Cobalt oxalate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-02-15 to 2016-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 2014-05-14

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt oxalate

EC Number:

212-409-3

EC Name:

Cobalt oxalate

Cas Number:

814-89-1

Molecular formula:

C2H2O4.Co

IUPAC Name:

cobalt(2+) oxalate

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: pink powder
- Particle size distribution:
D10% = 1.24 μm
D50% = 4.50 μm
D90% = 20.23 μm

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: container kept upright and tightly closed in a dry, cool and well-ventilated place, avoiding heat or direct sunlight.

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, Sulzfeld ,Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: approx. 8 weeks; females: approx. 9 weeks
- Weight at study initiation: males: 257 - 280 g; females: 233 - 260 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 3 animals (MAKROLON cages (type III plus)) during the 14-day observation period; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany)
- Diet: commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days
Animals 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):

>= 1.9 - <= 1.974 µm

Geometric standard deviation (GSD):

>= 2.5 - <= 2.55

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus /exposure chamber volume: dynamic inhalation apparatus (RHEMA-LABORTECHNIK) (air changes/h (≥ 12times)) with a nose-only exposure of the animals according to KIMMERLE & TEPPER.
The apparatus consists of a cylindrical exposure chamber, which holds the animals in pyrex tubes at the edge of the chamber in a radial position.
Actual dimensions of the inhalation chamber: inner diameter: 28.2 cm; height: 64.6 cm; volume: 40.3 L

- System of generating particulates/aerosols: dust of the test item was generated using a rotating brush dust generator (RBG 1000, PALAS GmbH Partikel und Lasermesstechnik). The generator was fed with compressed air (5.0 bar) from a compressor (ALUP Kompressorenfabrik).
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) 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: an analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor 6.0 L/min (TSE Systems GmbH).
The dust from the exposure chamber was drawn through the cascade impactor for 1 minute (constant flow rate: 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.

- Temperature, humidity, pressure in air chamber, oxygen content; carbon dioxide content: oxygen content in the inhalation chamber was 21% (determined at the beginning and at the end of the exposure; DRÄGER Oxygen-analysis test set)(DRÄGER Tube Oxygen 67 28 081). Carbon dioxide concentration did not exceed 1%.
Temperature (21.0°C ± 0.2°C (main study) or 21.1°C ± 0.1°C (satellite group)) and humidity (57.3% ± 0.1% (main study) or 51.4% ± 0.1% (satellite group)) 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. 8 minutes).
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) controlled by a rotameter. Dust samples were taken once every hour during the exposure. The filters were weighed before and after sampling (accuracy 0.1 mg).
- Samples taken from breathing zone: yes, at a constant flow of air of 5 L/min for 1 minute

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:

Main study (limit test):
- actual concentration: 5.06 ± 0.01 mg/L air
- nominal concentration: 24.44 mg/L air
Satellite group:
- actual concentration: 5.06 ± 0.03 mg/L air
- nominal concentration: 24.44 mg/L air

No. of animals per sex per dose:

Main study (limit test): 3 males / 3 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 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. Observations on mortality were made at least once daily (in the morning starting on test day 2).
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. 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 was carried out and all gross pathological changes were recorded:
- satellite animals: necropsy at 24 hours after cessation of exposure
- main study animals: necropsy at the end of the 14-day observation period

All main study and satellite animals were subjected to 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, and thickened mucous layer.
The following organs of all animals were fixed: nasal cavity (tip and 5 levels; tip and level 1: cut just anterior to the incisor teeth; level 2: cut approx. 2 mm posterior to free the tip of the
incisor teeth; level 3: cut through the incisive papilla; level 4: cut through the middle of the second palatal ridge, which is located just anterior to the molar teeth; level 5: cut through the middle of the molar teeth), nasopharynx, paranasal sinus, larynx, trachea, and lung.

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, June 01, 2010.

Assessment of respiratory tract irritation effects:
The assessment of respiratory tract irritation effects was conducted according to the criteria set forth in the OECD proposal document ENV/JM/HCL(2004)9/REV, where it is stated:
- There are currently no validated animal tests that deal specifically with respiratory tract irritation. However, useful information may be obtained from single and repeated inhalation toxicity tests. For example, animal studies may provide useful information in terms of toxicity (dyspnoea, rhinitis etc) and histopathology (e.g. hyperaemia, oedema, minimal inflammation, and thickened mucous layer) which are reversible and may be reflective of the characteristic clinical symptoms described above. Such animal studies can be used as part of weight of evidence evaluation.
- The special classification would occur only when more severe organ/systemic effects including the respiratory system were not observed.

Statistics:

not applicable

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.06 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

5.06 mg/L air (main study) and 5.06 mg/L air (satellite group): no animal died prematurely.

Clinical signs:

other: 5.06 mg/L air (main study) and 5.06 mg/L air (satellite group): slight dyspnoea (reduced frequency of respiration with increased volume) immediately after end of exposure until 3 hours post exposure was observed in 3 of 3 male and 3 of 3 female animals of

Body weight:

5.06 mg/L air (main study): all 3 of 3 male and 3 of 3 female animals appeared to be reduced in body weight gain throughout the whole recovery period.

Gross pathology:

No pathological findings were noted in the nasal cavity and in the lungs, neither for the main study animals (14-day sacrifice) nor for the satellite animals (24-hour sacrifice).

Other findings:

1) Histopathology (test item related changes):
- 5.06 mg/L air (satellite group): the turbinates of levels 2 to 5 of the nasal cavities showed an acute mild to moderate focal degeneration of the olfactory epithelium with focal pyknosis, karyorrhexis and loss of olfactory epithelial cells. In the neighborhood of these defects a normal olfactory epithelium was detected.
Inflammatory cells were not noted near the degenerative olfactory epithelium. A normal respiratory epithelium partially with cilia was observed for the levels 2 to 5 of the nasal cavity. The respiratory epithelium contained three normal major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells.

2) Histopathology (not test item related changes):
- 5.06 mg/L air (main study) and 5.06 mg/L air (satellite group):
Nose (five levels): the nasal cavity and the turbinates of the nose of levels 1 to 5 showed a normal squamous and respiratory epithelium and a normal olfactory epithelium for levels 2 to 5. The olfactory epithelium of the main study rats showed a complete reversibility after a recovery of 14 days. The respiratory epithelium contained three normal major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells without degenerative or inflammatory reactions.

Lungs (five levels): all 5 lung localizations per animal of the satellite animals and the main study rats showed a minimal to mild alveolar haemorrhage and a minimal to mild focal atelectasis. These minimal changes are interpreted as spontaneous incidental findings within the normal range.

Trachea and larynx : the trachea and larynx showed a focal minimal to mild subepithelial lymphohistiocytic infiltration without degeneration of the epithelial cells. The number of seromocous glands in the larynx without inflammatory reactions was morphologically normal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

LC50 (male and female rats; 4 hours) > 5.06 mg/L air (actual concentration)
According to the EC-Regulation 1272/2008 and subsequent regulations, cobalt oxalate is not classified as acute toxic via the inhalation route.

Based on the results of the macroscopic and histopathological investigations, cobalt oxalate appears to be marginally irritating for the nasal cavities, confirmed by marginal histopathological changes in form of an acute mild to moderate focal degeneration of the focal olfactory epithelium. The changes observed were completely reversible during the 14-day recovery period.