Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 14 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2013)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

EC Number:

500-288-2

EC Name:

Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Cas Number:

103213-20-3

Molecular formula:

Not applicable as UVCB

IUPAC Name:

Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: pale yellow liquid
- Expiration date of the lot/batch: 14 Feb 2017
- Storage condition of test material: room temperature in the dark

Test animals

Species:

human

Strain:

other: human-derived epidermal keratinocytes (EpiSkin)

Details on test animals or test system and environmental conditions:

TEST METHOD
Reconstructed Human Epidermis tissues are provided as kits (SkinEthic). The EpiSkin model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test item treated tissues relative to the negative control.

ADAPTATION TO CELL CULTURE CONDITIONS
On arrival, the human-derived epidermal keratinocyte tissues were transferred into pre-labeled 12-well plates (one for each culture condition) containing 2 mL of pre-warmed maintenance medium. The tissues were incubated overnight at 37 °C and 5% CO2 in air.

INCUBATION CONDITIONS (INCUBATOR)
Temperature (°C): 37
CO2 gas concentration (%): 5

Test system

Type of coverage:

other: in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with DPBS served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL:
Amount(s) applied (volume or weight with unit): 10 μL

NEGATIVE CONTROL SUBSTANCE:
Amount(s) applied (volume or weight with unit): 10 μL DPBS

POSITIVE CONTROL SUBSTANCE:
Positive control substance: 10 μL SDS, 5% (w/v)

Duration of treatment / exposure:

15 min

Observation period:

Post-treatment incubation period: 42 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Triplicate tissues were treated with the test item and the concurrent positive and negative controls for an exposure period of 15 min. At the end of the exposure period, each tissue was removed from the well and rinsed with DPBS containing calcium and magnesium. The rinsed tissues were transferred to other wells of the same 12-well plate containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C and 5% CO2 in air for 42 hours.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS:
For determining alterations in cell viability, MTT reduction assays were performed immediately after the 42 hour incubation period. Therefore, tissues were transferred to new wells of the same 12-well plate containing 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium. The tissues were incubated for 3 h at 37 °C and 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The epidermis was carefully separated from the collagen matrix and both parts (epidermis and collagen) placed into labeled 1.5 mL tubes containing 500 µL of acidified isopropanol. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a 96-well plate. The optical density was measured at 562 nm wave length in a microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (DPBS). Time point: 15 min . Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

15.3

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (SDS). Time point: 15 min . Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

108.1

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 15 min . Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean viability of the test item treated reconstructed human-derived keratinocyte tissues after a 15 min exposure period was 108.1% compared to the negative control. Therefore, the test item is considered to be a non-irritant.

Any other information on results incl. tables

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.767	0.767	0.05	100	100*	6.2
	0.719			93.7
	0.814			106.1
Positive Control Item	0.145	0.118	0.06	18.9	15.3	7.7
	0.158			20.6
	0.050			6.5
Test Item	0.813	0.829	0.01	106.0	108.1	1.9
	0.842			109.8
	0.833			108.6

SD: Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified