Spinels, chromium iron manganese brown

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-28 to 2012-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

During the MTT assay isopropanol / 2 N HCl 49:1 (v/v) should have been used as extractant solution. Due to inattention isopropanol without HCl was used as extractant solution. Due to Harlan CCR’s experimental experience concerning formazan salt extraction with and without acidifying of isopropanol, and due to the long extraction period of nearly three days, the risk of inadequate extraction is minimized. Therefore, the described deviation did not have a detrimental impact on the outcome of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010-07-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009-08-24

Deviations:

no

Test guideline 3

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Spinels, chromium iron manganese brown

EC Number:

271-411-2

EC Name:

Spinels, chromium iron manganese brown

Cas Number:

68555-06-6

Molecular formula:

(Mn, Fe)(Mn, Fe, Cr)2O4

IUPAC Name:

oxo[(oxochromio)oxy]chromium; oxo[(oxoferrio)oxy]iron; oxo[(oxomanganio)oxy]manganese

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Spinels, chromium iron manganese brown
- C.I. name: Pigment Brown 46
- Substance type: inorganic pigment
- Physical state:solid, black powder, odourless
- Storage condition of test material: at room temperature, keep dry in closed containers

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

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™ tissues (purchased from SkinEthic Laboratories)
- Tissue batch number: 12-EKIN-013
- Experiation date: 2012-04-02
- Delivery date: 2012-03-28
- Date of initiation of testing:2012-03-28

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 the tissues were rinsed with PBS to remove any residual test material. Then the inserts were placed in the plates with maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time with MTT: 3 hours
- Extraction of formazan: after the incubation period, MTT solution was aspirated from the wells and the wells were rinsed with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 69 hours in the refrigerator.
Per each tissue sample 2 x 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. Optical density (OD) was read with a spectrophotometer. Mean values were calculated from the 2 wells per tissue sample.
- Spectrophotometer: microplate reader (Versamax® Molecular Devices)
- Wavelength: 570 nm
- Filter: 1 nm

TEST FOR DIRECT MTT REDUCTION
To test for the ability of the test item to directly reduce MTT approx. 10 mg of the test item were added to 1 mL of MTT solution and 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 persumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues passed 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 HIV 1 and 2 antibodies, hepatitis C antibodies and absence of hepatitis B antigen HBs
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density 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 was 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 was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less than or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 10 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 20 µL of deionised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

Duration of treatment / exposure:

15 ± 1 minutes

Duration of post-treatment incubation (if applicable):

about 42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Test system

Vehicle:

water

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

92

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

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 (1.233, 1.178, and 1.129, respectively) were well within 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 21.4 % (acceptability criterion: positive control is ≤ 40 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviations between the % variabilities of the test item, positive and negative controls were below 7 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%).
Please refer to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

HISTORICAL DATA

Positive Control		Negative Control
Number of Studies	163	Number of Studies	163
Period	July 2007 – March 2012	Period	July 2007 – March 2012
Mean Viability	19.7%	Mean OD	1.012
Standard Deviation	10.0%	Standard Deviation	0.215
Range of Viabilities	0.7% - 39.7%	Range of ODs	0.590 – 1.680

Table 1: Results after treatment with spinels, chromium iron manganese brwon (Pigment Brown 46) and controls

Dose group	Treatment 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	Rel. Absor-bance [% of Negative Control]**
Negative Control	15 min	1.233	1.178	1.129	1.180	100.0
Positive Control	15 min	0.254	0.254	0.250	0.253	21.4
Test item	15 min	1.067	1.014	1.174	1.085	92.0

* Mean of two replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (meanabsorbance_testitem)/(mean absorbancenegative control)

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 EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.