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Description of key information

Acute inhalation toxicity: LC50 (rats; 4 hours) > 5.07 mg/L air (actual concentration) (OECD 436 (2009); GLP compliant)

Key value for chemical safety assessment

Acute toxicity: via inhalation route

Link to relevant study records
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-17 to 2012-12-10
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
according to guideline
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
adopted 2009-09-07
GLP compliance:
yes (incl. QA statement)
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
- 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
- Weight at study initiation: males: 247 - 260 g; females: 232 - 243 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. During the 14-day observation period, the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Diet: commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days

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.

- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
clean air
Details on inhalation exposure:
- 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 40 L) which holds the animals in pyrex tubes at the edge of the chamber in a radial position.

- 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) (air was taken from the surrounding atmosphere of the laboratory room and filtered using an in-line disposable gas-filter).
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.
The exhaust air was drawn through gas wash-bottles.

- Method of particle size determination: an analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor according to MAY (MAY, K. R. Aerosol impaction jets, J.Aerosol Sci. 6, 403 (1975), RESEARCH ENGINEERS Ltd., London N1 5RD, UK).
The dust from the exposure chamber was drawn through the cascade impactor for 5 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 32 µm particle size range and the filter (particle size range < 0.5 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 Mastersizer 2000 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, pressure in air chamber, oxygen content, 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°C ± 0.1°C (main study) or 21.0 ± 0.1 (satellite group)) and humidity 59.0% ± 0.3% (main study) or 63.3% ± 0.1% (satellite group)) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

The whole exposure system was mounted in an inhalation facility to protect the laboratory staff from possible hazards.

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).

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.

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.

- 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).
Individual chamber concentration samples did not deviate from the mean chamber concentration by more than 20%
- Samples taken from breathing zone: yes

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
Main study: 2.401 µm (GSD: 2.93)
Satellite group: 2.379 µm (GSD: 2.81)
Analytical verification of test atmosphere concentrations:
see above ("Details on inhalation exposure")
Duration of exposure:
4 h
Main study (limit test):
- actual concentration: 5.07 ± 0.03 mg/L air
- nominal concentration: 8.3 mg/L air
Satellite group:
- actual concentration: 5.06 ± 0.02 mg/L air
- nominal concentration: 8.3 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:
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 made and recorded systematically; individual records were maintained for each animal. Careful clinical examinations were made at least twice daily until all symptoms subsided, thereafter each working day. 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 human 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, rhinitis etc. The measurement of rectal temperatures might provide supportive evidence of reflex bradypnea or hypo-/hyperthermia related to treatment or confinement.
Individual weights of animals were determined once during the acclimatisation period, before and after the exposure on test day 1, on test days 3, 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 (3 + 3 males and 3+3 females) 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.
The following organs of all animals were fixed in 10% (nose, i.e. head without brain, eyes and lower jaw) or 7% (larynx, trachea, lungs) buffered formalin for possible histopathological examination. No further organs were preserved.
Since no animal died prematurely, the calculation of an LC50 was not required.
Dose descriptor:
Effect level:
> 5.07 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
No animal died prematurely.
Clinical signs:
other: Under the present test conditions, a 4-hour inhalation exposure to spinels, chromium iron manganese brown (Pigment brown 46) at a concentration of 5.07 mg/L air revealed slight ataxia and slight dyspnoea (reduced frequency of respiration with increased vo
Body weight:
All animals of the main study gained the expected body weight.
Gross pathology:
Marbled, dark discoloured and/or oedematous lungs were observed in all 3 of 3 male and 3 of 3 female animals of the main study as well as in all 3 of 3 male and 3 of 3 female satellite animals.
Interpretation of results:
not classified
Migrated information Criteria used for interpretation of results: EU
LC50 (rats; 4 hours) > 5.07 mg/L air (actual concentration)
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as acute toxic via the inhalation route.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the inhalation route.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The study fulfils the requirements for acute inhalation toxicity under REACH (Regulation (EC) 1907/2006).

Additional information

Acute inhalation toxicity

One reliable study described in Haferkorn (2013; OECD 436 (2009); GLP compliant) is considered to be reliable without restrictions and is used as key study for this endpoint. The LC50 was determined to be greater than 5.07 mg/L air (actual concentration).

Justification for selection of acute toxicity – inhalation endpoint
One reliable study described in Haferkorn (2013; OECD 436 (2009); GLP compliant) is considered to be reliable without restrictions and is used as key study for this endpoint. The LC50 was determined to be greater than 5.07 mg/L air (actual concentration).

Justification for classification or non-classification

Acute inhalation toxicity

The reference Haferkorn (2013) is considered as the key study for acute inhalation toxicity of spinels, chromium iron manganese brown and will be used for classification. Male and female rats were exposed to 5.07 mg/L air (main study, limit test, observation period 14 days) or 5.06 mg/L air (satellite group; observation period 24 hours) for 4 hours. No mortality occurred. The LC50 is > 5.07 mg/L air.

The classification criteria according to regulation (EC) 1272/2008 as acutely toxic are not met since the ATE is above 5.0 mg/L, hence no classification required.

Specific target organ toxicant (STOT) - single exposure: inhalation

The classification criteria according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – single exposure, inhalation (dust/mist/fume) are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value, inhalation (dust/mist/fume) for a Category 1 classification (C≤ 1 mg/L/4h). Based on the outcome of the limit test on acute inhalation toxicity (0 animals of 6 animals died at a concentration of 5.07 mg/L/4h and no systemic or local effects were observed) it can safely be assumed that no classification for Category 2 (5.0 ≥C > 1.0 mg/L/4h) is required.

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