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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May - 30 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed

In vitro test system

Test system:
human skin model
Source species:
other: human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): 16-EKIN-021
- Production date/ Shipping date: no data
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

PRE-TEST PROCEDURE
- Pre-incubation: On day of receipt the tissues were transferred into a 12-wells plate filled with maintenance medium. A different 12-wells plate was used for the test item and each control item. The tissue were incubated at 37°C, 5% CO2 in air overnight.
- Test item colour interference: To assess colour interference, 10 μL of test substance was added to 90 μL sterile water. The mixture was mixed for approximately 15 minutes. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to directly reduce MTT, 10 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in assay medium). The mixture was incubated in the dark for 3 hours at 37°C, 5% CO2 in air. Untreated MTT solution was used as a control. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature for 15 minutes, post-treatment incubation: 37 °C for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with DPBS with Ca++ and Mg++ to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in assay medium
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without reference filter)

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is > 50% of the mean viability of the negative controls.

ACCEPTANCE CRITERIA
- Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
- Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
- Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (100%)
Value:
5.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.3%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD562of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.265

1.293

0.045

97.8

100*

3.5

1.345

104.0

1.269

98.1

Positive Control Item

0.076

0.063

0.011

5.9

4.9

0.9

0.058

4.5

0.055

4.3

Test Item

0.062

0.077

0.017

4.8

5.9

1.3

0.096

7.4

0.072

5.6


OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

⊕ = The control groups were shared with Envigo - Shardlow study number TW67BS, MS48XJ, FC59GT, YB24MH, RS46VL, JD61DM and NL15CD.

Applicant's summary and conclusion

Interpretation of results:
other: Skin irritant, Category 2
Remarks:
according to EU CLP criteria (1272/2008) and its amendments.
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item treated tissues was determined to be 5.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, the test substance is considered to be irritating to the skin, Category 2 according to GHS.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro, using a reconstructed human epidermis model, according to OECD TG 439 guideline and GLP principles. Skin tissues were treated by topical application of 10 μL undiluted test substance for 15 minutes. After a 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a relative mean tissue viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% for test item, positive control and negative control treated tissues, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item treated tissues is determined to be 5.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, the test substance is considered to be irritating to the skin.