Registration Dossier

Administrative data

Description of key information

Acute toxicity, inhalation:
LC50 >5.01 mg/L air. MMAD: 3.656 µm (GSD: 3.08)

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-07 to 2010-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
2009-09-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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: 49 days; females: 63 days
- Weight at study initiation: males: 222- 228 g; females: 206 - 222 g
- Fasting period before study: Feeding was discontinued approx. 16 hours before exposure.
- 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 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany).
- Water (ad libitum): Drinking water
- Acclimation period: At least 5 days; The animals 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.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 3 °C (maximum range)
- Relative humidity: 55 % +/- 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
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 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).

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.

- Method of particle size determination: An analysis of the particulate 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). Delta of slides’ weight were determined.
The correct functioning of the dynamic separation of particles was controlled microscopically during spot-checks.
The 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 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.

- Treatment of exhaust air: The exhaust air was drawn through gas wash-bottles.

- Temperature, humidity, oxygen, carbon dioxide, air flow:: 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 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 (19.7°C ± 0.3°C) and humidity (52.0% ± 0.3%) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

TEST ATMOSPHERE - The inhalation chamber was equilibrated for at least 15 minutes (t95 approximately 8 minutes).
- 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). The correct loading of the filter was checked by the airflow via the rotameter and by a positive weight increase of the filter after the sampling period of 1 minute.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- MMAD: 3.656 µm (GSD: 3.08)
No further information on the inhalation exposure was stated.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
see above ("details on inhalation exposure")
Duration of exposure:
4 h
Remarks on duration:
Exposition started by locating the animals’ noses into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes.
Concentrations:
5.01±0.10 mg Colorante Negro/L air (actual concentration)
5.0 mg/l air (nominal 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: 14 days
- Frequency of observations and weighing: During and following exposure, observations were made. 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). 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.
- Necropsy of survivors performed: Yes
Necropsy of all animals was carried out and all gross pathological changes were recorded.
No microscopic examination was carried out as no pathological findings were noted at necropsy.
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: 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.
Changes in weight were calculated and recorded when survival exceeded one day.
No furtehr information on the study design was stated.
Statistics:
Since no mortality occurred, the calculation of an LC50 was not required.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.01 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Standard deviation: 0.10 mg/L air; The LC50 cut-off value is 'unclassified'.
Mortality:
No mortality occurred.
Clinical signs:
A 4-hour inhalation exposure to Colorante Negro at a concentration of 5.01 mg/L air revealed slight ataxia (0 - 60 minutes), slight to moderate tremor (0 minutes - 3 hours) and slight dyspnoea (0 minutes - 3 hours) in all 3 male and 3 female rats.
Body weight:
All animals gained the expected body weight throughout the study period.
Gross pathology:
No pathological findings were made.
Other findings:
No data
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the 4-hour inhalation LC50 of Colorante Negro is >5.01 mg/L air, and hence, the LC50 cut-off value 'unclassified'.
According to the EC Regulation 1272/2008 and subsequent regulations, the test material is not classified for acute inhalation toxicity.
According to the EC-Commission directive 67/548/EEC and subsequent regulations, the test material is not classified for acute inhalation toxicity.



Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

According to section 1 of REACH Annex XI, testing for acute oral toxicity is scientifically not relevant for this substance since this pigment can be considered as chemically inert due to the characteristics of the synthetic process (calcination at high temperatures, approximately1 000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. This assumption is supported by available bioaccessability data (Pardo Martinez, 2010) that indicate very low solubility of the contained elements under physiological conditions. With a 100 mg loading at pH 1.5 (gastric fluid conditions) and an assumed gastric phase of digestion of about 2 hours, 7.5±0.4 μg/L of Fe (0.02 wt-% based on the total Fe content) and <1 μg/L of Cr (below the limit of quantification of 1 μg/L )) were dissolved. Based on 100 mg of pigment, this results in 0.008 wt-% of dissollved Fe and less than 0.001 wt-% of Cr.

For acute inhalation toxicity there is one animal study which has been performed according to OECD TG 436 and which shows no signs of acute toxicity after inhalation exposure to chromium iron oxide, indicating a LC50 > 5.01 mg/L.

The 4-hour inhalation exposure to Colorante Negro at a concentration of 5.01 mg/L air revealed slight ataxia (0 - 60 minutes), slight to moderate tremor (0 minutes - 3 hours) and slight dyspnoea (0 minutes - 3 hours) in all 3 male and 3 female rats. No mortality occurred.

There are no reliable reports whatsoever on acute dermal toxicity in the public domain. However, the conduct of an acute dermal toxicity study is unjustified as inhalation of the substance is considered as major route of exposure and physicochemical properties of the substance do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006). This is underlined by the very low solubility and bioavailability (Pardo Martinez, 2010) of the elements out of this pigment, which can be considered as inert.

Justification for classification or non-classification

Acute inhalation toxicity:

The reference Haferkorn, J. is considered as key study for the endpoint acute inhalation toxicity and will be used for classification.

Under the conditions of this study, the 4-hour inhalation LC50 of Colorante Negro is >5.01 mg/L air. According to the EC Regulation 1272/2008 and subsequent regulations, the test material is not classified for acute inhalation toxicity. According to the EC-Commission directive 67/548/EEC and subsequent regulations, the test material is not classified for acute inhalation toxicity.

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

The classification criteria acc. 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 of 1.0 mg/L/4h and at the guidance value, inhalation dust/mist/fume for a Category 2 classification of 5.0 mg/L/4h. Therefore, no classification is required.

Finally, any category 3 classification should primarily be based on human data. However, such classification is also not warranted, since observations on respiratory irritation in test animals (rats) were not observed.