Manganese dioxide

Endpoint summary

Description of key information

REPEATED DOSE ORAL TOXICITY

Supporting Study on Read Across Substance MnSO4 (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.

 

Supporting Study on Target Substance (Komura & Sakamoto (1991)

Under the conditions of the study no NOAEL was identified.

 

REPEATED DOSE INHALATION TOXICITY

Supporting Study on Read Across Substance MnCl2 (Grieve, 2017)

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

 

Supporting Study on Read Across Substance MnCl2 (Camner, 1985)

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.

 

Supporting Study on Read Across Substance MnSO4 (Dorman, 2006)

MnSO4 inhalation affected the haematology and resulted in increased Mn concentrations in the brain of the monkey.

 

Supporting Study on Target Substance (Rylander, 1971)

In the animals exposed to MnO2, no significant changes in their particulate or bacterial clearance capacity as compared to the controls were observed.

 

Supporting Study on Target Substance (Rylander, 1973)

Exposure to MnO2 caused a slight increase in the number of macrophages in the infection-controlled group. The number of leukocytes was increased in both types of guinea pig.

 

Supporting Study on Target Substance (Singh, 1977)

 Histologically the lungs of most of the animals in the experimental group revealed normal structure of the pulmonary tissue with negligible amount of dust discernible. In some animals, small deposits of dusts persisted and cellular nodules composed of mononuclear cells, predominantly macrophages and thin reticulin fibres developed. The control animals did not reveal any comparable histologic change.

 

Supporting Study on Target Substance (Sylvestre, 1984)

Under the conditions of the study, all exposed groups suffered emphysema-like tissue lesions, most severe in mice dosed with gas mixture plus MnO2.

 

MnO2 will be proposed for classification as STOT RE2 (H373)- target organ brain, on the basis of the Roels et al. (1992) study on battery workers - exposure is mainly to MnO2. The route of exposure in the study was inhalation and the sub-clinical effects seen at the exposure concentrations in the study, are believed to indicate that significant toxicity could occur at moderate exposure levels. On the basis of this proposal, it is considered that conducting animal testing would be both scientifically unjustified and unethical.  Therefore, in accordance with Annex XI, section 1.1 further sub-acute, sub-chronic and chronic toxicity tests are not considered necessary.

 

REPEATED DOSE DERMAL TOXICITY

Short-term, sub-chronic and chronic toxicity studies via the dermal route do 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, testing via the inhalation route is not considered necessary.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reference 2

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)	Survival^a	Mean body weight and weight changes^brelative			Final Weight Feed to controls (%)	Consumption^c
		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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The study was not performed under GLP conditions, and the report contains unexplained inconsistencies. It is therefore not possible to ascertain the relibaility of the findings.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice were exposed to MnO2 in their diet for 100 days and compared to controls fed on a diet containing 130 mg Mn/kg. Thirty days into the exposure period, mice were tested for spontaneous motor activity. Mice were decapitated 24 hours after their last feed. Blood samples were collected and analysed. Furthermore, tissues were removed, weighed and analysed for Mn content.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 28.2 ± 0.7 g
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

100 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
2 g Mn / kg
Basis:
nominal in diet

No. of animals per sex per dose:

8 males per dose group.

Control animals:

other: Standard laboratory mouse chow containing 130 mg Mn/kg

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Daily
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- How many animals: all animals
- Parameters checked included: red blood cell count, white blood cell count, haemoglobin and haematocrit

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: the mice were tested for spontaneous motor activity after an interval of 30 days. Activity was measured using an ANIMEX Activity Meter. Two mice were placed in a plastic cage at night, and after 10 minutes of acclimatisation, their activity was measured for 30 minutes.

Sacrifice and pathology:

Mice were decapitated at 24 hours after their last feed.

Tissue samples were removed, weighed, and stored at -20°C until analysis. Hair was rinsed with ethyl alcohol and washed withsodium lauryl sulphate, and then dried. Tissues were digested by the wet ashing method and the resulting solutions were analysed for Mn content by atomic absorption spectroscopy with a flame atomizer.
Tissues sampled included: liver, kidney, pancreas, prostate gland, spleen, brain, hair, bone and muscle.

Statistics:

Data were statistically analysed using Student's t-test and analysis of variance.

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(Mn content of various tissues)

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN: MnO2 did not cause any effect on weight change compared to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was similar to controls. The mean daily food consumption was 3.6 ± 0.9 g in the control group and 3.8 ± 0.7 g in the MnO2 group.

HAEMATOLOGY: White blood cell count was significantly lower than the control

NEUROBEHAVIOUR: No locomotor differences were seen compared to the control

GROSS PATHOLOGY: manganese content in tissues was significantly greater than controls in the liver, kidney, spleen, hair, bone and muscle

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Manganese concentration in various tissues are shown below. Values represent the mean +/- SD for 8 mice

Organ	Control	MnO2
Liver	2.80±0.39	4.30±0.58^c)
Kidney	2.84±0.23	3.70±0.37^c)
Pancreas	2.04±0.23	2.75±1.01
Prostate gland	1.18±0.21	1.60±0.40
Spleen	0.40±0.06	0.85±0.30 b)
Brain	0.97±0.25	1.45±0.50
Hair	2.98±0.42	4.13±0.85 b)
Bone	1.66±0.32	2.75±0.25^c)
Muscle	0.37±0.04	0.63±0.10^c)

b) p < 0.05

c) p < 0.01 (Student’s t-test), i.e. statistically significantly different to control

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Intake rate decreased within the first month but then became constant in all groups.

Conclusions:

Due to inconsistencies in the study, the certainty of the results for MnO2 are bought into question, and as such it is difficult to make solid conclusions. Given the low bioavailability of MnO2 (actually, the lowest for all manganese substances tested, see Anderson K, IUCLID section 7.12) and its insoluble nature (0.073 mg/L, See IUCLID section 4.8) the raised Mn concentrations relative to the control could also be seen as unusual.

Executive summary:

The repeated dose toxicity of manganese dioxide was investigated in a study in which mice were exposed to MnO2 in the diet for 100 days and compared to controls fed on a diet containing 130 mg Mn/kg. The mice receiving MnO2 were fed an additional Mn dose of approximately 200 mg Mn/kg bw, daily. Thirty days into the exposure period, mice were tested for spontaneous motor activity. Mice were decapitated 24 hours after their last feed. Blood samples were collected and analysed. Furthermore, tissues were removed, weighed and analysed for Mn content.

There was no effect on food intake or body weight development between the controls and the group fed MnO2 during the course of the study. White blood cells (40.6 ± 9.9 x 10^2/mm³) showed a slight decrease compared to controls (59.3 ± 18.0 x 10^2/mm³); the difference was statistically significant (p < 0.05). Mn concentrations in the liver, kidney, spleen, hair, bone, and muscle were significantly increased in the MnO2 group compared to the controls. In the control group, motor activity increased from days 15-45, thereafter remaining constant. The MnO2 group showed significantly less activity than the control group.

Due to inconsistencies in the study, the certainty of the results for MnO2 are bought into question, and as such it is difficult to make solid conclusions; the findings from this study are therefore diregarded.

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

Reference

Reference 1

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.

Reference 2

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

Reference 3

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

Reference 4

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.

Reference 5

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.

Reference 6

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

Reference 7

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:

MnO2 will be proposed for classification as STOT RE2 (H373)- target organ brain, on the basis of the Roels et al (1992) study on battery workers - exposure is mainly to MnO2. The route of exposure in the study was inhalation and the sub-clinical effects seen at the exposure concentrations in the study, are believed to indicate that significant toxicity could occur at moderate exposure levels. On the basis of this proposal it is considered that conducting animal testing would be both scientifically unjustified and unethical. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.

Critical effects observed:

not specified

Reference 8

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Not conducted to GLP but follows basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The pulmonary clearance of E coli and inert particles (radioactive, killed E.coli) was determined in inhalation experiments. Guinea pigs were exposed to MnO2 on its own or in combination with SO2.

GLP compliance:

not specified

Species:

guinea pig

Sex:

male

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: 90 % of particles < 0.5 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: MnO2 was generated in a Rag Pe aerosol generation unit.
- Temperature, humidity, pressure in air chamber: temperature and humidity were kept constant throughout the exposure at 23°C and 50% relative humidity.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
5.9 mg/m³
Basis:

No. of animals per sex per dose:

10 animals

Control animals:

yes

Critical effects observed:

not specified

In the animals exposed to MnO2, no significant changes in their particulate or bacterial clearance capacity as compared to the controls were observed.

Conclusions:

The results indicate the importance of synergistic effects in the evaluation of airborne pollutants.

Reference 9

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Not conducted to GLP but follows basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of MnO2 on the respiratory tract was investigated by exposing guinea pigs for 28 days.

GLP compliance:

no

Species:

guinea pig

Strain:

other: conventional and infection controlled animals were used

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: tap water

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: > 0.5 µm < 5.0 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: MnO2 dust was generated in a Rag Pe aerosol generator.
- Air change rate: 4 air changes per hour.
- Method of particle size determination: Royco Model 225 particle counter was used. At 20mg MnO2/m³ approx 600 000 particles per minute were found to be >0.5µm and <5.0µm in diameter.

Duration of treatment / exposure:

28 days

Frequency of treatment:

No data

Remarks:

Doses / Concentrations:
20 mg/m3
Basis:

No. of animals per sex per dose:

No data

Control animals:

yes

Critical effects observed:

not specified

Exposure to MnO2 caused a slight increase in the number of macrophages in the infection controlled group. The number of leukocytes was increased in both types of Guinea-pig. MnO2 caused a slight increase in the irritation score when compared to controls. This was more apparent in infection controlled animals.

Conclusions:

Effects of MnO2 on the infection controlled group were not observed in the normal animals.

Reference 10

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Very brief methodology. Even though the text stated that the lungs from treated rats retained as high as 12-fold the normal lung content of manganese, the table of results showed: control lungs 33.2±5.2 manganese/g and treated lungs 30.3±5.1 manganese/g which contradicts the text. Furthermore, the animals were dosed by intratracheal injection which is not relevant in relation to exposure by inhalation. As such, the reliability of the data is compromised.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult male Wistar rats were intratracheally given a single dose of MnO2 dust in saline and sacrificed after 30 days. The lungs were removed, one part was used for histopathological studies, the other used for enzyme activity assays. Other tissues were removed and observed for manganese distribution.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 150 ± 5 g
- Housing: air conditioned rooms
- Diet: standard pellet diet
- Water: ad libitum

Route of administration:

other: intratracheal administration

Vehicle:

other: 1mL saline

Remarks on MMAD:

MMAD / GSD: The MnO2 dust was particle size < 5micrometre.

Duration of treatment / exposure:

A single dose, animals were exposed for 30 days (the end of which they were sacrificed for analysis)

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
50 mg dust suspension of manganese dioxide in 1 mL saline.
Basis:

No. of animals per sex per dose:

10 animals inoculated with test material, and 10 control animals.

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF DOSING SOLUTIONS:
50 mg of dust suspension in 1 mL saline. Control animals received 1 mL saline only.

Critical effects observed:

not specified

The highest concentration was observed in liver (152% above control), followed by pancreas (103% above control), testis (54%), brain (52%), whole blood (36%) and kidney (25%). The study stated that the lung retained as high as 12-fold the normal lung content of manganese (although this statement is contradicted by a lower value - 30.3 nanomoles Mn/g tissue in experimental animals versus 33.2 for the control- later in the study).

Histological examination of the lungs of most of the animals in the treated group revealed normal structure of the pulmonary tissue with negligible amount of dust discernable (again contradicting the earlier statement). In some animals, small deposits of dusts persisted and cellular nodules composed of mononuclear cells, predominantly macrophages and thin reticulin fibres developed. The control animals did not reveal any comparable histologic change.

The results suggest that manganese dioxide dust does not invoke significant biochemical and histopathological alterations in lungs.

Distribution of protein in the lung of normal and MnO2inoculated rats:   Protein content (mg/g fresh tissue) Control Experimental Whole homogenate 90.4 ± 2.53 96.4 ± 2.02 Mitochondrial fraction 9.2 ± 0.29 10.0 ± 0.44 Post-mitochondrial supernatant 53.4 ± 1.21 52.2 ± 0.97   Changes in enzyme activity of the lung in normal and MnO2inoculated rats:   Enzyme activity^a Control Experimental Whole homogenate Glutamic-oxaloacetate transaminase 132 ± 11.7 130 ± 9.1 Glutamic-pyruvate transaminase 82 ± 4.0 67 ± 1.0 Acid phosphatase 9 ± 1.0 10 ± 0.4 Alkaline phosphatase 110 ± 2.1 80 ± 10.0^b 5’Nucleotidase 4.2 ± 0.2 3.0 ± 0.1 Mitochondrial fraction Succinic dehydrogenase 59 ± 4.7 72 ± 9.0 Mg^2+-Adenosine triphosphatase 398 ± 27.1 400 ± 18.1 NADH-cytochrome c reductase 83 ± 8.0 103 ± 12.0^b Cytochrome c oxidase 49 ± 4.0 55 ± 2.2 Diaphorase 93 ± 6.1 89 ± 6.0 Malic dehydrogenase 492 ± 28.0 526 ± 26.0 Post-mitochondrial supernatant Lactic dehydrogenase 772 ± 40.0 796 ± 41.0 Malic dehydrogenase 844 ± 24.0 873 ± 40.0 Phosphoglucomutase 192 ± 17.0 295 ± 21.0^b Aldolase 87 ± 5.0 69 ± 1.0^c ^ananomoles of the respective substrate transformed/min/mg protein ^bP<0.02 ^cP<0.01   Tissues Mn content Control Experimental Brain 13.0 ± 2.1 24.9 ± 1.8^b Blood^a 17.1 ± 5.9 23.3 ± 4.5 Kidney 16.0 ± 0.9 20.0 ± 1.7 Liver 38.1 ± 5.9 96.2 ± 11.5^b Lung 33.2 ± 5.2 30.3 ± 5.1^c Pancreas 23.0 ± 3.3 46.9 ± 2.1^c Testis 10.0 ± 1.6 16.8 ± 1.9^d ^aµmol/L ^bP< 0.01 ^cP< 0.001 ^dP< 0.05     Recovery of protein: The protein content estimated from the whole homogenate, mitochondrial and post mitochondrial supernatant fractions remained unchanged.

Conclusions:

Since only a single dose was given, this cannot be considered as a repeat dose study as such. The dose given was not as an aerosol, and other mechanisms could have been operating that would not be seen in a modern study. Therefore the adequacy for risk assessment and classification is not sufficient.

Executive summary:

Adult male Wistar rats were intratracheally given a single dose of MnO2 dust in saline and sacrificed after 30 days. The lungs were removed, one part was used for histopathological studies, the other used for enzyme activity assays. Other tissues were removed and observed for manganese distribution.

 Histologically the lungs of most of the animals in the experimental group revealed normal structure of the pulmonary tissue with negligible amount of dust discernible. In some animals, small deposits of dusts persisted and cellular nodules composed of mononuclear cells, predominantly macrophages and thin reticulin fibres developed. The control animals did not reveal any comparable histologic change.

Even though the text stated that the lungs from treated rats retained as high as 12-fold the normal lung content of manganese, the table of results showed: control lungs 33.2 ± 5.2 manganese/g and treated lungs 30.3 ± 5.1 manganese/g which contradicts the text. Furthermore, the animals were dosed by intratracheal injection which is not relevant in relation to exposure by inhalation. As such, the reliability of the data is compromised and the data were disregarded.

Reference 11

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Summary only - very limited data available on materials and methods.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of mice were exposed to air pollutants, usually found in welding fume, and manganese dioxide for 30 days (exposure for 5 hours a day for 7 days a week) and the effects on the lungs evaluated.

GLP compliance:

no

Species:

mouse

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 25 days old
- Weight at study initiation: 26.76 g (average)

Route of administration:

inhalation: dust

Duration of treatment / exposure:

30 days

Frequency of treatment:

5 hours/day, 7 days/week

Critical effects observed:

not specified

All exposed groups suffered emphysema-like tissue lesions, most severe in mice dosed with gas mixture plus MnO2.

Executive summary:

Groups of mice were exposed to air pollutants, usually found in welding fume, and manganese dioxide for 30 days (exposure for 5 hours a day for 7 days a week) and the effects on the lungs evaluated.

Under the conditions of the study, all exposed groups suffered emphysema-like tissue lesions, most severe in mice dosed with gas mixture plus MnO2.

Reference 12

Endpoint:

chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

MnO2 will be proposed for classification as STOT RE2 (H373)- target organ brain, on the basis of the Roels et al (1992) study on battery workers - exposure is mainly to MnO2. The route of exposure in the study was inhalation and the sub-clinical effects seen at the exposure concentrations in the study, are believed to indicate that significant toxicity could occur at moderate exposure levels. On the basis of this proposal it is considered that conducting animal testing would be both scientifically unjustified and unethical. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.

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

Reference

Reference 1

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.

Reference 2

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

Reference 3

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

Reference 4

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.

Reference 5

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.

Reference 6

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

Reference 7

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:

MnO2 will be proposed for classification as STOT RE2 (H373)- target organ brain, on the basis of the Roels et al (1992) study on battery workers - exposure is mainly to MnO2. The route of exposure in the study was inhalation and the sub-clinical effects seen at the exposure concentrations in the study, are believed to indicate that significant toxicity could occur at moderate exposure levels. On the basis of this proposal it is considered that conducting animal testing would be both scientifically unjustified and unethical. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.

Critical effects observed:

not specified

Reference 8

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Not conducted to GLP but follows basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The pulmonary clearance of E coli and inert particles (radioactive, killed E.coli) was determined in inhalation experiments. Guinea pigs were exposed to MnO2 on its own or in combination with SO2.

GLP compliance:

not specified

Species:

guinea pig

Sex:

male

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: 90 % of particles < 0.5 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: MnO2 was generated in a Rag Pe aerosol generation unit.
- Temperature, humidity, pressure in air chamber: temperature and humidity were kept constant throughout the exposure at 23°C and 50% relative humidity.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
5.9 mg/m³
Basis:

No. of animals per sex per dose:

10 animals

Control animals:

yes

Critical effects observed:

not specified

In the animals exposed to MnO2, no significant changes in their particulate or bacterial clearance capacity as compared to the controls were observed.

Conclusions:

The results indicate the importance of synergistic effects in the evaluation of airborne pollutants.

Reference 9

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Not conducted to GLP but follows basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of MnO2 on the respiratory tract was investigated by exposing guinea pigs for 28 days.

GLP compliance:

no

Species:

guinea pig

Strain:

other: conventional and infection controlled animals were used

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: tap water

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: > 0.5 µm < 5.0 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: MnO2 dust was generated in a Rag Pe aerosol generator.
- Air change rate: 4 air changes per hour.
- Method of particle size determination: Royco Model 225 particle counter was used. At 20mg MnO2/m³ approx 600 000 particles per minute were found to be >0.5µm and <5.0µm in diameter.

Duration of treatment / exposure:

28 days

Frequency of treatment:

No data

Remarks:

Doses / Concentrations:
20 mg/m3
Basis:

No. of animals per sex per dose:

No data

Control animals:

yes

Critical effects observed:

not specified

Exposure to MnO2 caused a slight increase in the number of macrophages in the infection controlled group. The number of leukocytes was increased in both types of Guinea-pig. MnO2 caused a slight increase in the irritation score when compared to controls. This was more apparent in infection controlled animals.

Conclusions:

Effects of MnO2 on the infection controlled group were not observed in the normal animals.

Reference 10

Endpoint:

repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Very brief methodology. Even though the text stated that the lungs from treated rats retained as high as 12-fold the normal lung content of manganese, the table of results showed: control lungs 33.2±5.2 manganese/g and treated lungs 30.3±5.1 manganese/g which contradicts the text. Furthermore, the animals were dosed by intratracheal injection which is not relevant in relation to exposure by inhalation. As such, the reliability of the data is compromised.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult male Wistar rats were intratracheally given a single dose of MnO2 dust in saline and sacrificed after 30 days. The lungs were removed, one part was used for histopathological studies, the other used for enzyme activity assays. Other tissues were removed and observed for manganese distribution.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 150 ± 5 g
- Housing: air conditioned rooms
- Diet: standard pellet diet
- Water: ad libitum

Route of administration:

other: intratracheal administration

Vehicle:

other: 1mL saline

Remarks on MMAD:

MMAD / GSD: The MnO2 dust was particle size < 5micrometre.

Duration of treatment / exposure:

A single dose, animals were exposed for 30 days (the end of which they were sacrificed for analysis)

Frequency of treatment:

Single dose

Remarks:

Doses / Concentrations:
50 mg dust suspension of manganese dioxide in 1 mL saline.
Basis:

No. of animals per sex per dose:

10 animals inoculated with test material, and 10 control animals.

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF DOSING SOLUTIONS:
50 mg of dust suspension in 1 mL saline. Control animals received 1 mL saline only.

Critical effects observed:

not specified

The highest concentration was observed in liver (152% above control), followed by pancreas (103% above control), testis (54%), brain (52%), whole blood (36%) and kidney (25%). The study stated that the lung retained as high as 12-fold the normal lung content of manganese (although this statement is contradicted by a lower value - 30.3 nanomoles Mn/g tissue in experimental animals versus 33.2 for the control- later in the study).

Histological examination of the lungs of most of the animals in the treated group revealed normal structure of the pulmonary tissue with negligible amount of dust discernable (again contradicting the earlier statement). In some animals, small deposits of dusts persisted and cellular nodules composed of mononuclear cells, predominantly macrophages and thin reticulin fibres developed. The control animals did not reveal any comparable histologic change.

The results suggest that manganese dioxide dust does not invoke significant biochemical and histopathological alterations in lungs.

Distribution of protein in the lung of normal and MnO2inoculated rats:   Protein content (mg/g fresh tissue) Control Experimental Whole homogenate 90.4 ± 2.53 96.4 ± 2.02 Mitochondrial fraction 9.2 ± 0.29 10.0 ± 0.44 Post-mitochondrial supernatant 53.4 ± 1.21 52.2 ± 0.97   Changes in enzyme activity of the lung in normal and MnO2inoculated rats:   Enzyme activity^a Control Experimental Whole homogenate Glutamic-oxaloacetate transaminase 132 ± 11.7 130 ± 9.1 Glutamic-pyruvate transaminase 82 ± 4.0 67 ± 1.0 Acid phosphatase 9 ± 1.0 10 ± 0.4 Alkaline phosphatase 110 ± 2.1 80 ± 10.0^b 5’Nucleotidase 4.2 ± 0.2 3.0 ± 0.1 Mitochondrial fraction Succinic dehydrogenase 59 ± 4.7 72 ± 9.0 Mg^2+-Adenosine triphosphatase 398 ± 27.1 400 ± 18.1 NADH-cytochrome c reductase 83 ± 8.0 103 ± 12.0^b Cytochrome c oxidase 49 ± 4.0 55 ± 2.2 Diaphorase 93 ± 6.1 89 ± 6.0 Malic dehydrogenase 492 ± 28.0 526 ± 26.0 Post-mitochondrial supernatant Lactic dehydrogenase 772 ± 40.0 796 ± 41.0 Malic dehydrogenase 844 ± 24.0 873 ± 40.0 Phosphoglucomutase 192 ± 17.0 295 ± 21.0^b Aldolase 87 ± 5.0 69 ± 1.0^c ^ananomoles of the respective substrate transformed/min/mg protein ^bP<0.02 ^cP<0.01   Tissues Mn content Control Experimental Brain 13.0 ± 2.1 24.9 ± 1.8^b Blood^a 17.1 ± 5.9 23.3 ± 4.5 Kidney 16.0 ± 0.9 20.0 ± 1.7 Liver 38.1 ± 5.9 96.2 ± 11.5^b Lung 33.2 ± 5.2 30.3 ± 5.1^c Pancreas 23.0 ± 3.3 46.9 ± 2.1^c Testis 10.0 ± 1.6 16.8 ± 1.9^d ^aµmol/L ^bP< 0.01 ^cP< 0.001 ^dP< 0.05     Recovery of protein: The protein content estimated from the whole homogenate, mitochondrial and post mitochondrial supernatant fractions remained unchanged.

Conclusions:

Since only a single dose was given, this cannot be considered as a repeat dose study as such. The dose given was not as an aerosol, and other mechanisms could have been operating that would not be seen in a modern study. Therefore the adequacy for risk assessment and classification is not sufficient.

Executive summary:

Adult male Wistar rats were intratracheally given a single dose of MnO2 dust in saline and sacrificed after 30 days. The lungs were removed, one part was used for histopathological studies, the other used for enzyme activity assays. Other tissues were removed and observed for manganese distribution.

 Histologically the lungs of most of the animals in the experimental group revealed normal structure of the pulmonary tissue with negligible amount of dust discernible. In some animals, small deposits of dusts persisted and cellular nodules composed of mononuclear cells, predominantly macrophages and thin reticulin fibres developed. The control animals did not reveal any comparable histologic change.

Even though the text stated that the lungs from treated rats retained as high as 12-fold the normal lung content of manganese, the table of results showed: control lungs 33.2 ± 5.2 manganese/g and treated lungs 30.3 ± 5.1 manganese/g which contradicts the text. Furthermore, the animals were dosed by intratracheal injection which is not relevant in relation to exposure by inhalation. As such, the reliability of the data is compromised and the data were disregarded.

Reference 11

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Summary only - very limited data available on materials and methods.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of mice were exposed to air pollutants, usually found in welding fume, and manganese dioxide for 30 days (exposure for 5 hours a day for 7 days a week) and the effects on the lungs evaluated.

GLP compliance:

no

Species:

mouse

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 25 days old
- Weight at study initiation: 26.76 g (average)

Route of administration:

inhalation: dust

Duration of treatment / exposure:

30 days

Frequency of treatment:

5 hours/day, 7 days/week

Critical effects observed:

not specified

All exposed groups suffered emphysema-like tissue lesions, most severe in mice dosed with gas mixture plus MnO2.

Executive summary:

Groups of mice were exposed to air pollutants, usually found in welding fume, and manganese dioxide for 30 days (exposure for 5 hours a day for 7 days a week) and the effects on the lungs evaluated.

Under the conditions of the study, all exposed groups suffered emphysema-like tissue lesions, most severe in mice dosed with gas mixture plus MnO2.

Reference 12

Endpoint:

chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

MnO2 will be proposed for classification as STOT RE2 (H373)- target organ brain, on the basis of the Roels et al (1992) study on battery workers - exposure is mainly to MnO2. The route of exposure in the study was inhalation and the sub-clinical effects seen at the exposure concentrations in the study, are believed to indicate that significant toxicity could occur at moderate exposure levels. On the basis of this proposal it is considered that conducting animal testing would be both scientifically unjustified and unethical. Therefore in accordance with Annex XI, section 1.1 this test is not considered necessary.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

3.9 mg/m³

Study duration:

subchronic

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

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:

In accordance with Column 1 of REACH Annex VIII, repeated dose toxicity testing on the most appropriate route of administration shall be conducted having regard to the likely route of human exposure. For the registered substance, inhalation is deemed to be the most likely route of human exposure and therefore repeated dose toxicity testing via the dermal route is not considered scientifically necessary. The physiochemical properties of MnO2 suggest it is unlikely to be absorbed through the skin. In addition, MnO2 has a very low solubility in water, which coupled with its physical inorganic nature (powder) means that it is unlikely to be readily absorbed through the skin and therefore it is highly unlikely that it would be toxic via the dermal route. Furthermore, on animal welfare grounds, adaptation is appropriate in consideration of the extensive use of experimental animals that would be required.

Critical effects observed:

not specified