Registration Dossier

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 guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Pigment
- Physical state: Solid; black powder, odourless


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.