Registration Dossier

Administrative data

Description of key information

Skin irritation according to OECD TG 439: Not irritant

Eye irritation according to OECD TG 438: Not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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
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.

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.

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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jun 2017 - 13 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TISSUE SOURCE
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Age at study initiation: young cattle

OTHER: the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
triplicate
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32C. The corneas were incubated for the minimum of 1 hour at 32 C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Unexposed

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
Undiluted 10 min exposure time

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 2

- POST-EXPOSURE INCUBATION: yes 120 +/- 10 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea using an opacitometer
- Corneal permeability: microtiter plate reader (OD490) TECAN Infinite® M200 Pro Plate Reader
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to guideline
Irritation parameter:
in vitro irritation score
Run / experiment:
Main experiment (mean of triplicate)
Value:
-0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 53 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values:

Negative control
Opacity: -2.9 - 3.0, Mean=0.10, SD=1.04, n=78
Permeability: -0,016 - 0,042, Mean=0,01, SD=0,01, n=71
In vitro Irritancy Score: -2.8 - 3.0, Mean=0.20, SD=1.14, n=53

Positive control
In vitro Irritancy Score: 34.7-78.2, Mean=56.47, SD=12.48, n=53


Interpretation of results:
other: Not classified
Remarks:
Based on CLP (1272/2008/EC).
Conclusions:
Soybean oil, epoxidized, Me ester, reaction products with propylene glycol does not need to be classified for Eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The eye irritation hazard potential of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was as measured Acoording to OECDTG437 (BCOP test). The eye damage of

"Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol" was tested through topical application for 10 minutes.  The test item was applied as it is (750 µl) directly on top of the corneas. The negative and positive were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  The test item did not induce ocular irritation through both endpoints (no corneal opacity or permeability), resulting in a mean in vitro irritancy score of -0.3 after 10 minutes of treatment.  In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation

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).

Eye irritation

The eye irritation hazard potential of "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" was as measured according to OECDTG437 (BCOP test), under GLP. The eye damage of "Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol" was tested through topical application for 10 minutes.  The test item was applied as it is (750 µl) directly on top of the corneas.The negative and positive were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  The test item did not induce ocular irritation through both endpoints (no corneal opacity or permeability), resulting in a mean in vitro irritancy score of -0.3 after 10 minutes of treatment.  In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Justification for classification or non-classification

Based on the available data, "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" does not need to be classified for skin irritation and eye irritation in accordance with EU CLP (EC 1272/2008 and its updates).