Cobalt(2+) propionate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-02-22 to 2019-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

signed on 2017-05-08

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt(2+) propionate

EC Number:

216-333-1

EC Name:

Cobalt(2+) propionate

Cas Number:

1560-69-6

Molecular formula:

C3H6O2.1/2Co

IUPAC Name:

cobalt(2+) dipropanoate

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: pink crystalline powder
- Particle size parameter:
x10 = 4.43 µm
x50 = 23.80 µm
x100 = 365.00 µm
x: particle size; 10, 50, 100: percentage cumulative

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in original and tightly closed container in a dry, cool 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 study initiation: males: approx. 8 weeks; females: approx. 8 to 10 weeks
- Weight (shortly before exposure): males: 244 - 266 g; females: 207 - 246 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: kept by sex in groups of 2 or 3 animals in MAKROLON cages (type III plus); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum; refer for exception to "Fasting period before study" above): commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 5 adaptation days
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, 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

Mass median aerodynamic diameter (MMAD):

>= 1.58 - <= 1.624 µm

Geometric standard deviation (GSD):

2.06

Remark on MMAD/GSD:

The MMAD/GSD mentioned above are for the main study. The MMAD/GSD of the satellite group were as follows:
MMAD: 1.502 - 1.660 µm
GSD: 1.97 - 2.06

Details on inhalation exposure:

NOTE: the first tested concentrations were 5.07 and 5.09 mg/L air (satellite group and main study, respectively) followed by the concentration of 0.52 mg/L air (satellite group and main study) and by the concentration of 1.04 and 1.04 mg/L air (satellite group and main study).

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 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 (inner diameter: 23.9 cm; height: 63.0 cm; volume 28.5 L) which holds the animals in pyrex tubes at the edge of the chamber in a radial position.

- System of generating particulates/aerosols: dust of the test item 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).
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.

- Method of particle size determination: analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor (Cascade impactor 6.0 L/min (Article No. 700800-CI-060), TSE Systems GmbH, 61352 Bad Homburg, Germany).
The dust from the exposure chamber was drawn through the cascade impactor for 1 to 2 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (SARTORIUS BP 121 S, 120 g weighing capacity, precision: 0.1 mg, resolution: 0.1 mg, linearity: 0.2 mg). Deltas of slides’ weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear 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 physical particle size with a HELIOS (H3695) & RODOS/M, R5, by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, pressure in air chamber, oxygen content and carbon dioxide content: 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 (20.8 - 21.5°C) and humidity (56.5 - 67.6 %) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

Exposition started by locating the animals into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes (t95 approx. 6 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: 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 (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 to 10 minutes. The filters were weighed before and after sampling (accuracy 0.1 mg).
- Samples taken from breathing zone: yes
Individual chamber concentration samples did not deviate from the mean chamber concentration by more than 1%.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Actual concentrations:
Main study: 0.52 ± 0.01 mg/L air and 5.09 ± 0.02 mg/L air (nominal concentrations: 5.56 and 44.44 mg/L air, respectively)
Satellite group: 0.52 ± 0.01 and 5.07 ± 0.01 mg/L air (nominal cocnentrations: 5.56 and 44.44 mg/L air, respectively)

No. of animals per sex per dose:

Main study:
0.52 mg/L: 5 males / 5 females
5.09 mg/L air: 3 males / 3 females

Satellite group:
0.52 and 5.07 mg/L air: 3 males / 3 females

Control animals:

no

Details on study design:

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

- Frequency of observations and weighing: 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.
A clinical examination was made at least once each day, thereafter at least once daily until end of the study or at death (main study) or end of the 24-hour period before necropsy (satellite animals).
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.
Individual weights of animals were determined once during the acclimatisation period, before the exposure on test day 1, on test days 2, 4, 8 and 15 and at time of death. Changes in weight were calculated and recorded when survival exceeded one day.

- 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 or as soon as possible after exitus, also to assess whether any respiratory tract irritation persists 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 jaw) or 7% (other organs) buffered formalin:
- nasal cavity, nasopharynx and paranasal sinus (localisations: posterior part of upper incisors, incisive papilla, second palatine crest, and first molar teeth)
the 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 approximately 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. All sections were embedded face down to yield a section from the anterior section, except the nose tip was embedded posterior surface down.
- larynx (localisations: base of epiglottis, ventral pouch, and cricoid cartilage)
- trachea (one section, including the bifurcation, longitudinal horizontal)
- lung (localisations: 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.

The histopathology was conducted in consideration of the suggestions made in the OECD Guidance Document on Histopathology for Inhalation Toxicity Studies, Supporting TG 41 (Subacute Inhalation Toxicity: 28-day Study) and TG 413 (Subchronic Inhalation Toxicity: 90-day Study). OECD Series on Testing and Assessment No. 125, Document No. ENV/JM/MONO (2010) 16, 1 June 2010.

Statistics:

none

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

>= 1.03 - <= 5.09 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Main study:
- 5.09 mg/L air: all animals were found dead between test days 2 and 4.
- 0.52 mg/L and 1.03 mg/L air: no animal died prematurely.
Satellite group:
- 0.52, 1.04 and 5.07 mg/L air: no animal died prematurely.

Clinical signs:

other: Please refer to the field `Any other information on results incl. tables´.

Body weight:

Main study:
- 5.09 mg/L air: it was not possible to assess the body weight as all animals died within few days after administration.
- 0.52 and 1.03 mg/L air: the body weight gain of the animals was in the expected range at the end of the study.

Gross pathology:

Macroscopic changes in the nasal cavity and lungs:
Necropsy revealed marbled and/or slightly to markedly oedematous lungs in all main study and satellite animals.

Other findings:

Histopathology (nasal cavity and lungs):
1) Lesions related to the administration of the test item:
The histomorphological examination of the trachea, larynx, lungs and of the nose of male and female rats after inhalation of Cobalt Propionate revealed morphological changes in the nose of the male and female satellite animals and in the main study rats of group 1 (5.07/5.09 mg/L), group 3 (1.04/1.03 mg/L) and 2 (0.52 mg/L) which are considered to be test item-related. The lungs showed pathological findings in the high dose group 1 of the main study (5.09 mg/L) only.
a) Satellite animals (24-hour sacrifice):
Nose: The nasal cavity of level 1 revealed a normal squamous epithelium.
The level 2 revealed after 5.07 mg/L (group 1), and 1.04 mg/L (group 3) 0.52 mg/L (group 2) a dose dependant minimal to mild loss of cilia or ciliated cells with mixed cell infiltration. A minimal to moderate loss of olfactory cells with degeneration of the olfactory epithelium was noted in level 3 to 5 in all 3 groups. These changes begin at the transition from the respiratory to the olfactory epithelium. Vacuolization, membrane ruptures of the cells and a focal loss of the olfactory epithelial cells were noted.
The minimal to mild increase of the goblet cells between the cells of the respiratory epithelium is largely the same in all animals of the groups (0.52 mg/L, 1.04 mg/L and 5.07 mg/L).
Lungs: In most satellite animals a normal lung structure without pathological changes was histopathologically observed. Only a few male and female animals showed a minimal alveolar pulmonary oedema with perivascular oedema. These findings were interpreted as coincidental changes.
Larynx and trachea: A minimal focal lympho-histiocytic infiltration was noted in each rat. All the other rats showed a normal structure without inflammatory and degenerative changes.
b) main study animals (premature death test days 2 to 4):
The morphological findings of the main test rats at the nose in level 3 to 5 showed a mild to moderate loss of olfactory epithelium and a mild to moderate degeneration of olfactory epithelium. These findings were more pronounced in group 1 compared to the intermediate dose of group 3 and the low dose of group 2. Morphological differences between the female and male rats were not diagnosed.
In addition, a minimal to mild loss of the respiratory epithelium with focal degeneration was observed in the main study of group 1.
Lungs: The alveolar pulmonary oedema and the perivascular oedema were observed in all 5 lung localisations, most pronounced in the high dose group 1 (5.09 mg/L). The oedema fluid contains mild inflammatory cells and alveolar macrophages in the alveoli and perivascular.
Larynx: A mild glandular ectasia and a focal lympho-histiocytic subepithelial infiltration were noted in a few rats. The base of the epiglottis and the area of the cricoid cartilage showed a normal structure.
Trachea: No morphological differences were detected between the 3 groups (0.52, 1.03 and 5.09 mg/L).
Mortality of animals:
The cause in the premature death of the animals from the main group 1 was because of the test item related moderate lung changes. All animals showed moderate alveolar and perivascular inflammatory oedemas, haemorrhages and congestion. Furthermore, the epithelial cells in the nose showed a degeneration with a loss of the respiratory epithelial cells and a mild to moderate degeneration of the olfactory epithelium.
c) main study animals (terminal sacrifice, group 2 and 3):
The morphological findings of the main test rats at the nose in level 1 to 5 are comparable to those in the satellite groups. A minimal to mild loss of the olfactory epithelium with degeneration was noted in group 3 (1.03 mg/L) and 2 (0.52 mg/L) but minimal more pronounced in group 3. In addition, a minimal to mild loss of the respiratory epithelium with focal degeneration was observed in group 2 and 3.
Lungs: The alveolar pulmonary oedema and the perivascular oedema observed in group 3 (1.03 mg/L) and 2 (0.52 mg/L) were minimal and not test item related. The oedemas were interpreted as coincidetal findings.
Larynx: A mild glandular ectasia and a focal lympho-histiocytic subepithelial infiltration were noted in a few rats. The base of the epiglottis and the area of the cricoid cartilage showed a normal structure. All the other rats showed a normal structure without inflammatory and degenerative changes.
Trachea: No morphological differences were detected between group 3 (1.03 mg/L) and 2 (0.52 mg/L).

Any other information on results incl. tables

Clinicla signs:

Main study:

- 5.09 mg/L air: slight to moderate ataxia and slight to severe dyspnoea observed immediately after end of exposure until death, and slightly reduced motility and pilo-erection, observed from 1 day after exposure until death in all 3/3 male.

- 1.03 and 0.52 mg/L air: slight ataxia immediately until 60 minutes or 3 hours (0.52 or 1.03 mg/L, respectively) after end of exposure and slight dyspnoea immediately after end of exposure until 3 days post exposure in all 5/5 male and 5/5 female animals.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Under the present test conditions, the LC50 value (males and females combined) for rats following inhalation of Cobalt propionate for 4 hours was determined as follows (gravimetric concentration):
LC50: between 1.03 and 5.09 mg Cobalt propionate/L air.

According to regulation EC (1272/2008) and subsequent regulations, the test material is classified as acute toxic via the inhalative route (Categroy 4; H332).

Executive summary:

In an acute inhalation toxicity study, three rats per sex, dose and study type (main or satellite) were exposed for 4 hours to dust aerosol with concentrations of 0.52 ± 0.01, 1.03 ± 0.01 and 5.09 ± 0.02 mg Cobalt propionate/L air (main study) or 0.52 ± 0.01, 1.04 ± 0.01 and 5.07 ± 0.01 mg (satellite animals) Cobalt propionate/L air, using a dynamic nose-only exposure chamber.

For the inhalation chamber, a MMAD of 1.580 to 1.624 µm (main study) and 1.502 to 1.660 µm (satellite animal), and a GSD of 2.06 to 2.112 (main study) and 1.97 to 2.06 (24-hour sacrifice) were  measured and calculated.

A concentration of 5.09 mg/L air revealed slight to moderate ataxia and slight to severe dyspnoea observed immediately after end of exposure until death within 3 days after exposure, and slightly reduced motility and piloerection, observed from 1 day after exposure until death in all 3/3 male and 3/3 female animals. It was not possible to assess the body weight as all animals died within few days after administration.

A concentration of 1.03 and 0.52 mg/L air revealed slight ataxia immediately until 60 minutes or 3 hours (0.52 or 1.03 mg/L, respectively) after end of exposure and slight dyspnoea immediately after end of exposure until 3 days post exposure in all 5/5 male and 5/5 female animals. These concentrations did not result in premature death in any animal and the body weight gain was in the expected range at the end of the study.

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

The histomorphological examination of the trachea, larynx, lungs and the nose revealed particularly in the lungs and nose of the male and female main study rats in the high dose group 1 (5.09 mg/L) morphological changes which are considered to be test item-related. In the intermediate group 3 (1.03 mg/L) and low dose group 2 (0.52 mg/L) only the nose showed test item related findings.

Cobalt propionate caused test item-dependent changes in the nasal mucosa of group 1 (5.09 mg/L), 3 (1.03 mg/L) and 2 (0.52 mg/L). The lungs showed only test item-related oedemas in the male and female rats of group 1 (5.09 mg/L).

Under the conditions of this study, the 4-hour inhalation LC50 value for rats (males and females combined) of Cobalt propionate is between 1.03 and 5.09 mg/L air. According to regulation EC (1272/2008) and subsequent regulations, the test material is classified as acute toxic via the inhalative route (Category 4; H332).