Reaction mass of methyl (9E,12E,15Z)-9,12,15-octadecatrienoate and methyl (9E,12Z,15E)-9,12,15-octadecatrienoate and methyl (9Z)-9-octadecenoate and methyl (9Z,12E,15E)-9,12,15-octadecatrienoate and methyl (9Z,12Z)-9,12-octadecadienoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 75%) which does not affect the reliability of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 75%) which does not affect the reliability of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected March 2013; signature: May 2013

Test material

Test material information

Constituent 1

Reference substance name:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Molecular formula:

not applicable (reaction mass of constitutional isomers)

IUPAC Name:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: In refrigerator (2-8°C protected from light, container flushed with nitrogen
- Other: Colourless to pale yellow

Test animals

Species:

other: EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N).

Strain:

not specified

Details on test animals or test system and environmental conditions:

EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N).. The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

All incubations, with the exception of the test substance incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 75 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure.

Observation period:

Observations are made 3-minutes and 1-hour post-test item application

Number of animals:

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control.

Details on study design:

TEST SITE
- Area of exposure: 50 μl of the undiluted test substance was added on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Two tissues were washed after 3 minutes and two tissues were washed after 1 hour. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance.

SCORING SYSTEM: Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Tissue viability

Basis:

mean

Time point:

other: 3 minute exposure to test material

Score:

91

Max. score:

100

Reversibility:

no data

Remarks on result:

other: Score in terms of percentage of control

Results 2

Irritation parameter:

other: Tissue viability

Basis:

mean

Time point:

other: 1 hour exposure to test material

Score:

94

Max. score:

100

Reversibility:

no data

Remarks on result:

other: Scored in terms as percentage of control

Irritant / corrosive response data:

Table 1 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 94% respectively.

The relative mean tissue viability of the positive control was after the 3-minute and 1-hour treatments compared to the negative control tissues was 14% and 7% respectively.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test substance

	3-minute application (OD540)					1-hour application (OD540)
OD540measurement	A	B	Mean	SD	Mean tissue viability (% of control)	A	B	Mean	SD	Mean tissue viability (% of control)
Negative control	2.160	2.109	2.134	±0.036	100	2.033	2.098	2.065	±0.046	100
Test Material	2.009	1.873	1.941	±0.096	91	1.940	1.957	1.949	±0.012	94
Positive control	0.290	0.290	0.290	±0.000	14	0.129	0.166	0.148	±0.026	7

Values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.

Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. Fifty μl of the undiluted test substance was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 14% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 22% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 94%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. Under the conditions of this study the test material is not corrosive in the in vitro skin corrosion test.