Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-14 to 2010-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
adopted 2009-09-07
Deviations:
yes
Remarks:
; at one concentration in the main study 5 males / 5 females were used
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt carbonate
EC Number:
208-169-4
EC Name:
Cobalt carbonate
Cas Number:
513-79-1
Molecular formula:
CH2O3.Co
IUPAC Name:
lambda2-cobalt(2+) carbonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cobalt carbonate
- Molecular formula: CoCo3
- Molecular weight: 118.94 g/mol
- Physical state: pink-purple powder
- Storage condition of test material: in original container, tightly closed in a dry, cool and well ventilated place; the test item is hygroscopic
- Particle size parameter were determined with a CILAS 715 (non-GLP determination): d (0.1) = 3.89 µm; d(0.5) = 8.71 µm (median); d (0.9) = 18.72 µm
- Melting point: Decomposes at 280 °C
- Water solubility: 12.98 µg/L at 20 °C

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of administration: males: 50 - 52 days; females: 64 - 66 days
- Weight at start of administration: males: 237 - 274 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; kept by sex in groups of 2 -3 animals in MAKROLON cages (type III plus)
- Diet (please refer to "Fasting period before study" above): commercial diet, ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water(ad libitum): drinking water
- Acclimation period: at least 5 adaptation days; the animals were randomised before use. The animals were acclimatised to the test apparatus for approx. 1 hour on 2 days prior to testing. The restraining tubes did not impose undue physical, termal 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 & TREPPER. The apparatus consists of a cyclindrical 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).
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).
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 homogeneous 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.

- 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 (21.3 °C +/- 0.1 °C (main study- 5.08 mg/L air), 21.0 °C +/- 0.2 °C (main study- 1.05 mg/L air), 20.8 °C +/- 0.1 °C (satellite group- 5.08 mg/L air) or 19.7 °C +/- 0.1 °C (satellite group-1.07 mg/L air)) and humidity (52.5 % +/- 0.1 % (main study - 5.08 mg/L air), 62.2 % +/- 0.0 % (main study - 1.05 mg/L air), 48.4 % +/- 0.2 % (satellite group - 5.08 mg/L air) or 51.3 % +/- 0.1 (satellite group-1.07 mg/L air)) 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.

- 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 5 RD, 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 (SARTORIUS, type 1601 004, precision 0.1 mg). Deltas of slides' weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear 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 Geomteric 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 median physical particulate size with a CILAS 715 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

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 2 C (Membrane Pump, Vacuubrand GmbH + Co. KG, 97877 Wertheim/Main, Germany)) 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 minute. The filters were weighed before and after sampling (accuracy 0.1 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 minute.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
Main study:
5.08 +/- 0.02 mg/L air concentration: MMAD: 2.956 µm; GSD: 3.23 µm
1.05 +/- 0.03 mg/L air concentration: MMAD: 2.932 µm; GSD: 3.20 µm
Satellite group:
5.08 +/- 0.06 mg/L air concentration: MMAD: 3.379 µm; GSD: 3.51 µm
1.07 +/- 0.01 mg/L air concentration: MMAD: 2.809 µm; GSD: 3.28 µm
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: 5.08 +/- 0.02 mg/L air concentration and 1.05 +/- 0.03 mg/L air concentration
nominal concentration: 5.56 mg/L air and 1.39 mg/L air
Satellite group:
actual concentration: 5.08 +/- 0.06 mg/L air concentration and 1.07 +/-0.01 mg/L air concentration
nominal concentration: 5.56 mg/L air and 1.39 mg/L air
No. of animals per sex per dose:
Main study:
5.08 mg/L air concentration: 3 males / 3 females
1.05 mg/L air concentration: 5 males / 5 females
Satellite group:
5.08 mg/L air concentration: 3 males / 3 females
1.07 mg/L air concentration: 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 animals. 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 the exposure on test day 1, on test days 2, 8 and 15 and at 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 (8 + 6 males and 8 + 6 females) 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 persist or abates.
Autopsy and macroscopic inspections of rats which died prematurely were carried out as soon as possible after exitus.
Histopathology:
All main study and satellite animals 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 jar) 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 the 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.
Statistics:
Due to low mortality and small number of animals at the lethal concentration no LC50 calculation according to FINNEY was carried out.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.08 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Main study:
At the concentration of 5.08 mg/L air 2 of 3 male (test days 3 and 6) and 1 of 3 female animals (test day 3) died prematurely. No mortality occurred in the 1.05 mg/L air group.
Satellite group:
No mortality occurred in the 1.07 or 5.08 mg/L air group.

Clinical signs:
other: Main study: A 4-hour inhalation exposure to cobalt carbonate at a concentration of 5.08 or 1.05 mg/L air revealed slight ataxia and/or slight dyspnoea (reduced frequency of respiration with increased volume) on test day 1 immediately after end of exposure
Body weight:
Main study
Body weight gain of the surviving 3 animals at 5.08 mg/L appeared to be reduced. No weight inhibition was observed in the 1.05 mg/L air group.
Satellite group:
Also, the body weight gain of the satellite group animals appeared to be reduced.
Gross pathology:
Haemorrhagic lungs were observed at 5.08 mg/L in the prematurely deceased animals of the main study (14- day sacrifice) and 2 of 3 male and 2 of 3 female satellite animals (24-hour sacrifice). White discolourations of the lungs were observed in 4 of 5 male and 5 of 5 female animals at 1.05 mg/L (main study - 14-day sacrifice). Dark discolourations of the lungs were observed in 3 of 3 male and 2 of 3 female satellite animals at 1.07 mg/L (satellite animals - 24-hour sacrifice).
Other findings:
- Histopathology: a 4-hour inhalation exposure to cobalt carbonate at a concentration of 1.05±0.03 or 5.08±0.02 mg/L (main study – 14-day sacrifice) or 1.07±0.01 or 5.08±0.06 mg/L (satellite animals – 24-hour sacrifice) air revealed test item-related histopathological changes in the lungs and nasal cavity (please refer for results to "Attached background material" below).

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LC50 (male and female rats, 4 hours) > 5.08 mg/L air
Based on the results of the histopathological and macroscopic investigations, cobalt carbonate does not require classification for respiratory irritation.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, cobalt carbonate does not require classification either for acute inhalation toxicity or for respiratory irritation.
According to the EC Regulation No. 1272/2008 and subsequent regulations, cobalt carbonate does not require classification for acute inhalation toxicity or specific target organ toxicity - single exposure.