Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-14 to 2011-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012
Reference Type:
other: Amendment No.1 to final report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
adopted 2009-09-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cobalt
- Product name: Cobalt powder HMP
- Molecular formula: Co
- Molecular weight: 58.93 g/mol
- Physical state: Solid; very fine grey powder
- Storage condition of test material: at room temperature, in closed original container
- Particle size (determined by the Sponsor): D10 = 1.3 μm, D50 = 2.4 μm, and D90 = 4.6 μm
- Melting point: 1491°C
- Boiling point: 3100°C
- Water solubility: insoluble in water
- Relative density: 8.92

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: approx. 8 weeks; females: approx. 9 weeks
- Weight at study initiation: males: 235 - 262 g; females: 210 - 250 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week. During the 14-day observation period, the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Diet (ad libitum; for exception please refer to "Fasting period before study" above): Commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days; The animals were randomised before use. They were acclimatised to the test apparatus for approx. 1 hour on 2 days prior to testing. The restraining tubes did not impose undue physical, thermal or immobilization stress on the animals.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C±3°C (maximum range)
- Relative humidity: 55%±15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the study was carried out using a dynamic inhalation apparatus (RHEMA-LABORTECHNIK, 65719 Hofheim/Taunus, Germany)(air changes/h (≥ 12 times)) with a nose-only exposure of the animals according to KIMMERLE & TEPPER. The apparatus consists of a cylindrical exposure chamber (volume 40 L) which is able to hold 10 animals in pyrex tubes at the edge of the chamber in a radial position.

- System of generating particulates/aerosols: the dust of the test material was generated with a rotating brush dust generator (RBG 1000, PALAS GmbH Partikel und Lasermesstechnik,76229 Karlsruhe, Germany)(concentrations of 0.05 to 5 mg/L air). The generator was fed with compressed air (5.0 bar) from a compressor (ALUP Kompressorenfabrik, 73257 Köngen, Germany) (air was taken from the surrounding atmosphere of the laboratory room and filtered using an in-line disposable gas-filter).
The dust of the test material at the concentration of 0.05 mg/L air was generated with a Dust Generator acc. to Bundschuh (TSE Systems GmbH, 61352 Bad Homburg, Germany).
At the bottom of the exposure chamber, the air was sucked off at a lower flow rate than it was created by the dust generator in order to produce a homogenous distribution and a positive pressure in the exposure chamber (inflow 900 L/h, outflow 800 L/h).
A manometer and an air-flow meter (ROTA Yokogawa GmbH & Co. KG, 79664 Wehr/Baden, Germany) were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and corrected if necessary.
The exhaust air was drawn through gas wash-bottles.

- Method of particle size determination: an analysis of the particulate size distribution was carried out twice during the exposure period using a cascade impactor according to MAY (MAY, K.R. Aerosol impaction jets, J.Aerosol Sci. 6, 403 (1975), RESEARCH ENGINEERS Ltd., London N1 5RD, UK.).
The dust from the exposure chamber was drawn through the cascade impactor for 5 or 10 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (precision 0.1 or 0.01 mg). Deltas of slides’ weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of nonlinear regression analysis. The 32 μm particle size range and the filter (particle size range < 0.5 μm) were not included in the determination of the MMAD in order not to give undue weight to these values.
The Geometric Standard Deviation (GSD) of the MMAD was calculated from the quotient of the 84.1%- and the 50%-mass fractions, both obtained from the above mentioned non-linear regression analysis.
In addition, a sample of approx. 10 g test material was taken from the exposure chamber to determine the particle size distribution (volume) with a CILAS 715 Sizer by My-Tec GmbH, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, oxygen content, carbon dioxide content: the oxygen content in the inhalation chamber was 21%. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set (DRÄGER Tube Oxygen 67 28 081). Carbon dioxide concentration did not exceed 1%.
Temperature and humidity were measured once every hour with a climate control monitor (testo 175-HZ data logger).

The whole exposure system was mounted in an inhalation facility to protect the laboratory staff from possible hazards.

The exposure was initiated by placing the animals’ noses into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes (t95 approximately 8 minutes).

Before initiating the study with the animals, a pre-test was carried out with the exposure system in order to verify that under the experimental settings chosen, the limit concentration of 5 mg/L air could be achieved by gravimetric analysis.

The tests with the main study animals and the recovery animals were conducted in the same inhalation chamber but on different days. Between the exposure times the chamber was cleaned carefully.

TEST ATMOSPHERE
- Brief description of analytical method used: the actual dust concentration in the inhalation chamber was measured gravimetrically with an air sample filter (Minisart SM 17598 0.45 μm) and pump (Vacuubrand, MZ 2C8) controlled by a rotameter. Dust samples were taken once every hour during the
exposure. For that purpose, a probe was placed close to the animals' noses and air was drawn through the air sample filter at a constant flow of air of 5 L/min for 1 or 10 minutes. The filters were weighed before and after sampling (accuracy 0.1 or 0.01 mg). The correct loading of the filter was checked by the airflow via the rotameter and by a positive weight increase of the filter after the sampling period of 1 or 10 minutes.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
Main study:
- 0.05 ± 0.00 mg/L air: MMAD: 2.997 µm; GSD: 3.25
- 0.51 ± 0.02 mg/L air: MMAD: 3.211 µm; GSD: 2.77
- 1.05 ± 0.02 mg/L air: MMAD: 2.881 µm; GSD: 3.26
Satellite group:
- 0.05 ± 0.00 mg/L air: MMAD: 2.669 µm; GSD: 3.23
- 0.53 ± 0.01 mg/L air: MMAD: 3.461 µm; GSD: 2.75
- 1.03 ± 0.02 mg/L air: MMAD: 2.850 µm; GSD: 3.20
- 5.08 ± 0.07 mg/L air: MMAD: 2.920 µm; GSD: 2.99
No smaller GSD could be obtained with the test item supplied.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
please refer to "details on inhalation exposure" above
Duration of exposure:
4 h
Concentrations:
Main study:
actual concentration: 0.05 ± 0.00 , 0.51± 0.02 , and 1.05 ± 0.02 mg/L air
Satellite group:
actual concentration: 0.05 ± 0.00, 0.53 ±0.01 , 1.03 ± 0.02, and 5.08 ± 0.07 mg/L air
No. of animals per sex per dose:
Main study: 5 males/5 females
Satellite group: 3 males/3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 24 hours (satellite group) and 14 days (main study)
- Frequency of observations and weighing: during and following exposure, observations were made and recorded systematically; individual records were maintained for each animal. Careful clinical examinations were made at least twice daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily (in the morning starting on test day 2) to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.
Cageside observations included, but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, as well as somatomotor activity and behaviour pattern.
Particular attention was directed to observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The animals were also observed for possible indications of respiratory irritation such as dyspnoea, rhinitis etc..
Individual weights of animals were determined once during the acclimatisation period, before and after the exposure on test day 1, on test days 3, 8 and 15 and at time of death. Changes in weight were calculated and recorded when survival exceeded one day. At the end of the test, all surviving animals were weighed and sacrificed.
- Necropsy of survivors performed: yes
Necropsy of all main study and satellite animals was carried out and all gross pathological changes were recorded:
- Satellite animals: necropsy at 24 hours after cessation of exposure, as this is likely to be the time at which any signs of respiratory irritation would have manifested;
- Main study animals: necropsy at the end of the 14-day observation period, also to assess whether any respiratory tract irritation persists or abates.
Autopsy and macroscopic inspections of animals which died prematurely were carried out as soon as possible after exitus.
- Histopathology:
The main study and satellite animals of the concentrations of 0.5 and 0.05 mg/L air were subjected to the same level of histopathological examination upon necropsy at the end of the respective observation period. During histopathology, attention was paid to alterations that might be indicative of respiratory irritation, such as hyperaemia, oedema, minimal inflammation, thickened mucous layer.
The following organs of all animals were fixed in 10% (nose, i.e. head without brain, eyes and lower jaw) or 7% (other organs) buffered formalin for histopathological examination:
- Nose (5 levels of the nasal turbinates):
The tip and level 1 of the nose were taken from a cut just anterior to the incisor teeth. With tip removed, level 2 was taken approximately 2 mm posterior to free tip of the incisor teeth. Level 3 was cut through the incisive papilla. Level 4 was cut through the middle of the second palatal ridge, which is located just anterior to the molar teeth. Level 5 was cut through the middle of the molar teeth. All sections were embedded face down to yield a section from the anterior section, except the nose tip was embedded posterior surface down.
- Larynx
- Trachea
- Lungs (five levels)
Paraffin sections were prepared of all above mentioned organs and stained with haematoxylin-eosin.

An additional histopathological re-evaluation was conducted focusing on:
- interstitial oedema / perivascular oedema
- broncho-alveolar hypertrophy
- cellular infiltration
(Amendment dated 15.04.2013)
Statistics:
Due to high mortality no LC50 could be calculated.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
<= 0.05 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
<= 0.05 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
<= 0.05 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Main study:
- 0.05 mg/L air: all animals died prematurely within 11 days.
- 0.51 mg/L air: all animals died prematurely until test day 4.
- 1.05 mg/L air: all animals died prematurely until test day 4.
Satellite group:
- 0.05 mg/L air: 1/3 male animal died prematurely during the 24-hour observation period.
- 0.51 mg/L air: 2/3 male animals died prematurely during the 24-hour observation period.
- 1.05 mg/L air: 3/3 male and 1/3 female animals died prematurely during the 24-hour observation period.
- 5.08 mg/L air: 3/3 male and 3/3 female animals died prematurely within 24 hours.
Clinical signs:
other: - 0.05 mg/L air: Main study: slight ataxia and slight dyspnoea in 5/5 male and 5/5 female animals, slightly reduced muscle tone in 2/5 males and 5/5 females, tonic convulsions in 1/5 male and 3/5 females and slight tremor in 1/5 female. Satellite group:
Body weight:
Body weight gain could not be calculated as all animals died prematurely.
Gross pathology:
A concentration of 0.05 mg/L air revealed haemorrhagic lungs in 9 of 16 animals, lungs with blood-stained spots in 6 of 16 animals or a hardened, oedematous, red marbled lung in 1 of 16 animals. Haemorrhagic lungs were observed in all animals at concentrations of 0.51 to 5.08 mg/L air or a haemorrhagic lung with pale foci in 1 of 10 animals at a concentration of 1.05 mg/L air.
Other findings:
- Histopathology (only the main study and satellite animals at the concentrations of 0.5 and 0.05 mg/L air were subjected to histopathological examination and not all main and satellite animals, as all higher dosed animals died within 4 days):
0.05 mg/L air (main study and satellite group): test item-related histopathological changes were noted in the lungs but not in the nose, larynx or trachea.
The histopathological re-evaluation of the lung slides revealed a slight alteration of the intrepretation as follows:
The lungs showed a test item-related perivascular inflammatory oedema with neutrophilic granulocytes, lymphocytes and histiocytes and an interstitial pneumonia in the animals with premature deaths. The changes are classified as mild to moderate.
0.5 mg/L air (main study and satellite group): test item-related changes were noted in lungs, but not in the nose, larynx or trachea.
(Please also refer for results to "Attached background material" below).

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category I
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LC50 (4 hours) < 0.05 mg/L air (males, females and males and females combined)
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is classified as very toxic by inhalation
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as Category 1.