Fatty acids, tall-oil, reaction products with ethanolamine, ethoxylated

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-12 to 2012-08-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 13, 2004

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 22, 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 30 May 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

61791-19-3

Cas Number:

61791-19-3

IUPAC Name:

61791-19-3

Test material form:

other: liquid

Test animals

Species:

other: in vitro test

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Reconstructed human epidermal model EpiDermTM
- Source: MatTek In Vitro Life Science Laboratories, Bratislava

The EpiSkin SM model has been validated for irritation testing in an international trial and is considered to be suitable for this study.

QUALITY CONTROL:
- EpiSkin SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed via the MTT cell viability test using the cytotoxic test compound sodium dodecyl sulphate (SDS).

KIT CONTENTS:
- Units: 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells ∅ 1 cm
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava and Biochrom, Germany)
- Medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava /Sigma, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma,Germany), 1.0 mg / mL assay medium

Test system

Type of coverage:

other: Application on the skin model surface

Preparation of test site:

other: Human skin model

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive and negative control epidermis

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: a sufficient amount to cover evenly all the epidermal surface (30 μL per EpiSkin SM unit)

Observation period:

MTT test after 42 hours post-incubation

Number of animals:

3 EpiSkin SM for test item incubation
3 EpiSkin SM for positive control
3 EpiSkin SM for negative control

Details on study design:

Corrosion test:
Performance of the corrosion Study

Pre-incubation (day [-1]):
At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.

Application:
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, one killed tissue per exposure time was treated with the test substance and NC, respectively, in order to detect direct MTT reduction. Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC and killed tissue control, KC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC).

Rinsing:
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after tart of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed.

Post-incubation:
The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.

After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
Performance of the irritation Study

Pre-incubation (day [-1]):
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.

Application (day 0):
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.

Exposure (day 0):
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.

Rinsing (day 0):
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.

Post-incubation (day 0-2):
Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

MTT test after 42 hours incubation (day 2):
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: exposure time 3 min

Remarks on result:

other: Corrosion: test substance: 97 % Viability compared to untreated control

Results 2

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: exposure time 3min

Remarks on result:

other: Corrosion: positive control: 18 % Viability compared to untreated control

Results 3

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: exposure time 1 hours

Remarks on result:

other: Corrosion: test substance: 97 % Viability compared to untreated control

Results 4

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: exposure time 1 hour

Remarks on result:

other: Corrosion: positve control: 9 % Viability compared to untreated control

Results 5

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: 42 hours after 55-65 min exposure time

Remarks on result:

other: Irritation: test substance: 95 % Viability compared to untreated control

Results 6

Irritation parameter:

other: viability

Basis:

mean

Time point:

other: 42 hours after 55-65 min exposure time

Remarks on result:

other: Irritation: positive control: 2 % Viability compared to untreated control

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that Fatty acids, tall-oil, reaction products with ethanolamine, ethoxylated does not show a skin irritation/corrosion potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.