Linseed oil, ester with pentaerythritol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 24, 2017 to April 28, 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 23, 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Linseed oil, ester with pentaerythritol

EC Number:

271-985-4

EC Name:

Linseed oil, ester with pentaerythritol

Cas Number:

68648-28-2

Molecular formula:

UVCB

IUPAC Name:

2,3-dihydroxypropyl (7E,10E,13E)-hexadeca-7,10,13-trienoate; bis(3-[(7E,10E,13E)-hexadeca-7,10,13-trienoyloxy]-2-hydroxypropyl (9E)-octadec-9-enoate); 3-hydroxy-2-(hydroxymethyl)-2-[(octadec-9-enoyloxy)methyl]propyl (9E,12E,15E)-octadeca-9,12,15-trienoate; 3-hydroxy-2-{[(9E)-octadec-9-enoyloxy]methyl}-2-{[(9E,12E)-octadeca-9,12-dienoyloxy]methyl}propyl (9E,12E,15E)-octadeca-9,12,15-trienoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

The test system is a commercially available EpiDermTM-Kit (EPI-212-SIT, batch no: 25809, 2017), procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

- One plate (3 tissues) was used as negative control in which each tissue was treated with 30 μL DPBS buffer.
- One plate was used as positive control in which each tissue was treated with 30 μL 5% SDS-solution.
- One plate was used for treatment with the test substance in which 30 μL test substance were applied.
A nylon meshes were added to all plates in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2. 1 h after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 23 h and 30 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Duration of post-treatment incubation (if applicable):

Post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 h for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

Number of replicates:

From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls

Value:

ca. 96.9 - ca. 103.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative absorbance value was reduced to 99.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Any other information on results incl. tables

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability (in the form of formazan production) were calculated in comparison to the negative control:

 

Designation	linseed oil, ester with pentaerythritol	Positive Control
% Tissue viability (tissue 1)	99.5%	4.1%
% Tissue viability (tissue 2)	103.5%	3.6%
% Tissue viability (tissue 3)	96.9%	3.9%
% Tissue viability (mean)	99.9%	3.8%
± SD of mean tissue viability (%)	3.3%	0.2%

 

The relative absorbance value was reduced to 99.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test substance is considered as non-irritant to skin.

 

Validity and Acceptability

Validity criteria and results are stated in the following table:

 

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.7
% tissue viability of positive control SDS	≤ 20% of negative control	3.8%
SD of mean viability of the tissue replicates (%)	≤ 18%	1.0% (negative control) 0.2% (positive control) 3.3% (test item)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered as non-irritating to skin in the RhE test method.

Executive summary:

A study was conducted to determine skin irritation potential of the test substance in the Reconstructed Human Epidermis (RhE) according to EU Method B.46 and OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control, 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. Relative absorbance was reduced to 3.8% (required: ≤20%). Variation within the tissue replicates was acceptable (required: ≤18%). After the treatment with the test item, the relative absorbance value was reduced to 99.9%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance was considered as non-irritating to skin in the RhE test method (Andres, 2017).