Cobalt dihydroxide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-13 to 2016-11-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

2009-09-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2014-05-14

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt dihydroxide

EC Number:

244-166-4

EC Name:

Cobalt dihydroxide

Cas Number:

21041-93-0

Molecular formula:

CoH2O2

IUPAC Name:

cobalt(2+) dihydroxide

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: pink powder, hygroscopic
- Particle size distribution (determined with a Malvern Mastersizer 2000 (Non-GLP determination))
- D10% = 1.60 µm
- D50% = 31.43 µm
- D90% = 67.08 µm

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep in containers of same material as the original one. Store product in closed container. Keep containers tightly closed in a dry and well-ventilated place.

Test animals

Species:

rat

Strain:

other: Crl: 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
- Females nulliparous and non-pregnant: yes
- Age at start of administration: males: approx. 8 weeks; females: approx: 9 to 10 weeks
- Weight at start of administration: males: 242 - 330 g; females: 221 - 266 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: during the 14-day observation period: kept by sex in groups of 3 animals in MAKROLON cages (type III plus); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): 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
They were acclimatised to the test apparatus for approx. 1 hour on 2 days prior to testing.

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

Mass median aerodynamic diameter (MMAD):

>= 1.952 - <= 2.167 µm

Geometric standard deviation (GSD):

>= 2.31 - <= 2.55

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/Exposure chamber volume/Method of holding animals in test chamber: 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 holds the animals in pyrex tubes at the edge of the chamber in a radial position.

- System of generating particulates/aerosols: the dust of the test item was generated using 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).
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 particle size distribution was carried out twice during the exposure period using a cascade impactor 6.0 L/min.
The dust from the exposure chamber was drawn through the cascade impactor for 1 to 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 mg). Deltas of slides’ weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of nonlinear regression analysis. The 10.6 μm particle size range and the filter (particle size range < 0.55 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 median particle size with a Malvern Mastersizer 2000 by My-Tec, 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.

Exposition started by locating the animals 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.

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 2C) 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 to 10 minutes. The filters were weighed before and after sampling (accuracy 0.1 mg).
Individual chamber concentration samples did not deviate from the mean chamber concentration by more than 1%.
- Samples taken from breathing zone: yes

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 ± 0.01, and 5.06 ± 0.01 mg/L air (nominal concentrations: 1.11, 6.67, and 33.33 mg/L air, respectively)
Satellite group:
actual concentration: 0.05 ± 0.00, 0.53 ± 0.01, and 5.07 ± 0.01 mg/L air (nominal concentrations: 1.11, 6.67, and 33.33 mg/L air, respectively)

No. of animals per sex per dose:

Main study: 5 males / 5 females (exception: 3 males / 3 females for the 5.06 mg/L air concentration)
Satellite group: 3 males / 3 females

Control animals:

no

Details on study design:

1) Duration of observation period following administration: 24 hours (satellite group) and 14 days (main study)

2) Frequency of observations and weighing:
- clinical examinations: during the exposure period the animals were observed frequently. Following exposure, observations were made at least twice on the day of exposure and at least once each day thereafter and recorded systematically. A careful clinical examination was made at least once each day, thereafter at least once daily until death (main study) or end of the 24-hour period before necropsy (satellite animals). Individual records were maintained for each animal.
- mortality: observations 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.
- body weight: individual weights of animals were determined once during the acclimatisation period, before the exposure on test day 1, on test days 2 and 4, as all main study-animalds died before test day 8. Changes in weight were calculated and recorded when survival exceeded one day.

3) Necropsy:
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 as soon as possible after exitus as all animals died prematurely.

4) 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 jaw) or 7% (other organs) buffered formalin for histopathological examination:
- nasal cavity (5 levels: tip and Level 1 of the nose were taken from a cut just anterior to the incisor teeth. With the tip removed, Level 2 was taken approx. 2 mm posterior to free the 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).
- nasopharynx
- paranasal sinus
- larynx
- trachea
- lung (left lobe, right caudal lobe, right cranial lobe, right middle lobe, and accessory lobe)
Paraffin sections were prepared of all above listed organs and stained with haematoxylin-eosin.

Statistics:

not applicable

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

< 0.05 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

- 5.06 mg/L air: all animals died within 2 days after administration.
- 0.51 mg/L air: all animals died within 4 days after administration.
- 0.05 mg/L air: all animals died within 5 days after administration.

Clinical signs:

other: - 5.06 mg/L air: slight to moderate ataxia, slight to moderate tremor and slight to moderate dyspnoea (reduced frequency of respiration with increased volume) immediately after end of exposure until 1 day post exposure in 3 of 3 male and 3 of 3 female ani

Body weight:

It was not possible to assess the body weight as all animals died within few days after administration.

Gross pathology:

Necropsy revealed slightly to markedly oedematous lungs in all main study and satellite animals.

Other findings:

- Histopathology:
The histomorphological examination of the trachea, larynx, lungs and the nose of male and female rats after inhalation of the test item revealed morphological changes in the nose, lungs and larynx of the male and female satellite and main study rats (3 to 6 days, premature death) which are considered to be to the test item-related:

1) Satellite animals (24-hour sacrifice)
- nasal cavity: level 1 revealed a normal squamous epithelium and a normal respiratory epithelium. The normal respiratory epithelium partially containing cilia consisted of three major cell types: the basal cells above the basement membrane, the ciliated epithelial and the secretory goblet cells.
The levels 2 to 5 revealed a minimal to moderate degeneration of the olfactory epithelium. Vacuolization, membrane ruptures of the cells and a focal loss of the epithelial cells were noted. The changes were more pronounced in the high dose group (5 mg/L air). The animals of low dose group (0.05 mg/L air) showed the slightest changes. The focal detachment of the olfactoric epithelium was not only due to the degeneration, but was enhanced by a slight autolysis.
The minimal to mild increase of the goblet cells between the cells of the respiratory epithelium is largely the same in all three groups.

- lungs: the 5 lung localisations revealed a normal lung structure. All animals showed a minimal to moderate alveolar pulmonary oedema, a change that was frequently observed in all 5 localizations. The severity declined from the high dose in group (5 mg/L air) to low dose group (0.05 mg/L air). The oedema was covered by a reactive secondary alveolar emphysema. Since the animals died prematurely, they showed a distinct congestion. Pneumonic foci with macrophages and lymphocytes in the perivascular area and in the adjacent alveoli were most frequently observed in animals treated with low dose group (0.05 mg/L air).
A focal haemorrhage in some male and female animals was a coincidental finding and thus not test item-related.

- larynx: a minimal focal squamous cell metaplasia was observed in the base of epiglottis in level one of 2 female animals and in 1 male rat of 0.05 mg/L air group. The hig dose group (5 mg/L air) and mid dose group (0.5 mg/L air) had a normal structure. Level 2 showed a focal minimal to mild squamous cell metaplasia in the area of the cricoid cartilage in high dose group (5 mg/L air) in 2 male and 1 female animals, in 0.5 mg/L air and 0.05 mg/L air groups in each 1 animal.

- trachea: no morphological differences were detected between the 3 groups. In most animals, a minimal to mild autolysis was noted for the epithelium and normal lymphocytic follicles were observed.

2) Main study animals (premature death 3 to 6 days):

- nasal cavity: the morphological findings of the main test animals at the nose at level 1 to 5 are comparable to those of the satellite group. The slight to moderate degeneration of the olfactory epithelium and its detachment was most pronounced in the high dose group (5 mg/L air). In addition, a minimal to mild degeneration of the respiratory epithelium as well as an increase in the goblet cells was observed in the main study.

- lungs: the alveolar lungs oedema was most pronounced in all 5 lung localisations of the low dose of group (0.05 mg/L air). This could be due to the longer survival of the animals. This oedema is accompanied by an acute alveolar emphysema. The pneumonic foci with accumulations of macrophages and lymphocytes were predominantly observed in 0.05 mg/L air group.

- larynx: a squamous cell metaplasia was observed in the larynx only for the male and female animals of 0.5 mg/L air and 0.05 mg/L air groups. In level 1, base of epiglottis, squamous cell metaplasia was identified at the base of the epiglottis in the 0.5 mg/L air group in 2 male and 4 female animals as well as in 0.05 mg/L air group in 2 male and 3 female animals. One female animal showed a focal ulceration with mixed cell infiltrations.
Level 2 showed a focal minimal to mild squamous cell metaplasia in the area of the cricoid cartilage in the 0.5 mg/L air group in each 2 male and female animals and in the 0.05 mg/L air group in each 2 animals.
- trachea: no morphological differences was detected between the 3 groups. In most animals, a minimal to mild autolysis was detected for the epithelium and normal lymphocytic follicles were observed.

The premature death of the animals of the main group was based upon the test item related moderate lung changes. All animals showed moderate alveolar and perivascular inflammatory oedemas, reactive alveolar emphysemas, haemorrhages and congestion. Furthermore, the epithelial cells in the nose, trachea and larynx showed a degeneration with acute inflammatory reactions.

Applicant's summary and conclusion

Interpretation of results:

Category 1 based on GHS criteria

Conclusions:

LC50 (male and female rats; 4 hours) < 0.05 mg/L air (actual concentration)
According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is acutely toxic via the inhalative route (Category 1; H330).