Chromium iron oxide

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

draft Version

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test guideline 3

Qualifier:

according to guideline

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. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Chromium iron oxide

EC Number:

235-790-8

EC Name:

Chromium iron oxide

Cas Number:

12737-27-8

Molecular formula:

Fe(x)Cr(2-x)O3 0,65≤x≤1,75

IUPAC Name:

chromium(3+) iron(3+) trioxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium iron oxide
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

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

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.