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Description of key information

Chromium iron oxide has been tested in one in vitro skin irritation study (Commision regulation (EC) No. 440/2008 B.46) and one eye irritation study. All tests show a negative response, thus chromium iron oxide does not require classification either as skin or as eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-30 to 2010-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
draft Version
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4.
Version / remarks:
2009-12-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from moisture
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans (adult donors)
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ (source: Skinethic Laboratories, 06000 Nice, France)
- Tissue lot number: 10-EKIN-011
- Delivery date: 2010-03-30
- Date of initiation of testing: 2010-03-30

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed from the plate and rinsed with PBS to remove any residual test material. The inserts were placed in the plates with 2 mL
maintenance medium. The tissues were incubated for 42 ± 1 hour. Following incubation, the tissue viablity was measured.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL per well
- Incubation time with MTT: nearly 3 hours
- Extraction of Formazan: after a nearly 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Then, the tissue samples were immersed into extractant solution (0.5 mL extractant solution (isopropanol)). The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 2 hours 45 minutes while shaking (~120 rpm) at room temperature.
Per each tissue sample 2 × 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. Optical density was determined with a spectrophotometer. Mean values were calculated from the 2 wells per tissue sample.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter: no reference filter was used

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. To test for this ability approx. 15 mg of the test item were added to 1 mL of MTT solution. The mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: absence of bacteria, fungus, and mycoplasma as well as absence of HIV1- virus, Hepatitis B antigen HBs, and Hepatitis C antibodies
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The mean optical density (OD) of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (OD test item/ OD negative control) x 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model: for the current test, an irritation potential of a test item according to EU classification (Regulation 1272/2008/EC; Category 2) is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 15 mg of test item (approx. 39.47 mg/cm²) wetted with the vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 15 µL deionised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL deionised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL of a 5% sodium lauryl sulfate (SLS) solution
Duration of treatment / exposure:
15 ± 1 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hour
Number of replicates:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Irritation / corrosion parameter:
% tissue viability
Value:
100.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control the absorbance values (1.109, 0.993, and 1.034 (mean: 1.045)) were well above the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 27.4 % (acceptability criterion: positive control is ≤ 40 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviations between the % variabilities of the test item, the positive and negative controls were below 10 % (threshold of the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation": ≤ 18%)
Please refer to the field "Any other information on results incl. tables" below.

Results after treatment with Colorante negro

 

Dose group

Treat-ment Interval

Absor-bance 570 nm
Tissue 1

Absor-bance 570 nm
Tissue 2

Absor-bance 570 nm
Tissue 3

Mean Absor-bance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Standard Deviation in [%]

Rel. Absorbance

[% of Negative Control]

Nega-tive Con-trol

15 min

1.109

0.993

1.034

1.045

106.1
95.0
98.9

5.6

100.0

Posi-tive Con-trol

15 min

0.326

0.212

0.322

0.287

31.2
20.3
30.8

6.2

27.4

Test Item

15 min

1.133

1.070

0.948

1.050

108.4
102.4
90.7

9.0

100.5

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100 * (absorbance test item))/(absorbance negative control)

Historical data:

Positive Control

Negative Control

Number of Studies

73

Number of Studies

73

Period

July 2007 - March 2010

Period

July 2007 - March 2010

Mean Viability

16.5 %

Mean OD

1.081

Standard Deviation

11.0%

Standard Deviation

0.262

Range of Viabilities

3% - 36%

Range of ODs

0.7 – 1.6*

* The upper OD value is outside of the range of 0.6 - 1.5 recommended by the OECD guideline. Nevertheless since the OD value is only slightly above the required range, the historical data can still be considered as valid.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-26 to 2010-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, adopted 2002-04-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 5 - 6 months
- Weight at study initiation: Animal No. 1: 2.3 kg, Animal No. 2: 2.4 kg, Animal No. 3: 2.5 kg
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8 hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm X 425 mm X 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum): Commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): tap water
- Acclimation period: At least 20 adaption days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C (maximum range)
- Relative humidity: 30 % - 70 % (maximum range; aim was 50 % - 60 %)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of the animals. The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
1 hour after administration the eyes were rinsed
Observation period (in vivo):
1, 24, 48 and 72 hours after administration
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were rinsed with portions of 20 mL 0.9 % aqueous NaCl solution, each.
- Time after start of exposure: 1 hour after administration

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: The eyes were examined ophtalmoscopically with a slit lamp prior to the administration and 1, 24, 48, and 72 hours after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OTHER OBSERVATIONS: Any further lesions not covered by the scoring system were recorded. Body weight of all animals was measured at the beginning of the study and at the end of the study. Behaviour and food consumption were monitored.
No further information on the study design was stated.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24,48, and 72 hours
Score:
0
Max. score:
0
Irritant / corrosive response data:
Under the present test conditions, a single instillation of 100 mg Colorante Negro per animal into the conjunctival sac of the right eye of three rabbits caused the following changes:
Conjunctival redness (grade 1: some blood vessels hyperaemic (injected)) and secretion were observed in all animals 1 hour after instillation. These effects were reversible after 24 hours.
The cornea and the irises were not affected by the instillation of the test item and the fluorescein test performed 24 hours after instillation did not reveal any changes.
Other effects:
No systemic intolerance reactions were observed in all animals.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions Colorante Negro was non-irritating to eyes, hence, no labelling is required.
Also, according to the EC-Regulation 1272/2008 and subsequent regulations, the test item is non-irritatng to the eyes.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

One study has been performed on skin irritation (Heppenheimer, 2010, Commision regulation (EC) No. 440/2008 B.46) and one study was performed on eye irritating properties (Leuschner, 2010, OECD guideline 405).

After treatment with the test item Colorante negro the relative absorbance values for skin irritating properties did not decrease (100.5 %). Therefore, the test item is not considered to possess an irritant potential.

Under the present test conditions, a single instillation of 100 mg Colorante Negro per animal into the conjunctival sac of the right eye of three rabbits caused the following changes: Conjunctival redness (grade 1: some blood vessels hyperaemic (injected)) and secretion were observed in all animals 1 hour after instillation. These effects were reversible after 24 hours. The cornea and the irises were not affected by the instillation of the test item and the fluorescein test performed 24 hours after instillation did not reveal any changes.

Justification for classification or non-classification

Skin irritation in vitro:

relative absorbance values for skin irritating properties did not decrease, mean, after 15 min incubation: score=100.5%

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item chromium iron oxide is non irritant to skin and therefore, the test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008. Also, the test item should not be classified and labeled as skin irritant according to Directive 67/548/EEC.

Eye irritation in vivo:

conjunctivae score, mean animal #1, 24 -72h: score=0            

conjunctivae score, mean animal #2, 24 -72h: score=0          

conjunctivae score, mean animal #3, 24 -72h: score=0          

cornea score, mean animal #1, 24 -72h: score=0

cornea score, mean animal #2, 24 -72h: score=0

cornea score, mean animal #3, 24 -72h: score=0

iris score, mean animal #1, 24 -72h: score=0

iris score, mean animal #2, 24 -72h: score=0

iris score, mean animal #3, 24 -72h: score=0

chemosis score, mean animal #1, 24 -72h; score=0

chemosis score, mean animal #2, 24 -72h; score=0

chemosis score, mean animal #3, 24 -72h; score=0

According to the EC-Commission directive 67/548/EEC and its subsequent amendments on approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions chromium iron oxide was non-irritating to the eyes, hence no labelling is required. Also, according to the EC Regulation 1272/2008 and subsequent regulation, the test item is non- irritating.

Respiratory irritation

The classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) for further information.