Olivine, cobalt silicate blue

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-17 to 2017-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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 2017-05-08

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Olivine, cobalt silicate blue

EC Number:

269-093-5

EC Name:

Olivine, cobalt silicate blue

Cas Number:

68187-40-6

Molecular formula:

Co2SiO4

IUPAC Name:

silicon(4+) bis(λ²-cobalt(2+)) tetraoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment Blue 73)
- Substance type: inorganic pigment
- Storage condition of test material: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to + 25 °C

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
- Age at study initiation: males: approx. 8 weeks; females: approx. 9 weeks
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: males: 246 - 273 g; females: 218 - 252 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, the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany), the cages were changed and cleaned twice a week.
- Diet: commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water in bottles
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 10% (maximum range)
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.991 - <= 2.258 µm

Geometric standard deviation (GSD):

>= 2.26 - <= 2.5

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: 2.230 - 2.302 µm
GSD: 2.29 - 2.55

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 & TEPPER. The apparatus consists of a cylindrical exposure chamber (volume 28.5 L) 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 23.9 cm
Height 63 cm

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.
Air flow entrance (L/h): 900
Air flow exit (L/h): 800
Air changes (changes per hour): 31.6
The whole exposure system was mounted in an inhalation facility to protect the laboratory staff from possible hazards. The exhaust air was drawn through gas wash-bottles.

- 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).
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: an 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 or 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, 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 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 Malvern Mastersize 2000 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, pressure in air chamber, oxygen content and 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 (20.8 - 22.2°C (main study) or 20.5 - 22.4°C (satellite group)) and humidity (57.5 - 62.5% (main study) or 57.3 - 63.5% (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 approximately 8 minutes).

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 (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).
- Samples taken from breathing zone: yes

Analytical verification of test atmosphere concentrations:

yes

Remarks:

see above ("Details on inhalation exposure")

Duration of exposure:

4 h

Concentrations:

Main study:
- actual concentrations:
5.07 ± 0.03 mg/L air
1.08 ± 0.01 mg/L air
0.53 ± 0.01 mg/L air
- nominal concentrations:
22.22 mg/L air
5.56 mg/L air
2.78 mg/L air

Satellite group:
- actual concentrations:
5.06 ± 0.02 mg/L air
1.07 ± 0.01 mg/L air
0.52 ± 0.01 mg/L air

- nominal concentrations:
22.22 mg/L air
5.56 mg/L air
2.78 mg/L air

No. of animals per sex per dose:

Main study:
5.07 mg/L air: 3 males / 3 females
1.08 mg/L air: 5 males / 5 females
0.53 mg/L air: 5 males / 5 females
Satellite group:
5.06 mg/L air: 3 males / 3 females
1.07 mg/L air: 3 males / 3 females
0.52 mg/L air: 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 and following exposure, observations were recorded systematically; individual records were maintained for each animal. Careful clinical examinations were made at least once daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily with appropriate actions taken 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.
Individual weights of animals were determined once during the acclimatisation period, before the exposure on test day 1, on test days 4, 8 and 15 at the time of death. 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
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 tract 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, nasopharynx, paranasal sinus (posterior part of upper incisors, incisive papilla, second palatine crest, first molar teeth), larynx (base of epiglottis, ventral pouch, cricoid cartilage), trachea (incl. the bifurcation) and lung (left lobe, right caudal lobe, right cranial lobe, right middle lobe and accessory lobe)
Paraffin sections were prepared of all above mentioned 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, 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 and regulation (EC) 1272/2008, Annex I, Section 3.8.2.2.1:
- 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:

The LC50 was calculated according to FINNEY* (Probit Analysis).

*Finney, D. J., Ed. (1952). Probit Analysis. Cambridge, England, Cambridge University Press

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.75 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Main study:
5.07 mg/L air: all animals died preliminary (within 7 days).
1.08 mg/L air: all animals died preliminary (within 7 days).
0.53 mg/L air: no premature death was noted.

Satellite animals:
5.06 mg/L air: one male died within 24 h after exposure.
1.07 mg/L air: no premature death was noted.
0.52 mg/L air: no premature death was noted.

Clinical signs:

other: see remarks

Remarks:

In main and satellite animals: slightly to severely reduced motility, slight to severe ataxia, slight to severe dyspnoea

Body weight:

All 3 female animals at 0.53 mg/L air of the main study gained the expected body weight at the end of the study. All other animals of the main study died prematurely.

Gross pathology:

Necropsy revealed oedematous lungs in all animals of the main study and all satellite animals at all dose levels.
In addition, the lungs were also evaluated as marbled, at the dose level of 0.53 mg/L air in all 10 of 10 main study animals and in 1 of 5 male and in 1 of 5 female animals of this main study dose level showed an oedematous cervical lymph node.

Other findings:

- Histopathology:
Lesions related to the administration of the test item:
A 4-hour inhalation exposure to Olivine, Cobalt Silicate Blue revealed test item related histopathological changes in the nose and lungs of the rats.

Male and female satellite animals (24 hours sacrifice):
The nasal cavity and the turbinates in the nose of level 1 to 5 showed a normal squamous and respiratory epithelium. In the nasal turbinates, minimal to mild degeneration of the olfactory epithelium without inflammatory reactions was noted in most animals. In particular, the dorsal nasal conches showed these degenerations. The changes were more pronounced in the high dose and intermediate dose compared to the low dose.
The 5 lung localizations of the 3 test groups showed distinct morphological differences among themselves. In the high and intermediate dosed groups, the alveolar oedema and the perivascular oedema with inflammatory cells were significantly more pronounced then in the low dose group. Also, focal pneumonia with neutrophilic granulocytes in the alveolar lumen was
more distinct in high and intermediate dosed groups.
The trachea of a few male and female animals revealed minimal to mild subepithelial lympho-histiocytic infiltrations and a normal follicular lymphoid hyperplasia. There was no difference between the groups. The larynx showed loss of epithelial cells, in particular in male and female rats of high dose group.
Most of the respiratory epithelium of the larynx in intermediate and low dosed groups was normal without inflammatory or degenerative reactions.

Male and female animals of the main study (14 days sacrifice):
The histomorphological examination of respiratory tract organs (i.e. trachea, larynx, lungs and nose) of male and female rats after inhalation of Olivine, Cobalt Silicate blue revealed morphological changes in the nose and lungs of the male and female main study rats (14 days) of the high and intermediate dose groups). These changes are considered to be test item-related.
The nasal cavity of level 1 showed normal squamous and respiratory epithelia in all animals of all treatment groups.
In levels 2 to 5, normal respiratory epithelium partially with cilia was observed. The respiratory epithelium contained of three normal major cell types; the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells.
However, in levels 2 to 5 of the nasal cavities, the high and intermediate dose groups) showed minimal to moderate focal degeneration of olfactory epithelium with loss of olfactory epithelial cells in the turbinates.

Degenerated olfactory epithelium was detected 14 days after exposure to Olivine, Cobalt Silicate blue in male and female rats. These changes were similar to the changes detected in satellite animals after 24 hours exposure and were located in the same dorsal turbinates. Normal olfactory epithelium and no inflammatory cells were noted next to the degenerated cells.

The low dose group was less affected and showed minimal focal degeneration of olfactory epithelium. Most of the olfactory epithelium was normal.

All 5 lung localizations of the main study-animals in the high and intermediate dosed groups showed minimal to moderate perivascular inflammatory oedema with neutrophilic granulocytes, lymphocytes and histiocytes in the perivascular rooms. In addition, minimal to moderate multifocal mixed cellular pneumonia was observed in some rats. Furthermore, the animals showed a mild to moderate congestion and an alveolar oedema. These changes correlated with the macroscopic findings.

Dark granular to coarse-heavy material was found especially in the lumen of the bronchi, and less frequently in the bronchioles. There was no inflammatory reaction in the bronchial lumen.

The lungs of the low dosed animals were normal or showed only minimal to mild alveolar oedemas and perivascular oedemas or haemorrhages. Pneumonic foci are small foci with few neutrophilic granulocytes and lymphocytes in the alveolar lumen. The small number of pneumonic foci is interpreted as coincidental findings.

Only in one male rat of the low dosed group, minimal dark granular material was observed in the lumen of the bronchus.

The trachea showed focal minimal to mild subepithelial lympho-histiocytic infiltration. Loss of epithelial cells was observed due to autolysis. The larynx was normal in all 3 levels. Minimal to mild subepithelial lympho-histocytic infiltration was observed in a few animals. Loss of epithelial cells was observed due to autolysis.

In conclusion, the levels 2 to 5 showed focal degeneration of the olfactory epithelium with loss of epithelial cells in the nasal cavity without any subepithelial inflammatory reaction. No clear morphological difference was observed for the degeneration of the olfactory epithelium between the satellite and main study animals.

The primary changes of the lungs represent a disturbance of permeability with perivascular oedemas and congestions with secondary pneumonia.

These inflammatory oedema, pulmonary bleeding and pneumonia are the cause of death for the animals of the high and intermediate dose group.

The larynx and trachea showed an unobtrusive epithelium with unspecific subepithelial lympho-histiocytic infiltrations and regular lymphoid hyperplasia in all animals of all 3 groups. Loss of epithelial cells was observed due to autolysis. All other findings were spontaneous in nature and therefore not considered to be test item-related.

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

LC50 (male and female rats): 0.75 mg/L air
According to the EC Regulation 1272/2008 and subsequent regulations, the test item should be classified in the hazard category 3 and requires labelling with 'Danger' and 'H331: Toxic if inhaled'.

The classification criteria according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – single exposure, inhalation are not met since the toxic effects observed in the acute toxicity test via inhalation already leads to an acute inhalation toxicity classification. No additional effects in animals or humans are known that would justify a specific target organ toxicant (STOT) – single exposure: inhalation classification.

Executive summary:

Rats were exposed to a dry aerosol of Olivine, cobalt silicate blue at gravimetrically determined concentrations of 5.07 ± 0.03, 1.08 ± 0.01 or 0.53 ± 0.01 mg/L air (main study, 14-day sacrifice) or 5.06 ± 0.02, 1.07 ± 0.01 or 0.52 ± 0.01 mg/L air (satellite animals, 24-hour sacrifice) for 4 hours by inhalation using a dynamic nose-only exposure chamber. The aerosol was generated with the aid of a dry, rotating brush dust generator.

Under the present test conditions, a 4-hour inhalation exposure to Olivine, cobalt silicate blue at concentrations of 0.53 mg/L air (determined by gravimetric analysis) caused no premature death. A 4-hour inhalation exposure to Olivine, cobalt silicate blue at concentrations of 5.07 or 1.07 mg/L air (determined by gravimetric analysis) caused preliminary death of all animals.

LC50 value for males and females combined (14 days): 0.75 mg Olivine, cobalt silicate blue/L air/4 hours calculated according to Finney (1952).