Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-01-25 to 2011-04-06
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:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cobalt acetylacetonate
- Product name: Peconal H
- Physical state: salmon powder
- Storage condition of test material: kept away from moisture, heat and light
- Particle size parameter: determined with a CILAS 715 8non-GLP determination)
d(0.1) = 6.28 µm
d(0.5) = 27.38 µm (median)
d(0.9) = 70.64 µm

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: approximately 7 weeks; females: approximately 9 weeks
- Weight at study initiation: males: 237 - 265 g; females: 212 - 243 g
- Fasting period before study: feeding was discontinued approximately 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. During the observation period, the animals were kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Diet (ad libitum, except for fasting period as described 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 acclimatised to the test apparatus for approximately 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 & 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.

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 5RD, UK).
The dust from the exposure chamber was drawn through the cascade impactor for 5 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.

- Temperature, humidity, pressure in air chamber, 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- 5.09 mg/L air), 21.6°C +/- 0.1 °C (satellite group- 5.07 mg/L air)) and humidity (60.3% +/- 0.0 % (main study - 5.09 mg/L air), 60.2 % +/- 0.0 % (satellite group - 5.07 mg/L air)) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

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:
MMAD: 3.281 µm; GSD: 2.98 µm
Satellite group:
MMAD: 3.324 µm; GSD: 2.94 µm
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: 5.09 ± 0.05 mg/L air
- nominal concentration: 5.56 mg/L
Satellite group:
- actual concentration: 5.07 ± 0.06 mg/L air
- nominal concentration: 5.56 mg/L
No. of animals per sex per dose:
3 males / 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- 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, on the day of exposure prior to exposure, on test days 2, 4, 8 and 15. Changes in weight were calculated and recorded when survival exceeded one day. At the end of the test, all animals were weighed and sacrificed.
- Necropsy of survivors performed: yes
Necropsy of all main study and satellite animals (3 + 3 males and 3 + 3 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.
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:
Since no deaths occurred, the calculation of an LC50 was not required.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.09 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: SD = 0.05
Mortality:
Main study:
No deaths occurred.
Satellite group:
No deaths occurred.
Clinical signs:
other: Main study: A 4-hour inhalation exposure to cobalt acetylacetonate at a concentration of 5.09 mg/L air revealed slightly reduced motility and slight dyspnoea (reduced frequency of respiration with increased volume) in all 3 male and 3 female rats on test
Body weight:
Main study:
Body weight gain of 1 of the 3 female animals appeared to be reduced.
Gross pathology:
Main study:
No pathological findings were noted at necropsy.
Satellite group:
No pathological findings were noted at necropsy.
Other findings:
- Histopathology:
A 4-hour inhalation exposure to cobalt acetylacetonate at a concentration of 5.09±0.05 mg/L air (main study) or 5.07±0.06 mg/mL air (satellite group) 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) > 5.09 mg/L air
Based on the results of the histopathological and macroscopic investigations, cobalt acetylacetonate does not require classification for respiratory irritation.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, cobalt acetylacetonate 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 acetylacetonate does not require classification for acute inhalation toxicity or specific target organ toxicity - single exposure.