Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-12 to 2018-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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 2015-08-14
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium hexacyanocobaltate
EC Number:
237-742-1
EC Name:
Tripotassium hexacyanocobaltate
Cas Number:
13963-58-1
Molecular formula:
C6CoN6.3K
IUPAC Name:
tripotassium hexacyanocobalttriuide
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: bright yellow powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept dry in a bottle at room temperature

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx 7 to 8 weeks of age
- Weight at study initiation (means): males: 255.88 to 293.43 g; females: 184.28 to 203.18 g
- Housing: housed in Makrolon® (polycarbonate) cages type IV, three rats of the same sex per cage; bedding material: softwood bedding material (Lignocel BK8-15)
- Diet (fresh weekly or more often): Ssniff V1534 (Ssniff-Spezialdiäten (Soest, Germany))
- Water (fresh weekly or more often): tap water
- Acclimation period: approx. two weeks

For 2 weeks before exposure, rats were trained to the exposure tubes avoiding undue stress on the animals. Animal restraining tubes are constructed in such a way that hyperthermic effects on rats cannot occur.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Relative humidity: 55 % ± 15 %
- Air changes: at least 10 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.24 - <= 2.62 µm
Geometric standard deviation (GSD):
>= 1.76 - <= 2.23
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the aerosol was supplied to the rats by a flow-past nose-only inhalation exposure system. The rats are placed around the exposure cylinder in tapered acrylic glass tubes with adjustable backstops. Historical measurements have confirmed, that there are no differences in concentrations among the different outlets. The animal's snout protrudes in the anterior end of the tube, which is connected to the exposure cylinder by means of a push fit. The aerosol enters the nasal region of the animal through a small tube. The exposure cylinder is operated at slightly positive pressure with respect to the surrounding air. This ensures that a continuous air flow is passing through the animal's breathing zone. In this system, the aerosol is supplied to each rat individually, and exhaled air is immediately removed. Therefore, oxygen supply is sufficient and measurement of the oxygen concentration is not necessary. The exposure unit is capable to house up to 16 animals on one level (in this study 6/6 m/f will be exposed in step 1 etc.).

- System of generating particulates/aerosols: for aerosol generation, in both high dose groups the AeroNeb® system was used achieve the target MMAD values.
The airflow or aerosol flow to each rat is approx. 1 L/min which is assumed to be laminar. The total flow rate will be approx. 32 L/min; the total volume of the inhalation system including the mixing box ensures that the intended concentration of the test item is reached shortly after start of exposure (50%-value of concentration after approximately 4 min).
The test item will be aerosolized using a dispersion system optimized for the aerosolization of a liquid formulation and operated with pressurized air. This aqueous solution will be nebulized with pressurized air resulting in a dry aerosol after rapid evaporation of the water. The signal of an aerosol photometer will be used to control the feed rate of the dispersion system in order to keep the aerosol concentration in the inhalation unit constant.

- Method of particle size determination: MMAD of the aerosol phase was determined by measuring the K3[Co(CN)6] dry aerosol (Marple impactor; two MMAD determinations within the 4-hour exposure period).

- Temperature, humidity, exhaust air flow: air flow, temperature and relative humidity were measured continuously and recorded by 10-minute means.

TEST ATMOSPHERE
- Brief description of analytical method used: aerosol concentrations were determined gravimetrically using filter samples (2 to 4 times within the 4-hour exposure period).

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
48.9 mg/m³: 2.46 µm (GSD: 2.18)
489.8 mg/m³: 2.24 µm (GSD: 2.23)
1070.8 mg/m³: 2.62 µm (GSD: 1.76)
2113.5 mg/m³: 2.49 µm (GSD: 2.09)

CLASS METHOD
- Rationale for the selection of the starting concentration: due to the known strong inflammatory potential of ionic cobalt substances, a single exposure to a test concentration of approx. 0.05 mg/L for 4 hours was used as start concentration.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
target concentrations: 50, 500, 1000 and 2000 mg/m³
actual concentrations: 48.9, 489.8 ± 85.0, 1070.8 ± 83.8 and 2113.5 ± 101.9 mg/m³
The mean aerosol concentration of 2114 mg/m3 represents the maximum technically feasible concentration.
No. of animals per sex per dose:
3 males / 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 24 hours and 14 days
- Frequency of observations and weighing: on the day of treatment, all animals were observed several times for clinical symptoms (prior to, during and after exposure). Thereafter, all animals were clinically observed in their cages once a day. Once a week, they were inspected outside their home cages and carefully examined for abnormalities concerning their general condition.
Individual body weights were recorded on days 0, 1, 3 ,7, 10 and 14.

- Necropsy of survivors performed: yes/no
Following the exposure, 3 rats were sacrificed after 24 hours for histopathological examination, the other 3 rats were kept in Makrolon® cages under observation for additional 14 days; thereafter, these animals were sacrificed, too.
All animals at terminal sacrifice were killed by cutting the vena cava caudalis after anaesthesia with an overdose pentobarbital sodium and necropsied immediately.
The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. Heart, oesophagus, upper half of trachea, thymus and lung associated lymph nodes (mediastinal and tracheobronchial) were removed from the lung convolution.
The lung and the lower half of the trachea were weighed. For histopathology the lung was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion for a minimum of 2 hours, and used for histopathology.
The trimming was done according to Ruehl-Fehlert et al. (2003)*, Kittel et al. (2004)* and Morawietz et al. (2004)*.

The histopathology of all organs was performed in 3 males and 3 females of each dose step. Particular attention was paid to:
- histopathology of respiratory tract including bronchi and the lung-associated lymph nodes (mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including nasopharynx-associated lymphoid tissues)
- trimming: lungs 5 sections (acc. to OECD GD 125); nose 4 sections (acc. to Kittel et al. (2004)*); larynx: 3 sections; trachea: 1 section (longitudinal horizontal)

Tissues for histological examination were fixed for at least one week in buffered formalin (10%). Bones will be decalcified prior to embedding.
Lung lobes were fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with haematoxylin and eosin. In addition, masson trichrome staining was used for detection of connective tissue production (lower half).

*References:
- Ruehl-Fehlert, C., Kittel, B., Morawietz, G., Deslex, P., Keenan, C., Mahrt, C.R., Nolte, T., Robinson, M., Stuart, B.P., Deschl, U. Revised guides for organ sampling and trimming in rats and mice--Part 1. A joint publication of the RITA and NACAD groups. Exp Tox Path 55, 91-106 (2003).
- Kittel, B., Ruehl-Fehlert, C., Morawietz, G., Klapwijk, J., Elwell, M.R., Lenz, B., O'Sullivan, M.G., Roth, D.R., Wadsworth, P.F. Revised guides for organ sampling and trimming in rats and mice--Part 2. A joint publication of the RITA and NACAD groups. Exp Tox Path 55, 413-31 (2004).
- Morawietz, G., Ruehl-Fehlert, C., Kittel, B., Bube, A., Keane, K., Halm, S., Heuser, A., Hellmann, J., Revised guides for organ sampling and trimming in rats and mice--Part 3. A joint publication of the RITA and NACAD groups. Exp Tox Path 55, 433-49 (2004).


Statistics:
not applicable

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2 114 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: SD: 101 mg/m³
Mortality:
48.9, 489.8, 1070.8 and 2113.5 mg/m³: no unscheduled deaths occurred during the study period.
Clinical signs:
other: 48.9, 489.8, 1070.8 and 2113.5 mg/m³: upon cessation of exposure none of the rats exposed to the test item showed any signs of toxicity. Only usual signs of discomfort after exposure to particles were observed. Grooming activity started immediately after
Body weight:
48.9, 489.8, 1070.8 and 2113.5 mg/m³: body weight data were in the normal range of rats of the given age.
Gross pathology:
48.9, 489.8, 1070.8 and 2113.5 mg/m³: all animals were sacrificed at scheduled dates. Upon necropsy, test item- or dose-related macroscopical findings were not observed.
Other findings:
- Organ weights: lung weights were in the normal range of untreated rats at the given age.
- Histopathology: In the study, the respiratory tract such as nasal cavity, nasopharynx, larynx, laryngopharynx, trachea, lung, lung-associated lymph nodes and all gross lesions were examined of all dosed animals.
Exposure-related effects were detected only in the larynx of the animals of the 2114 mg/m³ dose level group sacrificed one day after exposure. Two of the three female and male animals showed at the base of the epiglottis a focal very slight erosion/ulceration, which was accompanied in three of the three male and female animals by a (multi)focal very slight subepithelial infiltration of inflammatory cells.
All other findings within the organs of the respiratory tract are commonly found background lesions and were interpreted to be unrelated to the treatment. These findings were dilatation of submucosal glands and subepithelial infiltration of mononuclear cells in the larynx, pulmonary hemorrhage alveolar histiocytosis, neuroendocrine cell hyperplasia, perivascular granulocytic cell infiltration, mixed inflammatory cell infiltration and osseous metaplasia in the lung, sinusoidal dilation in the lung-associated lymph nodes, mucous cell hyperplasia and subepithelial mixed inflammatory cell infiltration in the nasal cavity as well as dilatation of submucosal glands and subepithelial mixed inflammatory cell infiltration in the trachea. The nasopharynx and laryngopharynx were without any visible lesions.
Two females of the high dose group showed macroscopically an enlarged and discolored mandibular lymph node. These were histologically diagnosed as slight to moderate blood resorption.

Conclusion:
K3[Co(CN)6] exposure-related findings were detected within the larynx. These lesions at the specific location (base of epiglottis) represent rat specific findings.
No other treatment-related findings were detected in the respiratory tract.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
LC50 (rats; 4 hours) > 2114 ± 101 mg/m³ air (actual concentration; maximum attainable concentration)
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the inhalation route.