Reaction mass of cobalt olivine and crystalline silicon dioxide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-10 to 2020-01-20

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 2017-05-08

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of cobalt olivine and crystalline silicon dioxide

EC Number:

701-439-7

Molecular formula:

Co2SiO4∙xSiO2 (0,7≤x≤1,5)

IUPAC Name:

Reaction mass of cobalt olivine and crystalline silicon dioxide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment blue 73)
- new chemical name: Reaction mass of cobalt olivine and crystalline silicon dioxide
- Substance type: inorganic pigment
- Storage conditions: 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, keep dry in closed containers

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: 251 - 282 g; females: 219 - 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 were 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: 19 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.

The animals were randomised before use. They 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.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

2.157 µm

Geometric standard deviation (GSD):

2.323

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: 2.152 µm
GSD: 2.226

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: 63.0 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 tests with the main study animals and the satellite animals were conducted in the same inhalation chamber but on different days. Between the exposure times the chamber was cleaned carefully.
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 6.0 L/min (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 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 HELOS (H3695) & RODOS/M, R3, 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 (21.3 - 21.5°C (main study) or 20.7 - 21.3°C (satellite group)) and humidity (57.8 - 58.3% (main study) or 53.5 - 54.1% (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.


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 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:

see above ("Details on inhalation exposure")

Duration of exposure:

4 h

Concentrations:

Main study:
5.05 ± 0.01 mg/L air (actual concentration)
33.33 mg/L air (nominal concentration)

Satellites:
5.05 ± 0.08 mg/L air (actual concentration)
33.33 mg/L air (nominal concentration)

No. of animals per sex per dose:

Main study: 3 per sex
Satellites: 3 per sex

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 thereafter and recorded systematically. A careful 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). Individual records were maintained for each animal. 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.
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. The animals were also observed for possible indications of respiratory irritation such as dyspnoea.
Individual body 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:
The animals were sacrificed using a commercially available euthanasia chamber (Euthanex® SmartBox® Auto CO2 System, M1-VF Touchscreen) and an automatic program for adult rats. 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.

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 tract irritation, such as hyperaemia, oedema, minimal inflammation, thickened mucous layer. Trimming of the lungs was performed in accordance with OECD GD 125, trimming of the nasal cavity was performed according to Kittel et al. (2004).
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.

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.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

5.05 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No animal died prematurely.

Clinical signs:

other: see remarks

Remarks:

slight dyspnoea observed immediately after end of exposure until 3 h after exposure in all 3 of 3 male and 3 of 3 female animals.

Body weight:

The body weight gain of the animals was in the expected range at the end of the study.

Gross pathology:

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

Other findings:

- Histopathology:
The histomorphological examination of the nose, larynx, trachea and lungs of male and female rats revealed minimal morphological changes in the lungs and nose which are considered to be test item-related in the male and female satellite animals (24 hour sacrifice) and in the female and male main study animals (15 days sacrifice).

Main study:
The morphological findings of the main study animals at the nose in level 5 showed a minimal focal degeneration of the olfactory epithelium of 1/3 female animals. All other nasal cross-sections diagnosed a normal respiratory and olfactory epithelium.
Larynx was normal and the trachea showed mild lympho-histiocytic infiltrations in 1/3 male and 1/3 female animals.
Only minimal to mild perivascular oedema was found in the lungs of 2/3 male and 2/3 female animals. The alveolar lumen was free of oedema fluid and contained no inflammatory cells.

Satellite animals:
The nasal cavity of level 1 showed a normal squamous epithelium. In the level 2 a normal respiratory epithelium partially with cilia and a normal squamous cell epithelium were observed. The respiratory epithelium was containing three normal major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells. A normal olfactory epithelium covered the turbinates of the nose in the most areas of level 3 to 5. Only a focal minimal degeneration with artefacts of the olfactory epithelium was observed in the dorsal nasal conch (concha nasalis dorsalis) in levels 3 to 5 in 2/3 male (only level 5) and in 1/3 or 2/3 female (level 3 or level 4 and 5) animals. Inflammatory cells were not noted.
Most conches showed a healthy olfactory epithelium without inflammatory or degenerative changes. The epithelium of the larynx was normal in all male and female animals. The subepithelial minimal to mild lymphocytic infiltrations in 1/3 male and 1/3 female animals in the trachea were unspecific or spontaneous reactions and thus are not test item related. Minimal to mild perivascular oedema were observed in the lungs of all male and female animals.

- Potential target organs:
- Other observations:

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

LC50 (male and female rats): 5.05 mg/L air
According to the EC Regulation 1272/2008 and subsequent regulations, no classification for acute inhalation toxicity or specific target organ toxicant (STOT)- single exposure is warranted.

Executive summary:

Rats were exposed to an aerosol of Cobalt silicate olivine & silica polymorphs at gravimetrically determined concentrations 5.05 ± 0.01 mg/L (main study) or 5.05 ± 0.08 mg/L (satellite animals, 24-hour sacrifice) Cobalt silicate olivine & silica polymorphs/L air for 4 hours by inhalation using a dynamic nose-only exposure chamber. The aerosol was generated with the aid of a rotating brush dust generator.

Under the present test conditions, 4-hour inhalation exposure to Cobalt silicate olivine & silica polymorphs at the concentration of 5.05 mg/L air (determined by gravimetric analysis) did not result in premature death in any animal.

The LC50 value (males and females combined) for rats: 5.05 mg Cobalt silicate olivine & silica polymorphs/L air/4 hours