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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

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

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)

Species:

other: in vitro EpiSkin test

Strain:

other: human epidermal keratinocytes

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.

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)

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)

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)

 

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation

Remarks:

other: in vitro

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 an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437 of July 2013: Bovine Corneal Opacity and Permeability test method for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro test

Strain:

other: bovine eyes

Vehicle:

physiological saline

Amount / concentration applied:

750 µl of test substance, positive control solution (ethanol) or negative control solution (physiol. saline) was introduced into the anterior part of each cornea holder. Following application, turning of the holder into horizontal position and its slight rotation ensured uniform distribution of the test substance over the surface of the cornea.

Duration of treatment / exposure:

10 min (± 30 sec.).
Each cornea holder was incubated in a horizontal position at 32°C ± 1°C in a waterbath. Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (EMEM* with phenol red) was clear and there was no discolouration.

* EMEM, Eagles Minimal Essential Medium

Details on study design:

Eyes were maintained and transported to the laboratory and isolated corneas stored in sufficient HBSS* containing 1% penicillin-streptomycin solution (10’000 IU/ml and 10 mg/ml, respectively) until being used. The eyes were used within 4 hours of slaughter. Each treatment group (test substance, negative/positive controls) comprised 3 corneas from bovine eyes. Each cornea surrounded by ca. 2 to 3 mm of sclera was mounted to a holder. Adequate exposure of the epithelial surface of the cornea to the test substance was provided. Opacity of the cornea was determined by measurement of light transmission through the centre of each mounted cornea using a calibrated opacitometer. Permeability of the cornea was determined by spectrophotometric measurement at 490 nm (OD490) of medium from the posterior compartment of the cornea holder after addition of 1 ml of sodium fluorescein solution (4 mg/ml) to the anterior compartment, thus determining the degree of penetration of sodium fluorescein through all corneal cell layers. Prior to the spectrophotometric measurement, incubation with fluorescein solution had happened in horizontal position of the cornea holder at 32°C ± 1°C for 90 ± 5 minutes in a water bath. Then the medium in the posterior compartment was mixed by drawing approximately 2.5 ml gently up and down a 5 ml syringe, with a needle attached, three times. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Effects to the cornea (corresponding to possible eye damage) were measured as opacity and permeability, which when combined gave an In Vitro Irritancy Score (IVIS) for each treatment group. A substance that induces an IVIS greater than 55 is defined as a corrosive or severe irritant.

*HBSS Hanks Balanced Salt Solution containing Ca++ and Mg++ without phenol red

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

mean

Remarks:

calculated from the IVIS of each of 3 isolated bovine corneas

Score:

0.5

Remarks on result:

other: IVIS = Corrected Opacity Value + (15 x Corrected permeability OD490 Value)

Interpretation of results:

not irritating

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In both in vitro tests for skin irritation and eye irritation no irritating effects were observed. Therefore, classification of WS400136 for skin and eye irritation is not required [Regulation (EC) 1272/2008].