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Administrative data

Description of key information

Acute oral lethal dose (LD 50): > 2000 mg/kg bw.

Acute toxicity, inhalation: LC50  >5.14 mg/L air. MMAD: 3.455 µm (GSD: 2.87)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Minor deviations without an effect on the results: - According to the guideline, normally females are used for this test. In this study 3 males and 3 females were used for testing the acute toxicity. -According to the guideline, healthy young adults between 8 and 12 weeks of age should be used. The age of the animals was not stated in the study. It was only stated that the animals were young and healthy. - According to the guideline, the relative humidity in the animal room should be 30 - 70 %. The relative humidity in the study was slightly higher (55+/- 25 %). -According to the guideline, three animals are tested and depending on the outcome the next three animals are tested. In this study all animals were tested at the same time. -According to the guideline, the animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study. In the report, it was only stated that on the first day regular observations were performed, in the following days the animals were observed at least once until the 14 day. - According to the guideline, details on food and water quality must be included in the study report, which was missing in the report.
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
GLP compliance:
not specified
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: provided by an authorised supplier
- Weight at study initiation: Males: 135 g, 135 g and 133 g; Females: 113 g, 116g and 120 g
- Fasting period before study: The animals fasten during the previous night. After 3 hours of administration, feeding was restored.
- Housing: Housed in groups of three; Makrolon cages (48 X 27 X 20 cm), brand Tecniplast, with wooden bed
- Diet (ad libitum): Diet for experimental rats, provided by an authorised provider
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C (+/- 2°C)
- Relative humidity: 55 % (+/- 25 %)
- Air: Renewed 15 times per hour and prefiltered at 5 µm
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
2000 mg of sample was diluted in 20 mL of distilled apirogenic water.

MAXIMUM DOSE VOLUME APPLIED: 2 mL of the diluted sample per 100 g of alive weight, equivalent to the 2000 mg/kg of alive weight were administrated (sample was vigorously stirred before administration).

CLASS METHOD
- Rationale for the selection of the starting dose: The available information suggested that the sample is little toxic and the limit test was performed.
No further information on the oral exposure was stated.
Doses:
2000 mg/kg
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: The individual weight control was performed at the beginning of the study, before administration, after 7 days of administration and at the end of the study or in case of having had a death, right after death. A general clinical exam was performed every working day in which modification were observed. The first day regualr observations were performed, in the following days the animals were observed at least once until the 14 days. the observed signs of toxicity were observed, at the moment of appearance and also the time for recovery.
- Necropsy of survivors performed: Yes
After the study, the surviving animals were sacrificed by means of a humanitarian method (cervical dislocation). All the animals in the study were subjected to macroscopic necropsy. Macroscopic pathology changes for each animal were noted.
- Other examinations performed: During clinical examination modification were observed and noted in among others: Skin, hair, eyes, mucosity, respiratory track, circulatory system, central and autonomous nervous system, motion activity and behavioural lines of action. With special care for: shaking, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Statistics:
No data
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All the animals administrated with the sample survived normally until the 14 days of observation.
Clinical signs:
Neither abnormal effects nor any toxicity signs that could be attributed to the sample were observed.
Body weight:
The registered weight variations can be regarded as normal for these animals (males and females).
Gross pathology:
Necropsies were performed after 14 days of administration of the sample in the test animals, without observation of relevant macroscopic changes in any of them.
Interpretation of results:
GHS criteria not met
Conclusions:
The sample submitted as "VERDE D7" (B2240) (Hematite chromium green black) , studied under the described method, presents an acute toxicity -LD50 by oral route in rat: above 2000 mg/kg.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not classified as acute toxic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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:
2010-07-01 to 2010-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: 53 days; Females: 67 days
- Weight at study initiation: Males: 217 - 221 g; Females: 213 - 228 g
- Fasting period before study: Feeding was discontinued approx. 16 hours before exposure.
- Housing: During the 14-day observation period the animals are kept by sex in groups of 3 animals in MAKROLON cages (type III plus). Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- 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 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
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.455 µm
Geometric standard deviation (GSD):
2.87
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).
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 (22.5°C ± 0.2°C) and humidity (60.5% ± 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: 3.455 µm (GSD: 2.87)
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:
5.14 ± 0.06 mg Colorante Verde/L air (actual concentration)
5.13 mg/L air (nominal concentration)
No. of animals per sex per dose:
3 male rats / 3 female rats
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.
- 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.

Statistics:
not applicable
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.14 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Standard deviation: 0.06 mg/L air
Mortality:
No mortality occurred.
Clinical signs:
other:
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:
GHS criteria not met
Conclusions:
Under the conditions of this study, the 4-hour inhalation LC50 of Colorante Verde is >5.14 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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

One acute oral toxicity study according to EU Method B.1 tris. (Acute Oral Toxicity - Acute Toxic Class Method) has been performed. This study indicates that oral LD50 is >2000 mg/Kg bw in rats. No deaths occured.

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 Colorante Verde, indicating a LC50 > 5.14 mg/L.

Slight tremor (0 min - 3 h) was seen in all males and in all females (0 - 60 min) . Slight dyspnoea (0 min - 3 h) was observed in all males and females (0 min - 3 h). No mortality occured.

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 toxicity oral:

The sample submitted as "VERDE D7" (B2240) (Hematite chromium green black) , studied under the described method, presents an acute toxicity -LD50 by oral route in rat: above 2000 mg/kg. According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not classified as acute toxic.

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

The classification criteria acc. to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – single exposure, oral 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, oral for a Category 1 classification of 300 mg/kg bw and at the guidance value, oral for a Category 2 classification of 2000 mg/kg bw. No classification required.

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 Verde is >5.14 mg/L air. According to the EC Regulation 1272/2008 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.