Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-4-28 to 2010-5-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
GLP compliance:
yes
Test type:
fixed concentration procedure
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimanganese tetraoxide
EC Number:
215-266-5
EC Name:
Trimanganese tetraoxide
Cas Number:
1317-35-7
Molecular formula:
Mn3O4
IUPAC Name:
dimanganese(3+) manganese(2+) tetraoxidandiide
Test material form:
solid: particulate/powder
Details on test material:
- Molecular formula of test material: Mn3O4
- Appearance: Brown powder
- Physical state: Solid
- Composition of test material, percentage of components: Mn:71.2 %
- Purity test date: 26/09/08
- Lot/batch No.: 08100
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: HsdHan: WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK, Ltd. Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: The bodyweight varied between 200g to 350 g
- Housing: Animals were housed in groups of three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet : With the exception of the exposure period, free access to food (Harlan 2014 Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study.
- Water: With the exception of the exposure period, free access to mains drinking water was allowed throughout the study.
The diet, drinking water, bedding and chew blocks were routinely analysed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: A minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Consisted of a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber

- Exposure chamber volume: The cylindrical exposure chamber had a volume of approximately 30 litres (dimensions: 28 cm diameter x 50 cm high).

- Method of holding animals in test chamber: Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber 'O' ring. Only the nose of each animal was exposed to the test atmosphere

- Source and rate of air: The SAG 410 was connected to a meter compressed air supply.

- Method of conditioning air: Homogeneity of the test atmosphere within the chamber was not specifically determined during the study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals breathing zone with a wide variety of test materials (Green J D et al, 1984).

- System of generating particulates/aerosols: A dust atmosphere was produced from the test material using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany). A particulate separator was introduced before the aerosol entered the exposure chamber in order to remove large particles and thereby increase the inhalable portion of the generated aerosol.

- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (8.2, 5.7, 2.9, 1.4, 0.85 and 0.37 µm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test material, collected at each stage, calculated by difference. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.2, 5.7, 2.9, 1.4, 0.85 and 0.37 µm was calculated.

- Treatment of exhaust air: The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system.

- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/ humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacent port in the animals' breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough, East Sussex) located in a vacant port in the animals' breathing zone of the chamber and recorded every thirty minutes through out the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.


TEST ATMOSPHERE
- Samples taken from breathing zone: yes. The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fibre filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.



TEST ATMOSPHERE
- Particle size distribution: The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4µm (considered to be the inhalable fraction) was determined. See table 2.



Analytical verification of test atmosphere concentrations:
yes
Remarks:
Gravimetric analysis
Duration of exposure:
4 h
Concentrations:
5.17 mg/L
No. of animals per sex per dose:
3 Male and 3 Female
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for fourteen days. Any deaths or evidence of overt toxicity was recorded at each concentration. Individual bodyweights were recorded prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes. At the end of the fourteen day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
Data evaluations included the relationship, if any, between the animals' exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, necropsy findings, bodyweight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test material was made.

Results and discussion

Preliminary study:
No preliminary study
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.17 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.17 mg/L for four hours
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. Generalised fur stai
Body weight:
Individual bodyweights, together with weekly bodyweight changes, are given in table 9.
Variations in bodyweight gain are frequently seen for female animals of this strain and age during this type of study and, in isolation, are considered not to be significant.
All animals exhibited a slight bodyweight loss during the first three days post-exposure but recovered to show normal bodyweight development during the course of the study.
Gross pathology:
With the exception of one female animal, all animals exhibited dark patches on the lungs at necropsy

Any other information on results incl. tables

Table 3: The mortality data were summarised as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

5.17

0/3

0/3

0/6

 

Table 4: Exposure Chamber Atmosphere Concentration

Duration of Exposure (minutes)

Net Weight of Sample (mg)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

13.13

2

50

6.57

14

10.05

2

50

5.03

29

10.52

2

50

5.26

45

10.10

2

50

5.05

61

10.18

2

50

5.09

75

9.63

2

50

4.82

93

10.05

2

50

5.03

105

10.02

2

50

5.01

120

10.08

2

50

5.04

135

10.57

2

50

5.29

150

9.24

2

50

4.62

167

11.70

2

50

5.85

180

10.00

2

50

5.00

194

9.10

2

50

4.55

210

10.81

2

50

5.41

226

10.16

2

50

5.08

237

10.27

2

50

5.14

 

Mean achieved atmosphere concentration (mg/L) = 5.17

Standard deviation = 0.46

Test Material Used (g)

120

Air Flow (L/min)

50

Total Generation Time (mins)

252*

Nominal Concentration (mg/L)

7.40

 * Test atmospheres were generated for a total of 12 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle size distribution

 

Table 5: Cascade Impactor Data

Impactor Stage Number

Cut Point (µm)

Amount Collected (mg) per sample number

Mean Amount Collected (mg)

1

2

3

3

8.2

0.26

0.08

0.10

0.15

4

5.7

0.55

0.23

0.29

0.36

5

2.9

1.01

0.61

0.82

0.81

6

1.4

0.86

0.56

0.73

0.72

7

0.85

0.30

0.23

0.21

0.25

8

0.37

0.13

0.07

0.09

0.10

Back-up filter

 < 0.37

0.08

0.00

0.11

0.06

Total Mean Amount of Test Material Collected

2.45

 

Table 6: Calculation

Cut Point (μm)

Log10 Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

%

Probit

8.2

0.914

2.30

93.9

6.55

5.7

0.756

1.94

79.2

5.81

2.9

0.462

1.13

46.1

4.90

1.4

0.146

0.41

16.7

4.04

0.85

-0.071

0.16

6.53

3.49

0.37

-0.432

0.06

2.45

3.03

 

Mortality Data

Mean Achieved Atmosphere concentration

(mg/L)

Sex

Deaths

During

Exposure

Deaths Post Exposure

(1 Hour)

Deaths During Day of Observation

Total Deaths

1

2

3

4

5

6

7

8-14

5.17

Male

0

0

0

0

0

0

0

0

0

0

0/6

Female

0

0

0

0

0

0

0

0

0

0

 

Key To Clinical Observations

Fs = generalised brown fur staining by the test material

H = hunched posture

P = pilo-erection

Rd = decreased respiratory rate

Ri = increased respiratory rate

RI = laboured respiration

Wf= wet fur

L = Lethargy

 

Table 7: Individual Clinical Observations (Day of Exposure)

Mean achieved atmosphere concentration(mg/L)

Animal number and sex

Hours during exposure

On removal

From chamber

One hour post-exposure

1

2

3

 

 

 

 

 

 

 

 

 

 

 

 

5.17

1 Male

WfRi

WfRi

WfRi

Wf H P Ri Fs

 Wf H P Ri Fs

2 Male

WfRi

WfRi

WfRi

Wf H P Ri Fs

Wf H P Ri Fs

3 Male

WfRi

WfRi

WfRi

Wf H P Ri Fs

Wf H P Ri Fs

4 Female

WfRi

WfRi

WfRi

Wf H P Ri Fs

Wf H P Ri Fs

5 Female

WfRi

WfRi

WfRi

Wf H P Ri Fs

Wf H P Ri Fs

6 Female

WfRi

WfRi

WfRi

Wf H P Ri Fs

Wf H P Ri Fs

 

Table 8: Individual Clinical Observation (Recovery Period)

Mean achieved atmosphere concentration (mg/L)

Animal number and sex

Days post exposure

1

2

3

4

5

6

7

8-14

 

 

 

 

 

 

 

 

 

5.35

1 Male

H P Ri Fs

H P Rd RI L Fs

 H P Rd RI L Fs

 H P Rd RI Fs

 H P Ri Fs

 H P Ri Fs

H Ri

H Days 8 and 9 Ri Days 8 - 11

2 Male

H Ri Fs

H Ri Fs

 H Ri Fs

 H Ri Fs

 H Ri Fs

 H Ri

 H Ri

H Days 8 and 9 Ri Days 8 - 11

3 Male

H Ri Fs

H Ri Fs

H Ri Fs

 H Ri Fs

H Ri Fs

H Ri Fs

H Ri

H Days 8 and 9 Ri Days 8 - 11

4 Female

H P Ri Fs

H P Ri Fs

 H P Ri Fs

 H P Ri Fs

 H Ri Fs

 H Ri Fs

 H Ri

H Day 8 Ri Days 8 and 9

5 Female

H P Ri Fs

H P Ri Fs

 H P Ri Fs

H P Ri Fs

 H Ri Fs

 H Ri Fs

 H Ri

H Day 8 Ri Days 8 and 9

6 Female

H P Ri Fs

H P Ri Fs

 H P Ri Fs

 H P Ri Fs

 H Ri Fs

 H Ri

 H Ri

H Day 8 Ri Days 8 and 9

 

 

Table 9: Individual Bodyweights

Mean Achieved

Atmosphere

Concentration

(mg/L)

Animal Number

and Sex

Bodyweight (g) on Day:

Increment (g) During Days:

-22

0

1

3

7

14

-22-0

0-1

1-3

3-7

7-14

 5.17

1 Male

213

276

252

227

246

281

63

-24

-25

19

35

2 Male

228

304

290

268

283

310

76

-14

-22

15

27

3 Male

227

326

315

301

318

341

99

-11

-14

17

23

4 Female

192

216

211

207

210

219

24

-5

-4

3

9

5 Female

194

225

218

204

217

226

31

-7

-14

13

9

6 Female

205

229

218

205

225

230

24

-11

-13

20

5

 

 

Table 10: Individual Necropsy Findings

Mean Achieved

Atmosphere

Concentration

(mg/L)

Macroscopic Observations

Animal number and sex

5.17

Lungs: Dark Patches

1. Male

2. Male

3. Male

4. Female

5. Female

6. Female

 

P

P

P

 

P

P

 

 

 

 

N

 

 

 

 Table 11: Temperature and Relative Humidity in Exposure Chamber

Time (Minutes)

Chamber Temperature(°c) During Exposure

Chamber Relative Humidity (%) During Exposure

0

21

55

30

21

55

60

21

53

90

21

54

120

21

53

150

21

55

180

21

58

210

21

61

240

21

62

 

Table 12: Air Flow and Oxygen Concentration in Exposure Chamber

Time (Minutes)

Air Flow (L/min) During Exposure

Oxygen Concentration(%) During Exposure

-12*

50

-

0

50

20.8

30

50

-

60

50

-

90

50

-

120

50

20.8

150

50

-

180

50

-

210

50

-

240

50

20.8

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.17 mg/L for four hours.
It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Mn3O4, in the HsdHan:WIST stain rat, was greater than 5.17 mg/L.
Executive summary:

The acute toxicity of the test material via the inhalatory route was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 403 and EU Method B.2.

No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.17 mg/L for four hours.

It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Mn3O4, in the HsdHan:WIST stain rat, was greater than 5.17 mg/L.