep.

Sheep were maintained on a diet containing varying ppm levels of MnO for 84 days. Feed consumption, bodyweight, Haematological parameters and Mn organ concentrations were recorded. No mortalities were observed and no adverse clinical signs were recorded. Average daily food intake was reduced by high levels of dietary Mn. Haemoglobin and haematocrit were not affected by dietary Mn. Mn levels in the liver, spleen and kidney were increased at the high dose level. Manganese in the heart was increased in all treatment groups with the exception of the 500 ppm dose group. Mn concentrations in bone increased with dose level.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and according to peer reviewed methods.
Qualifier:
according to guideline
Guideline:
other: NTP peer reviewed methods
Principles of method if other than guideline:
Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500 or 25000 ppm manganese sulphate. Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses. A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
GLP compliance:
yes
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Stone Ridge, NY)
- Age at study initiation: 50 days



Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 1600, 3130, 6250, 12500 or 25000 which was equivalent to doses from 110 to 1700 mg/kg in males and 115 to 2000 mg/kg in females
Basis:
nominal in diet
No. of animals per sex per dose:
10 male and 10 female per dose group
Control animals:
yes
Observations and examinations performed and frequency:
Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses.
Sacrifice and pathology:
A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
-BODY WEIGHT AND WEIGHT GAIN: The mean bodyweight gain in males receiving 3130 ppm was marginally lower than that of the controls and was significantly lower in the three highest female dose groups than the controls.
-FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by exposed rats was similar to that of the controls. Females ingested an average of 20% more manganese sulphate than males in the corresponding exposure groups.
-HAEMATOLOGY: Neutrophil counts were significantly higher in all exposed male groups. Lymphocyte counts were significantly lower in the three highest dose groups.
In females: leukocyte counts were significantly lower in the three highest dose groups.
A significant increase in the percent haematocrit and erythrocyte counts occurred in males exposed to the three highest dose levels.
-ORGAN WEIGHTS: Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than the controls.
Dose descriptor:
NOAEL
Effect level:
1 700 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
Critical effects observed:
not specified

No clear relationship between observed differences and the ingestion of manganese sulphate has been defined.

Table 1.Survival, Body Weights, and Feed Consumption of Rats in the Rats in the 13-Week Feed Study of Manganese (II) Sulphate Monohydrate

 

Concentration (ppm)

Survivala

Mean body weight and weight changesbrelative

Final Weight Feed to controls (%)

Consumptionc

Initial

Final

Change

Week 1

Week 13

 

Male

 

 

 

 

 

 

 

0

10/10

136±5

291±4

155±4

 

14.9

13.1

1, 600

10/10

142±4

294±5

152±4

101

14.5

13.5

3,130

10/10

149±3

291±4

141±4

100

14.8

13.6

6, 250

10/10

148±2

294±3

146±3

101

15.0

9.6

12, 500

10/10

150±11

290±6

140±11

99

14.9

14.9

25, 000

10/10

140±4

284±6

144±4

97

14.1

14.4

Female

 

 

 

 

 

 

 

0

10/10

99±1

184±2

84±2

 

10.7

9.2

1, 600

10/10

103±1

181±2

79±2

99

10.8

9.3

3,130

10/10

96±1

175±2*

80±3

95

10.9

9.2

6, 250

10/10

101±1

176±2*

75±1**

96

10.7

14.3

12, 500

10/10

106±1**

178±1*

73±2**

97

10.7

10.5

25, 000

10/10

104±1**

174±3**

70±2**

95

12.1

10.3

* Significantly different (P≤0.05) from the control group by Williams’ or Dunnett’s test

** P≤0.01

a Number of animals surviving at 13 weeks/ number initially in group

b Weight given as mean ± standard error

c Feed consumption is expressed as grams per animal per day

 

Conclusions:
The high level of MnSO4 consumed on a daily basis for 13 weeks in this study, without mortality, supports the lack of acute toxicity. Although some changes in lung weight and certain haematological parameters were significant compared to controls, the lack of clinical and histopathological findings despite the very high daily oral dose over a sub-chronic period, is an indication of the relatively low toxicity of MnSO4, at least for the parameters studied in this report.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
1 700 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
Critical effects observed:
not specified
Endpoint:
chronic toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
MnO is mostly used in industrial settings where good industrial hygiene is employed. Outside these industrial settings, the registered substance is used by trained professionals. Exposure via the oral route is therefore implausible. However, in the unlikely event of accidental consumption, the acute oral toxicity test for MnO ( IUCLID 5 section 7.2.1) confirms no evidence of toxicity via this route. In addition there was no indication of any significant toxicity in the 100 day mouse study on MnCO3 (Komura et al, 1991), which can be read across to MnO, so a further study would appear scientifically unjustified. Therefore in accordance with Annex XI section 1.1, for the above reasons and animal welfare, this test is not considered necessary.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 700 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Manganese oxide is not acutely toxic by the oral route and has relatively low bioavailability (see Andersen K, section 7.12) in both artificial lung fluid and simulated gastric fluid. The particle size distribution test (IUCLID section 4.5) indicates that inhalation is a possible route of exposure, but has low bioavailability in artificial lung fluid. In addition, MnO is not acutely harmful by the inhalation route. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
LOAEC
Effect level:
3.9 mg/m³ air
Based on:
other: Concentration of Mn in test material
Sex:
male
Critical effects observed:
not specified
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.
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
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)

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)
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
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.
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.
Dose descriptor:
LOAEC
Effect level:
3.9 mg/m³ air
Based on:
other: concentration of manganese in test material
Sex:
male
Critical effects observed:
not specified
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.

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.
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
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Details on test animals or test system 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.
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
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
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.
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)
Critical effects observed:
not specified

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.

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.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Critical effects observed:
not specified
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
other: two-generation study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOEL
Remarks:
F0 and F1
Effect level:
20 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
other: two-generation study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2 July 2012 to 4 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A two-generation reproduction inhalation toxicity study conducted under GLP conditions and in accordance with a standardised guideline. Since animals were dosed over two generations, for a period of approximately 17 weeks, findings are considered to be indicative of repeated dose toxicity via the inhalation route. Since 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.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing period of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26°C
- Humidity (%): 33 - 69%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: See 'Exposure Conditions' under "Any other information on materials and methods incl. tables" for information.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and that the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test material aerosols were generated using a Wright Dust Feed generator device (Wright Dust Feed Mark II, BGI Industries, USA). Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powdercake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using appropriately sized modular nose only stainless steel flow past exposure chamber. Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.


TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4°C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at Charles River, Edinburgh under Study No. 428133 (Method No. 2813). Full details of the analytical methodology are contained within that report.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. For F0 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. For F1 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
Frequency of treatment:
Daily (ca 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of lactation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca 1 hour per day
From Day 3-4 of lactation: ca 2 hours per day
From Days 5-20 of lactation until prior to termination (ca Day 21 of lactation): ca 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Remarks:
Doses / Concentrations:
0, 5, 10, 20 µg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 6, 15, 25 µg/L
Basis:
other: analytical conc. (F0 generation)
Remarks:
Doses / Concentrations:
0, 4, 10, 17 µg/L
Basis:
other: analytical conc. (F1 generation)
No. of animals per sex per dose:
- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:
yes, concurrent vehicle
Details on study design:
- Selection and Weaning of F1 Animals
From each group, at least 24 males and 24 females were selected for post-weaning assessments. The selected pup(s) were the median’th weight pup(s) of that sex in the litter on Day 21 of lactation. These pups were removed from their mother on Day 21 of lactation, individually identified and housed in a new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- All animals were checked for early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of
a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHERS (REPROTOXICITY):
- Observation of females with litters during lactation, seuxal maturation, estrous cyclicity, sperm parameters, litter observations.

Sacrifice and pathology:
> PARENTAL ANIMALS
SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10% formalin. The reproductive tracts of all females were also examined.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices; cranial; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix), vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Other examinations:
SACRIFICE / GROSS NECROPSY (OFFSPRING)
Pups that were not selected for post-weaning assessments were killed at the same time as their mother. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

Pups that died prematurely were sexed and checked for external abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination.
At planned termination of offspring, 3 male and 3 female pups were necropsied and examined for external abnormalities followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved and the weights of the brain, spleen and thymus were recorded from one of the 3 pups of each sex; these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also preserved.
The following were weighed for all animals: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY (PARENTAL ANIMALS)
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejenum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 751 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.

CLINICAL SIGNS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental. Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

BODY WEIGHT (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 μg/L and target 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 μg/L and target 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05) following covariance analysis brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed
to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration ofthe test material.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

OTHER FINDINGS (PARENTAL ANIMALS)
There were no treatment-related effects on estrous cyclicity, sperm parameters or on mating performance, fertility or duration of gestation in either generation.
Dose descriptor:
NOEL
Remarks:
F0 and F1
Effect level:
20 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed
Critical effects observed:
not specified

Blood Analysis Results

F0 Males

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

6

Prior to mating

6

13

23

27

Prior to Necropsy

6

19

27

29

F0 Females

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

7

Prior to mating

6

16

28

39

Prior to Necropsy

7

16

24

33

At target 20 μg/L, manganese levels prior to mating were 350% higher than controls in males and 550% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 383% and 371% for males and females.

At target 10 μg/L, manganese levels prior to mating were 283% higher than controls in males and 367% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 350% and 243% for males and females.

At target 5 μg/L, manganese levels prior to mating were 117% higher than controls in males and 167% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 217% and 129% for males and females.

F1 Males

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

12

16

16

17

Prior to mating

6

9

13

19

Prior to Necropsy

6

9

14

21

F1 Females

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

13

12

15

15

Prior to mating

6

10

16

23

Prior to Necropsy

7

10

16

21

At target 20 μg/L, manganese levels prior to mating were 217% higher than controls in males and 283% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 250% and 200% for males and females.

At target 10 μg/L, manganese levels prior to mating were 112% higher than controls in males and 133% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 167% and 129% for males and females.

At target 5 μg/L, manganese levels prior to mating were 50% higher than controls in males and females at the pre-mating timepoint. At terminal necropsy, these values were 50% and 43% for males and females.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Conclusions:
Under the conditions of the study the No Observed Effect Level (NOEL) of manganese chloride, was determined to be 20 µg/L, the highest concentration tested.
Executive summary:

The subchronic reproductive toxicity of manganese chloride was investigated in a two generation study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed with manganese chrloride for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment to inhalation exposure of manganese chloride were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls, however this reflected a slightly smaller litter size at this level. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

Inhalation of manganese chloride was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.No test substance-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations. However, these effects on the respiratory tract are specific to manganese chloride because it dissolves and recrystalises as sharp, irritating, crystals in the respiratory tract. This physico-chemical property of the chloride is not common to the carbonate and hence it can be concluded that the effects on the respiratory tract would not be apparent following exposure to manganese carbonate.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test substance through the mother’s milk during lactation resulted in an increased exposure to the test substance in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, the No Observed Effect Level was considered to be the target dose level 20 μg/L.

Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Manganese oxide has low bioavailability (IUCLID section 7.12) in artificial lung fluid. The particle size distribution test (IUCLID section 4.5) indicates that inhalation is a possible route of exposure, but has low bioavailability in artificial lung fluid. In addition, MnO is not acutely harmful by the inhalation route (IUCLID section 7.2.2). Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3.9 mg/m³
Study duration:
subchronic
Species:
rabbit

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Manganese oxide is not acutely toxic by the oral route and has relatively low bioavailability (see Andersen K, section 7.12) in both artificial lung fluid and simulated gastric fluid. The particle size distribution test (IUCLID section 4.5) indicates that inhalation is a possible route of exposure, but has low bioavailability in artificial lung fluid. In addition, MnO is not acutely harmful by the inhalation route. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
LOAEC
Effect level:
3.9 mg/m³ air
Based on:
other: Concentration of Mn in test material
Sex:
male
Critical effects observed:
not specified
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.
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
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)

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)
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
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.
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.
Dose descriptor:
LOAEC
Effect level:
3.9 mg/m³ air
Based on:
other: concentration of manganese in test material
Sex:
male
Critical effects observed:
not specified
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.

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.
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
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Details on test animals or test system 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.
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
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
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.
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)
Critical effects observed:
not specified

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.

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.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Critical effects observed:
not specified
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
other: two-generation study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOEL
Remarks:
F0 and F1
Effect level:
20 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
not specified
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
other: two-generation study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2 July 2012 to 4 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A two-generation reproduction inhalation toxicity study conducted under GLP conditions and in accordance with a standardised guideline. Since animals were dosed over two generations, for a period of approximately 17 weeks, findings are considered to be indicative of repeated dose toxicity via the inhalation route. Since 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.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing period of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26°C
- Humidity (%): 33 - 69%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: See 'Exposure Conditions' under "Any other information on materials and methods incl. tables" for information.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and that the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test material aerosols were generated using a Wright Dust Feed generator device (Wright Dust Feed Mark II, BGI Industries, USA). Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powdercake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using appropriately sized modular nose only stainless steel flow past exposure chamber. Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.


TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4°C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at Charles River, Edinburgh under Study No. 428133 (Method No. 2813). Full details of the analytical methodology are contained within that report.
Duration of treatment / exposure:
F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. For F0 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. For F1 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
Frequency of treatment:
Daily (ca 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of lactation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca 1 hour per day
From Day 3-4 of lactation: ca 2 hours per day
From Days 5-20 of lactation until prior to termination (ca Day 21 of lactation): ca 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.
Remarks:
Doses / Concentrations:
0, 5, 10, 20 µg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 6, 15, 25 µg/L
Basis:
other: analytical conc. (F0 generation)
Remarks:
Doses / Concentrations:
0, 4, 10, 17 µg/L
Basis:
other: analytical conc. (F1 generation)
No. of animals per sex per dose:
- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L
Control animals:
yes, concurrent vehicle
Details on study design:
- Selection and Weaning of F1 Animals
From each group, at least 24 males and 24 females were selected for post-weaning assessments. The selected pup(s) were the median’th weight pup(s) of that sex in the litter on Day 21 of lactation. These pups were removed from their mother on Day 21 of lactation, individually identified and housed in a new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- All animals were checked for early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of
a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHERS (REPROTOXICITY):
- Observation of females with litters during lactation, seuxal maturation, estrous cyclicity, sperm parameters, litter observations.

Sacrifice and pathology:
> PARENTAL ANIMALS
SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10% formalin. The reproductive tracts of all females were also examined.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices; cranial; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix), vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Other examinations:
SACRIFICE / GROSS NECROPSY (OFFSPRING)
Pups that were not selected for post-weaning assessments were killed at the same time as their mother. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

Pups that died prematurely were sexed and checked for external abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination.
At planned termination of offspring, 3 male and 3 female pups were necropsied and examined for external abnormalities followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved and the weights of the brain, spleen and thymus were recorded from one of the 3 pups of each sex; these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also preserved.
The following were weighed for all animals: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY (PARENTAL ANIMALS)
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejenum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 751 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.

CLINICAL SIGNS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental. Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

BODY WEIGHT (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 μg/L and target 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 μg/L and target 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05) following covariance analysis brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed
to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration ofthe test material.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

OTHER FINDINGS (PARENTAL ANIMALS)
There were no treatment-related effects on estrous cyclicity, sperm parameters or on mating performance, fertility or duration of gestation in either generation.
Dose descriptor:
NOEL
Remarks:
F0 and F1
Effect level:
20 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed
Critical effects observed:
not specified

Blood Analysis Results

F0 Males

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

6

Prior to mating

6

13

23

27

Prior to Necropsy

6

19

27

29

F0 Females

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

7

7

7

7

Prior to mating

6

16

28

39

Prior to Necropsy

7

16

24

33

At target 20 μg/L, manganese levels prior to mating were 350% higher than controls in males and 550% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 383% and 371% for males and females.

At target 10 μg/L, manganese levels prior to mating were 283% higher than controls in males and 367% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 350% and 243% for males and females.

At target 5 μg/L, manganese levels prior to mating were 117% higher than controls in males and 167% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 217% and 129% for males and females.

F1 Males

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

12

16

16

17

Prior to mating

6

9

13

19

Prior to Necropsy

6

9

14

21

F1 Females

Time-point

Blood Mn conc (ppb w/v (ng/mL))

Group 1 (Control)

Group 2 (5 µg/L)

Group 3 (10 µg/L)

Group 4 (20 µg/L)

Pre-treatment

13

12

15

15

Prior to mating

6

10

16

23

Prior to Necropsy

7

10

16

21

At target 20 μg/L, manganese levels prior to mating were 217% higher than controls in males and 283% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 250% and 200% for males and females.

At target 10 μg/L, manganese levels prior to mating were 112% higher than controls in males and 133% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 167% and 129% for males and females.

At target 5 μg/L, manganese levels prior to mating were 50% higher than controls in males and females at the pre-mating timepoint. At terminal necropsy, these values were 50% and 43% for males and females.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Conclusions:
Under the conditions of the study the No Observed Effect Level (NOEL) of manganese chloride, was determined to be 20 µg/L, the highest concentration tested.
Executive summary:

The subchronic reproductive toxicity of manganese chloride was investigated in a two generation study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed with manganese chrloride for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment to inhalation exposure of manganese chloride were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L. At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls, however this reflected a slightly smaller litter size at this level. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment. There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L). In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

Inhalation of manganese chloride was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.No test substance-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations. However, these effects on the respiratory tract are specific to manganese chloride because it dissolves and recrystalises as sharp, irritating, crystals in the respiratory tract. This physico-chemical property of the chloride is not common to the carbonate and hence it can be concluded that the effects on the respiratory tract would not be apparent following exposure to manganese carbonate.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test substance through the mother’s milk during lactation resulted in an increased exposure to the test substance in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, the No Observed Effect Level was considered to be the target dose level 20 μg/L.

Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
Manganese oxide has low bioavailability (IUCLID section 7.12) in artificial lung fluid. The particle size distribution test (IUCLID section 4.5) indicates that inhalation is a possible route of exposure, but has low bioavailability in artificial lung fluid. In addition, MnO is not acutely harmful by the inhalation route (IUCLID section 7.2.2). Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3.9 mg/m³
Study duration:
subchronic
Species:
rabbit

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
A short-term toxicity study via the dermal route does not need to be conducted as the physiological properties of the substance do not suggest a significant rate of absorption through the skin and no systemic effects or other evidence of absorption were seen in the skin or eye irritation studies (IUCLID section 7.3) and furthermore the water solubility of the substance is very poor (IUCLID section 4.8), and therefore a limited amount of potential substance is available for systemic absorption via the dermal route. Therefore in accordance with Annex XI, section 1.1, this test is not considered necessary.
Critical effects observed:
not specified