Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul 2017 - 31 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Test item 207811/B
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 17 May 2018 (expiry date)
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Not indicated

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: not applicable
Justification for test system used:
According to testing guideline OECD Guideline 439
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): 17-EKIN-030
- Date of initiation of testing: 25 Jul 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure/ post-treatment incubation: 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Wavelength: 570 nm

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 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 incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL
Duration of treatment / exposure:
15 ± 0.5 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:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
5.9
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: color change was observed by adding MTT-medium and it was concluded that the test item did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
- Colour interference with MTT: No colour changes observed

ACCEPTANCE OF RESULTS:
-The positive control had a mean cell viability of 5.9% after 15 ± 0.5 minutes exposure ( % viability should be ≤18)
- The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
-The relative mean tissue viability of the test item compared to the negative control tissues was 94% and the standard deviation value of the viability was 7.9%.
-The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.9%, indicating that the test system functioned properly.
-The non-specific reduction of MTT by the test item was 4.69% of the negative control tissues.

Any other information on results incl. tables

Mean absorption and tissue viability in the in vitro Skin irritation test with Soybean oil, epoxidized, Me ester, reaction products

Test Group Mean Absorbance of 3 Tissues  Mean Absorbance per Tissues Standard deviation (%) Mean tissue viability (% of negative control)
Negative Control 0.684 0.721 / 0.632 / 0.699 6.8 100.0
Positive Control 0.040 0.057 / 0.031 / 0.033 2.1

5.9

Test Item*

0.643

0.675 / 0.581 / 0.673

7.9

94

*The test item values are corrected for the non-specific MTT reaction..

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Remarks:
Based on CLP (1272/2008/EC).
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item was determined as 94%. Since this value is above the threshold for irritancy of ≤ 50%, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol) does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The possible skin irritation potential of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours post-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. The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Soybean oil, epoxidized, Me ester, reaction products was 4.69% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

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 mean cell viability of 5.9 % after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 94 %. This value is above the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Soybean oil, epoxidized, Me ester, reaction products with propylene glycol is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).