Tall oil, compd. with diethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2012 to 3 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, compd. with diethanolamine

EC Number:

268-452-3

EC Name:

Tall oil, compd. with diethanolamine

Cas Number:

68092-28-4

Molecular formula:

Example formula, based on oleic as the fatty acid: C18H35COOH.N(CH2CH2OH)2

IUPAC Name:

Tall oil, compd. with diethanolamine

Test material form:

semi-solid (amorphous): gel

Test animals

Species:

other: in vitro Reconstructed Human Epidermis (RHE) Model

Details on test animals or test system and environmental conditions:

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 the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit
Supplier: SkinEthic Laboratories, Lyon, France

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro controls were used

Amount / concentration applied:

10 µL

Duration of treatment / exposure:

A treatment period of 15 minutes was followed by a post-exposure incubation period of 42 hours.

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

MATERIAL PREPARATION
-Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control.
-Sodium Dodecyl Sulphate (SDS) 5 % w/v aqueous dilution was used as the positive control.
-A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
-A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

PRE-TEST
Assessment of Direct Test Material Reduction of MTT

MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
The test material was checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO₂ in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue, it is presumed to have reduced the MTT.

The test material was shown to directly reduce MTT. There was a possibility that if the test material could not be totally rinsed off the tissues, any residual test material present on or in the tissue may have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues. This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues.

Water-killed tissues were prepared by placing untreated EPISKIN tissues in a 12-well plate containing 2.2 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5 % CO₂ in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed, the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use, each tissue was thawed by placing in 2.2 mL of maintenance medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the test material was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

PRE-INCUBATION (Day 0: Tissue Arrival)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 °C, 5 % CO₂ in air overnight.

MAIN TEST
APPLICATION OF TEST MATERIAL AND RINSING (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test material for an exposure period of 15 minutes. It was applied topically to the corresponding tissues, ensuring uniform covering. 10 µL of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control material, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes of contact time, the SDS solution was re-spread with a pipette tip to maintain the distribution for the remainder of the contact period. The plate(s) were kept in a biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO₂ in air for 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (Day 3)
Following the 42 hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO₂ in air. At the end of the 3 hour incubation period, each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. 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.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (Day 6)
At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce an homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
-Quantitative MTT Assessment (percentage tissue viability)
For the test material, the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD540 of test material / mean OD540 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability according to the following:

Criteria for in vitro interpretation EU DSD Classification EU CLP Classification
Relative Mean Tissue Viability is ≤50 % Irritant (I) R38 Category 2
Relative Mean Tissue Viability is >50 % Non-Irritant (NI) No Category

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

-Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40 % relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18 %.
-Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18 %.
-Test Material
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18 %.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

OD540 0.061

Value:

7.9

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

OD540 0.083

Value:

10.7

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

OD540 0.092

Value:

11.9

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

An assessment found that the test material was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed that a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

 

Quality Criteria

-The relative mean tissue viability for the positive control treated tissues was 8.9 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.9 %. The positive control acceptance criterion was therefore satisfied.

 -The mean OD540 for the negative control treated tissues was 0.774 and the standard deviation value of the percentage viability was 15.0 %. The negative control acceptance criterion was therefore satisfied.

-The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 2.1 %. The test material acceptance criterion was therefore satisfied.

 

Table 1 Mean OD540 Values and Percentage Viabilities for the Negative and Positive Controls and the Test Material

Material	OD540 of Tissues	Mean OD540 of Triplicate Tissues	± SD of OD540	Relative Individual Tissue Viability (%)	Relative Mean Viability (%)	± SD of Relative Mean Viability (%)
Negative Control	0.674	0.774	0.116	87.1	100*	15.0
	0.747			96.5
	0.901			116.4
Positive Control	0.052	0.069	0.015	6.7	8.9	1.9
	0.079			10.2
	0.077			9.9
Test Material	0.061	0.079	0.016	7.9	10.2	2.1
	0.083			10.7
	0.092			11.9

*The mean viability of the control group is set at 100 %

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: Skin irritant Cat. 2

Remarks:

according to the criteria set up on the OECD guideline 439

Conclusions:

Skin irritant

Executive summary:

The skin irritation potential of the test material was evaluated in vitro using the EPISKIN reconstructed human epidermis model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before being incubated for 42 hours, after which each tissue was taken for MTT loading. After MTT loading, a total biopsy of each epidermis was made and formazan crystals were extracted out of the MTT-loaded tissues. Duplicate tissues treated with Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++ served as the negative control and duplicate tissues treated with 5 % w/v aqueous Sodium Dodecyl Sulphate served as the positive control. The optical density of all treated tissues was measured at 540 nm.

The relative mean viability of the test material treated tissues was 10.2 % after the 15 minute exposure period.

Under the conditions of this study, the test material was determined to be a Category 2 irritant in accordance with the criteria set out in Annex I of Regulation (EC) No 1272/2008 (CLP). It also classified as irritant according to EU labelling regulations Commission Directive 2001/59/EC.