Cobalt sulphide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-19 to 2016-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2019

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

2009-09-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-08-14

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tricobalt tetraoxide

EC Number:

215-157-2

EC Name:

Tricobalt tetraoxide

Cas Number:

1308-06-1

Molecular formula:

Co3O4

IUPAC Name:

215-157-2

Constituent 2

Reference substance name:

Cobalt sulphate

EC Number:

233-334-2

EC Name:

Cobalt sulphate

Cas Number:

10124-43-3

Molecular formula:

Co.H2O4S

IUPAC Name:

cobalt(2+) sulfate

Test material form:

other: 1) Tricobalt tetraoxide: solid: particulate/powder; 2) Cobalt sulfate heptahydrate: solid: crystalline

Details on test material:

1) Tricobalt tetraoxide (test item)
- State of aggregation: grey-black powder (odourless)
- Particle size (laser difffraction): d50: 1.6 µm
- Relative density: 6.11 (at 25 °C)
- Water solubility: 1.62 mg/L (at 20 °C)

2) Cobalt sulfate heptahydrate (reference item)
- State of aggregation: reddish crystals (odourless)
- Relative density: 1.95 (at 25 °C)
- Water solubility: 376.7 g/L (at 20 °C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
1) Tricobalt tetraoxide (test item)
- Storage condition of test material: at room temperature, dry, protected from light

2) Cobalt sulfate heptahydrate (reference item)
- Storage condition of test material: at room temperature, dry, at a dark place

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*Reference:
- Vu, V., Barrett, J.C., Roycroft, J., Schuman, L., Dankovic, D..Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212 (1996)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males: approx. 250 g; females: approx. 175 g
- Housing: housed in Makrolon® (polycarbonate) cages type III, two rats per cage; bedding material: absorbing softwood (Lignocel BK 8-15')
- Diet (ad libitum): commercial chow in pellet form (Ssniff "V1534"; ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 2 - 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
- food quality: a certificate of feed analysis is issued by the supplier periodically. The analysis is done at the LUFA-ITL in Kiel, Germany, i.e. in a certified lab for this kind of analysis (DIN EN ISO/IEC 17025:2000).
- water quality: a certificate of water analysis issued by the water supplier (Stadtwerke Hannover) is sent periodically to Fraunhofer ITEM.

ENVIRONMENTAL CONDITIONS
- Temperature: 22° + 2° C
- Relative humidity: 55% + 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.51 - <= 2.14 µm

Remarks on MMAD:

MMAD (tricobalt tetraoxide): 1.81 µm - 2.14 µm
MMAD (cobalt sulfate heptahydrate): 1.51 µm - 1.59 µm
GSD (tricobalt tetraoxide): 1.97 - 2.26
GSD (cobalt sulfate heptahydrate): 2.07 - 2.23

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus / Method of holding animals in test chamber : the aerosol is given to the rats by a flow-past nose-only inhalation exposure system. Aerosols are supplied to each rat individually. For exposure to the test item the rats were restrained in acrylic tubes with adjustable backstops. The exposure tubes are arranged around a cylinder capable to take up 16 tubes per platform. The rat nose is located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (1x clean air control, 3x treatment Co3O4, 2x treatment CoSO4 * 7H2O) are located each under a separate hood to prevent contamination among different dose groups.

For a period of 2 - 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes.

- Air flow rate: airflow to each rat is approx. 1 L/min which is calculated to be laminar (rat minute volume: 0.2 L). Therefore measurement of the oxygen concentration is not necessary.

- System of generating particulates/aerosols:
1) Tricobalt tetraoxide: the particulate sample aerosols were generated by dispersing the dry powder of Co3O4. Dispersion is achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by the laboratory. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test substance under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer

2) Cobalt sulfate heptahydrate: the particulate sample aerosols were generated by nebulising an aqueous solution of CoSO4 x 7 H2O using pressurised air. Due to rapid evaporation of the droplets the rats were exposed to solid particles.

- Temperature, humidity, air flow: air flow, temperature and relative humidity were measured continuously and recorded by 20-minute means. The limits were set at 22° C + 2° C for temperature and 55 % + 15 % for relative humidity. Animal room lighting was on a 12 hour light/dark cycle controlled by an automatic timing device.

- Method of particle size determination: the MMAD was determined twice during the 28-day exposure period for each test/reference item exposure unit (6 units) by a cascade impactor (Marple impactor).

TEST ATMOSPHERE
- Brief description of analytical method used: the actual aerosol concentrations was measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration is determined throughout the study by comparing to gravimetric concentrations.
Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol. The evaluation of filter samples was done by gravimetrical analysis. As a permanent control of the aerosol concentrations is guaranteed by photometers the scheduled filter sampling frequency is sufficient (in agreement with OECD guideline 412).
- Samples taken from breathing zone: yes
The measured mean concentrations were very close to the target concentration for all dose groups of the test and reference item, i.e. amounted to 102%, 103% and 101% in the Co3O4 and to 101% and 101% in the CoSO4 groups.

Prior to the 28-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

Tricobalt tetraoxide:
Nominal aerosol concentrations of 5, 20, and 80 mg/m³ were used for the test item in the low, mid and high dose groups, respectively. According to the MPPD model, in the low, mid and high dose groups deposited masses of approx. 0.37, 1.5 and 6 mg/lung could be expected for an aerosol with an MMAD of approx. 1.75 µm (5.2% deposition fraction), thus inducing a particle overload situation in the high dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations were very close to the target concentration for all dose groups of tricobalt tetraoxide, i.e. amounted to 102, 103 and 101 % in the low, mid and high dose groups.
The mean concentrations were very close to the target concentration for all dose groups of cobalt sulfate heptahydrate, i.e. amounted to 101 % for each dose group.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5.12 mg/m³ air (analytical)

Remarks:

SD: 0.87; tricobalt tetraoxide (test item)

Doses / concentrations 2

Dose / conc.:

20.5 mg/m³ air (analytical)

Remarks:

SD: 2.18; tricobalt tetraoxide (test item)

Doses / concentrations 3

Dose / conc.:

80.78 mg/m³ air (analytical)

Remarks:

SD: 7.70; tricobalt tetraoxide (test item)

Doses / concentrations 4

Dose / conc.:

2.04 mg/m³ air (analytical)

Remarks:

SD: 0.21; cobalt sulfate heptahydrate (reference item)

Doses / concentrations 5

Dose / conc.:

10.19 mg/m³ air (analytical)

Remarks:

SD: 0.83; cobalt sulfate heptahydrate (reference item)

No. of animals per sex per dose:

main study: 10 males / 10 females
recovery group: 10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a 14-day dose range finding study was conducted in male and female Crl: WI rats using nose-only exposure. The test substances were tricobalt tetraoxide (13.41, 46.14 and 219.16 mg/m³) and cobalt sulfate heptahydrate (1.81 and 8.94 mg/m³). Groups of eight males and eight females were used. A negative control group (clean air) was run concurrently. The following endpoints were investigated: clinical observation, body weight, gross pathology, histopathology, immunohistochemistry and bronchoalveolar lavage.

All male and female test animals survived treatment and effects indicating systemic toxicity were not observed. Sex-specific differences were not detected. Furthermore, body weight development did not show any statistically significant changes as compared to concurrent controls.
Lung weights showed statistically significant changes as compared to concurrent controls in the mid/high Co3O4 and the high CoSO4 dose groups. In addition, bronchoalveolar lavage analysis showed a statistically significant increase in the polymorphonuclear neutrophil (PMN) percentages in the Co3O4 mid and high dose groups of both sexes and in the female CoSO4 high dose group.
The histopathological examination did not show any adverse effects for Co3O4 in the respiratory tract. In lungs, a dose-dependent alveolar/interstitial accumulation of particle-laden macrophages was observed. However, CoSO4 exposed animals exhibited adverse changes in the larynx in both exposure groups.
Lastly, immunohistochemistry investigation did not showed any increase of 8-OH-dG-positive cells in lung parenchyma.

- Post-exposure recovery period in recovery groups: 3 months
Upon cessation of exposure the animals of the recovery groups were kept in Makrolon® cages.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (main study and recovery group)
- Time schedule: at least once a day (cages); at least before and after exposure and sometimes also during exposure
- Cage side observations checked: clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes (main study and recovery group)
- Time schedule: once a week

BODY WEIGHT: Yes (main study and recovery group)
- Time schedule for examinations: twice a week in the first 4 weeks and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION: Yes (main study and recovery group):
- Time schedule for examinations: weekly during the study period (including post-exposure observation period) using 10 and after day 1 5 animals per dose and sex group

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes (main study and recovery group)
- Time schedule for examinations: weekly during the study period (including post-exposure observation period) using 10 and after day 1 five animals per dose and sex group

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (main study and recovery group)
- Time schedule for collection of blood: day 1 after exposure (exception: blood for prothrombin and thromboplastin times were taken only before sacrifice) an after additional 91 days after exposure
- Anaesthetic used for blood collection: Yes, slight isoflurane anesthesia
- Animals fasted: Yes, 16-hour fasting period (tap water ad libitum)
- How many animals: 5 animals/group/sex
- Parameters checked: red blood cells, hemoglobin, hematocrit, reticulocytes, mean cell volume, mean hemoglobin/erythrocyte, mean hemoglobin concentration/erythrocyte, prothrombin time, total white blood cells, differential white cell count, platelets,

CLINICAL CHEMISTRY: Yes (main study and recovery group)
- Time schedule for collection of blood: day 1 after exposure (exception: blood for prothrombin and thromboplastin times were taken only before sacrifice)
- Animals fasted: Yes 16-hour fasting period (tap water ad libitum)
- How many animals: 5 animals/group/sex
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-Glutamyl transpeptidase, urea, triglyceride, total bilirubin, creatinine, total protein, albumin, globulin, ALB/GLB, glucose, cholesterol, sodium, calcium, potassium, phosphorus and chloride

URINALYSIS: Yes (main study and recovery group; 5 males / 5 females/dose group))
- Time schedule for collection of urine: day 1 after exposure
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, and leukocytes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: Yes
Oxidative damage of the lung tissue can result in genotoxicity in situ effects, e.g. the occurrence of 8-hydroxy-deoxyguanosine - 8-OH-dG. The immunohistochemically stained cells were quantified in the lung tissue and the epithelium of the terminal bronchioles. Examination was done in the left lung lobe of 5 animals per time-point and group in the control groupand high dose groups (80.78 mg Co3O4/m³ and 10.19 mg CoSO4 *H2O/m³ groups) 1 day upon cessation of exposure.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (main study and recovery group)
All animals were subjected to a complete necropsy, which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The rats used for bronchoalveolar lavage investigations (BAL) were anesthetized with an overdose pentobarbital sodium and killed. For rats used for histopathological investigations an overdose of carbon dioxide was used.
The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) were removed from the lung convolution.
The lung and the lower half of the trachea were weighed, and used for BAL or histopathology. For histopathology the lung was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion, and used for histopathology.
The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart. The respiratory tract was preserved as follows: nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 412 were prepared for histopathology.


HISTOPATHOLOGY: Yes (main study and recovery group)
The histopathology of all organs (respiratory tract, adrenal glands, bone marrow of the femur, brain (cerebrum and cerebellum), esophagus, femur with knee joint, heart, kidneys, liver, ovaries, seminal vesicles, spinal cord (cervical, thoracic and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands and uterus) was performed in all animals per group and sex at days 1 and day 91 post-exposure (with the exception of the 5.12 mg Co3O4/m³, 20.50 mg Co3O4/m³ and 2.04 mg CoSO4 * 7H2O/m³ groups); particular attention was paid to:
• Histopathology of respiratory tract including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) (respiratory tract will be examined in groups 5.12 mg Co3O4/m³, 20.50 mg Co3O4/m³ and 2.04 mg CoSO4 + H2O/m³ groups, too);
• Trimming: lungs 5 sections; nose 4 sections; larynx: 3 sections; trachea: 1 section
Tissues for histological examination will be fixed for at least one week in buffered formalin (10%). Bones will be decalcified prior to embedding.
Lung lobes will be fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H & E). In addition, masson trichrome staining will be used for detection of connective tissue production (lower half).

Other examinations:

BRONCHOALVEOLAR LAVAGE (main study and recovery group)
Bronchoalveolar lavage was performed in 5 male and 5 female rats per group after end of exposure (day 1) and following end of recovery (day 91). The method of Henderson et al. (1987)* was used with minor modifications.
Following preparation, the lungs were lavaged with saline using two lavages each. The lavage fluid was collected in calibrated tubes and the harvested volume was recorded. The leukocyte concentration of the lavagate was determined using a counting chamber and two cytoslides were prepared with a cytocentrifuge for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes).
After centrifugation of the lavage fluid, biochemical indicators relevant for diagnosis of lung damage were determined in the supernatant (lactic dehydrogenase - LDH, β-glucuronidase, total protein). These parameters were analyzed according to routine clinical chemistry protocols using a Cobas Fara device.

The justification of the parameters is given below:
Cytological parameters
• total cell count (recruitment of lung leukocytes)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions; a total of 400 leukocytes per rat will be evaluated)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells)
Cytokine levels (MCP-1, IL-8 alias CINC-1, HIF-1α) were analysed using ELISA. Along with the samples to be analysed, a standard curve of the respective cytokine was included on each plate. The plates were read on a microplate reader, i.e. a Tecan InfiniteF200Pro. Cytokine levels were determined using a four-parameter logistic curve fit to the standard curve.
• Monocyte chemoattractant protein-1 (MCP-1)
• Interleukin-8 (IL-8 alias CINC-1)
• Hypoxia-inducible factor 1-α (HIF-1α)
The HIF1-α analysis was expanded to the BALF cell sediments (gained by centrifugation) to juxtapose the two compartments (BALF supernatant and BALF cell sediment).

*Reference:
- Henderson, R.F., Mauderly, J.L. Pickrell, J.A., Hahn, R.F., Muhle, H., and Rebar, A.H. Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329 342 (1987)

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test (Dunnett, 1955; 1964)*.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the PROVANTIS system.

*References
- Dunnett C. W. A multiple comparison procedure for comparing several treatments with a control, J Am Stat Assoc, 50, 1096–1121 (1955)
- Dunnett C. W. New tables for multiple comparisons with a control, Biometrics, 20, 482–491 (1964)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Tricobalt tetraoxide
80.78 mg/m³: one day after end of exposure, in females, the relative lung weights were statistically significantly increased (treatment group (lung/bw g/kg): 8.68; control group (lung/bw g/kg): 6.41; p < 0.01). At day 91 of the recovery period absolute and relative lung weights in the male and female Co3O4 high dose groups came out with statistically significant increases as compared to controls.
Males:
Absolute lung weight (g): treatment group: 3.07; control group: 2.00; p< 0.01
Relative lung weight (lung/bw g/kg): treatment group: 7.47; control group: 4.95; p< 0.01
Females:
Absolute lung weight (g): treatment group: 2.08; control group: 1.19; p< 0.01
Relative lung weight (lung/bw g/kg): treatment group: 9.43; control group: 5.36; p< 0.01

Absolute and relative lung weights of rats used for bronchoalveolar lavage showed a statistically significant increase at day 1 in the 80.78 mg Co3O4/m³ and 10.19 mg CoSO4 * 7H2O/m³ dose groups (males and females). At day 91 of the post-exposure observation period the findings in the Co3O4 groups persisted whereas in the CoSO4 * 7H2O groups all values had returned to normal values.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Tricobalt tetraoxide
5.12, 20.50 and 80.78 mg/m³:
a) Lung (after 28 days of exposure; 5 animals/sex7dose):
Within the lung, there was a dose-dependent multifocal very slight to slight alveolar accumulation of particle-laden macrophages in the low, mid and high dose exposed male (all very slight in low and mid dose and all slight in the high dose group). In addition, there was a very slight multifocal interstitial accumulation of particle-laden macrophages in 2/5 mid dose males and females and in all high dose males and females. Furthermore, a very slight accumulation of particle-laden macrophages in the bronchus-associated lymphoid tissue (BALT) was seen in 2/5 mid dose males and females and in all high dose males and females. Particles free in the alveoli were observed dose-dependent in all mid dose males and females (all very slight) and all high dose males and females (all slight). The free particles were not within cells but attached to eosinophilic granular material, which was interpreted as proteinaceous material. A very slight lipoproteinosis was visible in 2/5 mid dose males and females and in all high dose males and females. The lipoproteinosis is most likely cellular detritus from alveolar macrophages but might also represent exudate from pulmonary vessels. In 1/5 high dose males and females a very slight multifocal alveolar granulocytic cell infiltration was found. There was a very slight multifocal mononuclear cell infiltration in the lung parenchyma at the terminal bronchus in 3/5 high dose males and in 1/5 mid dose females and in 4/5 high dose females.

b) Lung (after additional 90 days of recovery; 5 animals/sex/dose)
Within the lung, there was a dose-dependent multifocal very slight to slight alveolar accumulation of particle-laden macrophages in the low, mid and high dose exposed male (all very slight in low and mid dose and all slight in the high dose group). In addition, there was a very slight multifocal interstitial accumulation of particle-laden macrophages in 3/5 mid dose males and females and in all high dose males and females. Furthermore, an accumulation of particle-laden macrophages in the bronchus-associated lymphoid tissue (BALT) was seen in 2/5 low dose males and females (very slight), in all mid dose males and females (all very slight) and in all high dose males and females (4/5 very slight and 1/5 slight in males and in females, respectively). Particles free in the alveoli were observed dose-dependent in all mid dose males and females (all very slight) and all high dose males and females (all slight). A lipoproteinosis was visible in 3/5 mid dose males and females (males 2/5 very slight and 1/5 slight; females 3/5 very slight) and in all high dose males and females (slight in 1/5 and moderate in 4/5 males and females, respectively). The lipoproteinosis is most likely cellular detritus from alveolar macrophages but might also represent exudate from pulmonary vessels. A multifocal alveolar granulocytic cell infiltration was found very slight in 2/5 mid dose males and in 1/5 mid dose females and, in addition, in 4/5 high dose males (2/5 very slight and 2/5 slight) and in 3/5 high dose females (all three very slight). There was a very slight multifocal mononuclear cell infiltration in the lung parenchyma at the terminal bronchus in 2/5 mid dose males and females (all very slight) and in 5/5 high dose males and females (in 4/5 males very slight and in 1/5 males slight as well as in all females very slight). A very slight (multi)focal bronchio-alveolar hyperplasia of the bronchiolar type was visible in 4/5 high dose males and in 1/5 low dose, 2/5 mid dose and 5/5 high dose females. A very slight (multi)focal interstitial fibrosis was observed in 1/5 mid dose males and females as well as in 4/5 high dose males and in 5/5 high dose females. There was a focal slight alveolar macrophage aggregation in 1/5 males and a multifocal moderate in 1/5 males (both high dose group).

c) Lung-associated lymph nodes (after 28 days of exposure; 5 animals/sex/dose):
After 28 days of exposure, in the lung-associated lymph nodes (LALN) a very slight to slight accumulation of particle-laden macrophages was visible in mid and high dose exposed animals (male mid dose: 4/5 very slight, male high dose: 2/5 very slight and 2/5 slight; female mid dose: 4/5 very slight, female high dose: 1/5 very slight and 3/5 slight).

d) Lung-associated lymph nodes (after additional 90 days of recovery; 5 animals/sex/dose):
After additional 90 days recovery following exposure, in the lung-associated lymph nodes (LALN) a very slight to moderate accumulation of particle-laden macrophages was visible in low, mid and high dose exposed animals (male low dose: 2/5 very slight, male mid dose: 2/5 very slight, 2/5 slight and 1/5 moderate, male high dose: 1/5 very slight, 2/5 slight and 2/5 moderate; female low dose: 2/5 very slight, female mid dose: 1/5 very slight and 2/5 slight, female high dose: 2/5 very slight, 1/5 slight and 1/5 moderate).
The diffuse sinusoidal dilatation represented a single event and was interpreted as unrelated to the treatment.

e) Larynx (after 28 days of exposure and after additional 90 days of recovery; 5 animals/sex/dose/time point):
A very slight focal subepithelial accumulation of particle-laden macrophages was visible in one mid dose male and one high dose female after 28 days of exposure as well as in one high dose male after additional 90 days recovery following exposure.

f) Remaining organs of the respiratory tract (after 28 days of exposure and after additional 90 days of recovery; 5 animals/sex/time point):
There was a dose-dependent very slight accumulation of particle-laden macrophages in the nasal associated lymphoid tissue in the animals after 28 days exposure (2/5 low dose males and females; 4/5 mid dose males and females and 5/5 high dose males and females) as well as after additional 90 days recovery (2/5 low dose males and females; 3/5 mid dose males and 4/5 mid dose females as well as 5/5 high dose males and females).
Single animals of the exposed groups showed focally a very slight accumulation of particle-laden macrophages at the tracheal bifurcation.
The mononuclear cell infiltrations in different organs of the respiratory tract in some animals of all groups are commonly found background lesions and were interpreted as unrelated to the treatment. Other detected lesions such as dilatation of submucosal glands, focal granulomatous inflammation and inflammatory cell infiltration represented single events and were interpreted as unrelated to the treatment.

2) Cobalt sulfate heptahydrate:
2.04 and 10.19 mg/m³:
a) Lung (after 28 days of exposure; 5 animals/sex/dose):
Within the lung, there was a dose-dependent multifocal very slight lipoproteinosis in all high dose animals (males and females).

b) Lung (after additional 90 days of recovery; 5 animals/sex/dose)
Within the lung, there was very slight focal cholesterol granuloma in 1/5 high dose males and females. Furthermore, there was a very slight focal alveolar macrophage aggregation in 1/5 low dose males as well as in 1/5 males and in 2/5 females of the high dose group.
A perivascular granulocytic cell infiltration was visible in some animals of all groups, including the control animals. The perivascular granulocytic cell infiltration is a commonly found background lesion, which is unrelated to the treatment. Other detected lesions such as neuroendocrine cell hyperplasia, inflammatory cell infiltration, pulmonary hemorrhage and osseous metaplasia represented single events and were interpreted as unrelated to the treatment.

c) Lung-associated lymph nodes (after 28 days of exposure; 5 animals/sex/dose):
No treatment related findings were detected in the lung-associated lymph nodes after exposure.

d) Larynx (after 28 days of exposure; 5 animals/sex/dose):
In the larynx accentuated at the base of the epiglottis, a focal ulcerative inflammation was visible in 1/5 low dose males (slight) and in 5/5 high dose males (1/5 slight, 3/5 moderate and 1/5 severe) as well as in 5/5 high dose females (3/5 moderate and 2/5 severe). At the same location a focal chronic-active inflammation was seen in 3/5 low dose males (2/5 very slight and 1/5 slight) as well as in 2/5 low dose females (very slight). In addition, at the base of the epiglottis was a focal squamous metaplasia in the low and high dose exposed animals (male low dose: 2/5 very slight and 2/5 slight, male high dose: 1/5 very slight and 4/5 slight; female low dose: 4/5 very slight, female high dose: 5/5 slight). At the same location a very slight focal epithelial alteration was observed in 1/5 low dose males and females.

e) Larynx (after additional 90 days of recovery; 5 animals/sex/dose):
Accentuated at the base of the laryngeal epiglottis, there was a focal squamous metaplasia in the high dose exposed animals (male high dose: 1/5 very slight and 1/5 slight; female high dose: 2/5 very slight). Furthermore, at the same location a very slight focal epithelial alteration was observed in 2/5 high dose males and a very slight focal erosion was seen in 1/5 high dose males. In addition, a focal chronic inflammation was found in the low and high dose exposed animals (male low dose: 2/5 very slight and 1/5 slight, male high dose: 1/5 very slight and 1/5 slight; female high dose: 3/5 very slight). The tissue at the base of the laryngeal epiglottis showed focally chronic granulation tissue in the high dose exposed animals (male high dose: 3/5 very slight and 2/5 slight; female high dose: 1/5 very slight and 2/5 slight). The chronic granulation tissue is part of a reparation process, which might finally end in fibrosis. Furthermore, two high dose exposed females exhibited a degeneration of the cartilage at this location.

f) Remaining organs of the respiratory tract
No treatment related findings were detected in the remaining organs of the respiratory tract after exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: one day after exposure, statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the mid (19%/13%; M/F; p < 0.001 or p < 0.05) and high dose groups (31%/35%; M/F; p < 0.001) of both sexes; in the low dose groups, PMN showed the same levels as clean air controls (<1%). Following 91 days of recovery, the PMN levels had increased moderately in the mid dose groups (27%/18%; M/F; p < 0.001) and persisted at similar levels in the high dose groups (34%/34%; M/F; p < 0.001).

Also, for lactic dehydrogenase (LDH), ß-glucuronidase and total protein statistically significant increases were detected in the mid (LDH and total protein; M) and high dose groups (LDH, β-glucuronidase and total protein; M+F) at day 1 after end of exposure. Overall, a clear dose-dependency of observed effects was found.
At day 91 of the post-exposure observation period, the statistically significant level of LDH persisted in the mid dose group (M) and was detected also in females (mid dose). LDH, β-glucuronidase and total protein levels persisted in the high dose groups (M+F).

At day 1 after end of exposure, MCP-1 and IL-8 concentrations in the BALF supernatant showed a dose-dependent increase (statistically significant in the mid and high dose groups; p < 001 or p < 0.05); persisting after recovery).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after exposure, statistically significant increases of the polymorphonuclear neutrophils (PMN) percentages were detected in both doses and sexes (low dose: 12%/12%; M/F; p < 0.05 (males only) - high dose: 25%/25%; M/F; p < 0.001). Following 91 days of recovery, all the effects observed in the CoSO4 high groups (M+F) had returned to normal.

One day after exposure, in the low dose group lactic dehydrogenase (LDH) (males) and in the high dose groups (males and females) LDH, ß-glucuronidase and total protein were statistically significantly increased. Overall, a clear dose-dependency of observed effects was found.
At day 91 of the post-exposure observation period, all the effects observed in the high groups (M+F) had returned to normal.

At day 1 after end of exposure, MCP-1 and IL-8 concentrations in the BALF supernatant showed statistically significant increases in the high dose group (p < 0.001 and p < 0.05, respectively) and partially low dose groups (only IL-8; p < 0.05); in the recovery periods these effects returned to normal.

Details on results:

CLINICAL SIGNS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: effects indicating systemic toxicity were not observed. Sex differences were not detected.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: effects indicating systemic toxicity were not observed. Sex differences were not detected.

MORTALITY
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no unscheduled deaths occurred during the study period.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no unscheduled deaths occurred during the study period.

BODY WEIGHT AND WEIGHT CHANGES
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: relevant statistically significant changes were not observed in the treatment groups as compared to controls.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: relevant statistically significant changes were not observed in the treatment groups as compared to controls.

FOOD CONSUMPTION
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12 and, 20.50 mg/m³: no relevant statistically significant changes were observed
- 80.78 mg/m³: relevant statistically significant changes were observed only in the female Co3O4 high dose group (decrease as compared to controls; findings not considered as having no toxic relevance).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no relevant statistically significant changes were observed

WATER CONSUMPTION
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12 and, 20.50 mg/m³: no relevant statistically significant changes were observed
- 80.78 mg/m³: relevant statistically significant changes were observed only in the female Co3O4 high dose group (decrease as compared to controls; findings not considered as having no toxic relevance).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no relevant statistically significant changes were observed

HAEMATOLOGICAL FINDINGS
1) Tricobalt tetraoxide (main study and recovery group)
- 80.78 mg/m³: one day after exposure, the mean cell volumes were statistically significantly increased in males and females (males: treatment group: 57.20 fl; control group: 59.74 fl; p < 0.05) (females: treatment group: 57.00 fl; control group fl: 59.78; p < 0.05).
- 5.12 and 80.78 mg/m³: one day after eyposure, lymphocytes (calc.) showed statistically significant decreases in males (low dose group: 4.554 G/L; control group: 5.904 G/L; p < 0.05)(high dose group: 4.396 G/L; control group: 5.904 G/L; p < 0.05).
- 20.50 mg/m³: at day 91 of the recovery period, lymphocytes (%) were statistically significantly decrease in males (treatment group: 62.2 %; control group: 81.8 %; p < 0.05)(not considered a toxicological relevant finding)

Changes in the other parameters did not reach statistical significance.
Overall, no clear dose-dependency was observed; neither showed both sexes conclusive results.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 mg/m³: one day after exposure, lymphocytes (calc.) showed statistically significant decreases in males (treatment group: 3.926; control group: 5.904 G/L; p < 0.01). Correspondingly, also lymphocytes (%) showed significant effects in males (treatment group: 72.6 %; control group: 85.6 %; p < 0.01). Furthermore, one day after exposure, segmented neutrophils (calc.) were statistically significantly increased in females (treatment group: 0.918 G/L; control group: 0.420 G/L; p < 0.05). Correspondingly, also segmented neutrophils (%) showed significant effects in in males and females (males: treatment group: 24.6 %; control group: 11.0 %; p < 0.01)(females: treatment group: 18.8 %; control group: 10.4 %; p < 0.05).
- 2.04 and 10.19 mg/m³: one day after exposure, the thromboplastin time was statistically significantly decreased in males (low dose group: 14.68 seconds; control group: 17.24 seconds; p < 0.01)(high dose group: 14.84 seconds; control group: 17.24 seconds; p < 0.05). This finding persisted until day 91 of the recovery period, the thromboplastin time values were still statistically significantly decreased in the low and high groups (males; low dose group: 17.70 seconds; high dose group: 17.70 seconds; control group: 20.64 seconds; p < 0.05) as well as in the in the high group of females (high dose group: 17.38 seconds; control group: 19.86 seconds; p < 0.05).

Changes in the other parameters did not reach statistical significance.
Overall, no clear dose-dependency was observed; neither showed both sexes conclusive results.

CLINICAL CHEMISTRY
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significantly increases/decreases were detected for any parameter, except for albumin that was statistically significantly reduced in males of the mid dose group on day 91 of the recovery period (mid dose group: 34.04 g/L; control group: 36.54 g/L; p < 0.05)( considered not to be a relevant finding).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after exposure, urea was statistically significantly decreased in males (low dose group: 4.293 mmol/L; control group: 5.834 mmol/L; p < 0.01)(high dose group: 4.600 mmol/L; control group: 5834 mmol/L; p < 0.05). Furthermore, one day after exposure, total bilirubin was statistically significantly increased in females of the high dose (high dose group: 1.88 µmol/L; control: 1.14 µmol/L; p< 0.01).

Overall, no clear dose-dependency was observed.

URINALYSIS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significantly increases/decreases were detected.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no statistically significantly increases/decreases were detected.

ORGAN WEIGHTS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: one day after end of exposure lung weights (absolute and relative) of the male Co3O4 groups did not show statistically significant increases as compared to concurrent controls. In addition, females of the low and mid doses did not show statistically significant increases in absolute and relative lung weights as compared to concurrent controls.
Furthermore, no statistically significant increases or decreases were observed for any other organ.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after end of exposure lung weights (absolute and relative) of the male and females did not show statistically significant increases as compared to concurrent controls.
Furthermore, no statistically significant increases or decreases were observed for any other organ.

GROSS PATHOLOGICAL FINDINGS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: all animals were sacrificed at scheduled dates. Upon necropsy, test item- or dose-related macroscopical findings were not observed. Only some incidental macroscopical observations were obtained

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: all animals were sacrificed at scheduled dates. Upon necropsy, test item- or dose-related macroscopical findings were not observed.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
Tricobalt tetraoxide and cobalt sulfate heptahydrate (main study and recovery group; 5 animals/per sex /dose/time point)
Within the other investigated organs, only incidental findings were detected. These represented a mononuclear cell infiltration in different organs. Furthermore, there were a focal hypertrophy and a focal fatty degeneration in the cortex of the adrenal gland, a focal mineralization in the brain, a submucosal inflammatory cell infiltration in the glandular stomach, an inflammatory cell infiltration accompanied by myofiber degeneration in the heart, a lymphoid hyperplasia in the spleen, a tubules and cords hyperplasia and a congenital cyst in the thymus and an interstitial fibrosis and ectopic thymic tissue in the thyroid gland. In the kidney, a focal cortical tubular basophilia, a cortical cysts, cortical and medullary cysts, unilateral hydronephrosis, interstitial nephritis, pyelonephritis and reactive hyperplasia of the pelvic epithelium was visible in single animals. The liver exhibited in single animals a granulocytic cell infiltration and a periportal inflammatory cell infiltration. An estrous related luminal dilatation of the uterus was visible in some animals.

One animal (5.12 mg Co3O4/m³ dose group) showed macroscopically a 2 mm in diameter sized nodule in the epididymis. This nodule represented histologically a focal spermatic granuloma.
One animal (5.12 mg Co3O4/m³ dose group) exhibited macroscopically alopecic areas at the head and front paws. These areas showed histologically a mixed inflammatory cell infiltration in the hair follicles with accompanying hair cycle arrest.
One animal (10.19 mg CoSO4 * 7H2O/m³ dose group) showed macroscopically an attachment of pancreas, liver stomach and omentum with 3mm in diameter sized brown nodules. These areas showed histologically chronic granulation tissue with hemosiderosis consistent with a previous hemorrhage.
One animal (10.19 mg CoSO4 * 7H2O/m³ dose group) exhibited macroscopically a slight herniation of the liver, which represented histologically a hepatodiaphragmatic nodule.
All lesions occur occasionally in rats and are considered to be unrelated to the exposure.

BRONCHOALVEOLAR LAVAGE
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: the females of the high dose group showed a statistically significant increase in lymphocytes. This finding is considered as incidental because all other groups remained at control levels.

Variation in the HIF-1α results observed in both the BALF supernatant and BALF cell sediment for the tricobalt tetraoxide groups cannot be fully determined, resulting in difficulty in concluding on the significance of the results. A study with a longer duration of exposure (i.e. 90-day repeated-dose) may provide further clarification on this endpoint.

IMMUNOHISTOCHEMISTRY - 8-OH-dG ANALYSIS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significant changes compared to concurrent controls were observed

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no statistically significant changes compared to concurrent controls were observed

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

5.12 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt tetraoxide

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

2

Key result

Dose descriptor:

LOAEC

Effect level:

20.5 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt tetraoxide

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

3

Key result

Dose descriptor:

LOAEC

Effect level:

2.04 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt sulfate heptahydrate

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

Remarks on result:

other: equivalent to 1.13 mg cobalt sulfate/m³

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this repeated dose inhalation toxicity study, the administration of cobalt tetraoxide and cobalt sulfate heptahydrate to male and female Wistar rats at doses of 5.12, 20.5 and 80.78 mg/m³ and 2.04 and 10.19 mg/m³, respectively, for a duration of 28 days produced no unscheduled deaths or signs of systemic toxicity. Furthermore, no effects on body weight, food consumption, water consumption, haematology, clinical chemistry, urinanalysis, immunohistochemistry (8-OH-dG) or macroscopical findings were observed.

After administration of 80.78 mg Co3O4/m³, the relative lung weights of females were statistically significantly increased compared to controls. Following a recovery period of 90 days, it was observed that the absolute and relative lung weights in the males and females were statistically significantly increased compared to the controls. One day after exposure, no effects were observed for the 5.12 and 20.50 mg/m³ dose groups regarding lung weights.Lastly, after administration of 2.04 and 10.19 mg cobalt sulfate heptahydrate no effects were observed for organ weights.

One day after the end of cobalt tetraoxide exposure, histopathological evaluation revealed exposure-related findings in lungs. Interpreted as adverse findings were: the alveolar infiltration of granulocytic cells, the interstitial mononuclear cell infiltration at the terminal bronchus, the lipoproteinosis and the interstitial fibrosis, which were seen in the 80.78 mg/m³ and partially 20.50 mg/m³ dose exposed animals (males and females). After additional 91 days of recovery the following findings occurred statistically significantly: alveolar infiltration of granulocytic cells, the interstitial mononuclear cell infiltration at the terminal bronchus, the lipoproteinosis and the interstitial fibrosis in the 80.78 mg/m³ dose exposed males and females.

In addition, for the cobalt sulfate heptahydrate treatment groups exposure-related findings were found in the lung as well as in the larynx. In the 10.19 mg/m³ group (males and females), lipoproteinosis occurred statistically significantly. Mononuclear cell infiltration was detected in the 10.19 mg/m³ group (females). In the larynx the squamous cells metaplasia, the ulcerative inflammation, the chronic-active inflammation, the chronic inflammation, the erosion and chronic granulation tissue and the degeneration of the cartilage were interpreted as adverse findings, which were seen in the 10.19 mg/m³ and partially 2.04 mg/m³ dose exposed animals. The squamous cells metaplasia and the ulcerative inflammation occurred statistically significantly in the 10.19 mg/m³ group (males and females). After additional 91 days of recovery the chronic granulation tissue occurred statistically significantly in the 10.19 mg/m³ group (males).

In this study a bronchoalveolar lavage was carried out. At day 1 after end of exposure statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the 20.50 (19%/13%; males/females) and 80.78 mg Co3O4/m³ dose groups (31%/35%; males/females) of both sexes; in the low dose groups, PMN showed the same levels as clean air controls (<1%). The PMN influx persisted after 91 days of recovery. In the two CoSO4 7H2O groups statistically significant increases of the PMN percentages were detected in both sexes (2.04 mg/m³ dose: 12%/12%; males/females - 10.19 mg/m³ dose: 25%/25%; males/females). After 91 days of recovery the effect disappeared. For lactic dehydrogenase, ß-glucuronidase and total protein statistically significant increases were detected in the 20.50 mg Co3O4/m³ (LDH and total protein; males) and 80.78 mg Co3O4/m³ dose groups (LDH, β-glucuronidase and total protein; males and females) at day 1 after end of exposure. In the 2.04 mg CoSO4 7H2O/m³ dose group (LDH; males) and in the 10.19 mg CoSO4 7H2O/m³ dose group all three parameters were statistically significantly increased (males and females). Cytokine concentrations (MCP-1, IL-8 alias CINC-1) at day 1 after end of exposure showed a dose-dependent increase in the Co3O4 groups (statistically significant in the mid and high dose groups; persisting after recovery); in the CoSO4 groups statistically significant increases were observed in the high dose group and partially low dose groups (only IL-8); in the recovery periods these effects returned to normal. Overall, a clear dose-dependency of observed effects was found.

Absolute and relative lung weights of rats used for bronchoalveolar lavage showed a statistically significant increase at day 1 in the 80.78 mg Co3O4/m³ and 10.19 mg CoSO4 7H2O/m³ dose groups (males and females). At day 91 of the post-exposure observation period the findings in the Co3O4 groups persisted whereas in the CoSO4 groups all values had returned to normal values.

The HIF1-α analysis was measured in BALF and the BALF cell sediments (gained by centrifugation) Variation in the HIF-1α results observed in both the BALF supernatant and BALF cell sediment for the tricobalt tetraoxide groups cannot be fully determined, resulting in difficulty in concluding on the significance of the results. A study with a longer duration of exposure (i.e. 90-day repeated-dose) may provide further clarification on this endpoint.

Based on the finding of an increase of polymorphpnuclear neutrophils, the NOAEC and LOAEC of cobalt tetraoxide in the Wistar rat for both sexes are considered to be 5.12 mg/m³ and 20.5 mg/m³, respectively. In addition, it has to be mentioned that after 28 days of exposure to 5 mg/m³, the cobalt tetraoxide low dose group behaves toxicologically very similar as the well-characterised TiO2 inert dust (“Bayertitan T”; rutile modification; after a 90-day exposure of rats to 5 mg/m3; historical data).

Based on the finding of an increase of polymorphpnuclear neutrophils, the LOAEC of cobalt sulfate heptahydrate in the Wistar rat for both sexes is considered to be 2.04 mg/m³ (equivalent to 1.13 mg cobalt sulfate/m³).