Tall-oil fatty acids salts of condensation products of tall-oil fatty acids with diethanolamine and triethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

Reconstructed Human Epidermis Test Method of 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKIN, EPISKIN Skin Irritation Test 15min - 42 hours, Standard Operating Procedure: February 2009 Version 1.8.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test animals

Species:

other: in vitro EpiSkin test

Strain:

other: human epidermal keratinocytes

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Types of Treatment in the Main Test
- Negative control: Dulbecco's Phosphate Buffered Saline (DPBS), dose volume 10 µl/tissue sample
- Test Substance: BYK-LP N 22655, dose volume 10 µl/tissue sample
- Positive control: 5% Sodium Dodecyl Sulphate (SDS) in purified water, dose volume 10 µl/tissue sample

Duration of treatment / exposure:

15 ± 0.5 minutes with the test substance, negative or positive control at room temperature, followed by thorough rinsing of each epidermis unit with Dulbecco's phosphate buffered saline

Details on study design:

Test System
EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes and forming a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum (matrix: collagen type 1 coated with type IV collagen).

Principle of the Test – Main Test
Irritant substances are sufficiently cytotoxic to cause cell deaths in the cell layers. Therefore, cell viability of the multilayers was determined by measurement of mitochondrial dehydrogenase activity assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, coloured, formazan salt. The degree of formazan salt formation (positively correlated with the degree of cell viability) was measured photometrically (i.e. determination of the optical density of formazan extracts from tissue at 540 nm).

Depending on the percentage of tissue viability attained (compared to negative control viability) a test substance is classified as skin irritating or not skin irritating.

Pre-Tests – Checking for Interference of the Test Substance with the Assay
It was found, that the test material BYK-LP N 22655 itself interacted with MTT. Therefore, additional (killed) tissue controls were used. The test substance BYK-LP N 22655 did not show any potential for colouring water.

Main Test
Each treatment group (test substance, negative/positive controls) comprised 3 live (viable) tissue samples placed into wells of 12 well plates.

Incubation of these tissues before treatment in maintenance medium: ≥ 24 h at 37°C, 5% CO2 in air, in humidified atmosphere , in wells
each containing 2 mL fresh pre-warmed maintenance medium.
Test material administration: Spreading of thin even layer over the epidermal surface.
Termination of 15 ± 0.5 minute exposure period: Removal of residual test material or positive control substance by thorough
rinsing of each epidermis unit with Dulbecco's PBS;
removal of remaining PBS by blotting on absorbent paper.
Posttreatment incubation (all tissue samples) : 42 ± 1 h at 37°C , 5% CO2 in humidified atmosphere, in wells each containing
2 mL fresh pre-warmed maintenance medium
Then MTT incubation: 3 hours (± 5 minutes) at 37°C , 5% CO2 in humidified atmosphere, in wells each containing 2 mL of 0.3 mg/mL MTT.
Formazan extraction (all tissue samples) : Further processing of tissue samples & formazan extraction by vortexing in
acidic isopropanol, 500 µL/sample. The vortexed samples were stored at 2-8°C
protected from light for 48 - 70 hours.
Qantitative determination of optical density: At 540 nm with acidified isopropanol (0.04 N HCl final concentration,
6 x 200 µL) as blanks.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Negative Control Tissue viability as % of OD of negative controls

Value:

100

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 15 ± 0.5 minutes control exposure to DPBS, 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 48 - 70 h formazan extraction. Max. score: 100.0. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: BYK-LP N 22655 treated "true" tissue viability as % of OD of negative controls

Value:

93.7

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 15 ± 0.5 minutes exposure to BYK-LP N 22655, 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 48 - 70 h formazan extraction. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction. . (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Positive Control Tissue viability as % of OD of negative controls

Value:

11.3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: Following 15 ± 0.5 minutes exposure to 5% SDS, 42 ± 1 h incubation in fresh maintenance medium, 3 h incubation in MTT solution and 48 - 70 h formazan extraction. Reversibility: other: not applicable to the EPISKIN skin irritation assay. Remarks: Mean OD of replicate blanks has been accounted for by subtraction.. (migrated information)

Any other information on results incl. tables

 

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU