Tall oil, compd. with ethanolamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

16 August 2010 to 16 September 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The study was conducted in accordance with the draft guideline OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, compds. with ethanolamine

EC Number:

268-640-5

EC Name:

Fatty acids, tall-oil, compds. with ethanolamine

IUPAC Name:

Fatty acids, tall-oil, compds. with ethanolamine

Test material form:

other: liquid (unspecified)

Details on test material:

- Appearance: untransparent, yellow liquid
- Storage Conditions: ambient temperature

Test animals

Species:

other: in vitro Reconstructed Human Epidermis (RHE) Model

Details on test animals or test system and environmental conditions:

The EpiDerm EPI-200 skin model consists of normal human epidermal keratinocytes (NHEK) from one single donor, derived from neonatal-foreskin tissue. The keratinocytes are plated on chemically modified, collagen-coated, 8 mm ID cell culture inserts (surface area 0.5 cm²). The skin models are commercially available.
Upon arrival at the testing laboratory, the EPI-200 skin models were pre-incubated in 0.9 mL assay medium provided with the kit for 60 minutes (at ca. 37 °C and ca. 5 % CO₂). At the end of the first pre-incubation period, the tissues were additionally pre-incubated overnight for 21 to 24 hours (at ca. 37 °C and ca. 5 % CO₂).

EPI-200 skin model supplier: MatTek Corporation, USA

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:

30 µL

Duration of treatment / exposure:

A treatment period of 60 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:

REFERENCE MATERIALS
- Positive control: 5 % Sodium Lauryl Sulphate (SLS) in demineralised water (= 5 % Sodium Dodecyl Sulphate (SDS))
- Negative control: Phosphate Buffered Saline (PBS)

PRELIMINARY TEST
The tissue staining potential of the test material was investigated prior to the main study by incubating 30 µL in 300 µL of demineralised water for 1 hour (at ca. 37 °C). At the end of the exposure time, the presence and intensity of the staining (if any) was evaluated. The test material did not change the colour of the solution; therefore, it was concluded that the test material did not have the potential to stain the tissue.
Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the main study, 30 µL of the test material was incubated in 1 mL of 1 mg/mL MTT solution for 3 hours (at ca. 37 °C) and the formation of a blue formazan product was assessed. The test material did not show MTT reducing capacity, i.e. the solution did not turn blue/purple.
Furthermore, the test material showed no interaction with a nylon mesh. Therefore, nylon mesh was used to facilitate equal distribution of the test material.

EXPOSURE TO THE TEST MATERIAL
After overnight pre-incubation, the skin membranes were exposed to 30 µL of the test material, positive or negative controls in triplicate for 60 ± 2 minutes. The test material was manually shaken just before application to facilitate homogenisation. Immediately after application, a nylon mesh (provided with the EPI-200 kit) was placed onto the tissue surface to facilitate an equal distribution of the test material.

Exposure was performed at ambient temperature in a flow cabin in four series, including one series of frozen controls. After dosing the last tissue of each series, the plates containing the skin membranes were transferred to a humidified incubator (ca. 37 °C and ca. 5 % CO₂). After 35 ± 1 minutes, the plates were removed from the incubator and placed in the flow cabin until the exposure period of 60 minutes was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using an excess of phosphate buffered saline (PBS).
Subsequently, the insert was blotted dry and the tissue surface was carefully dried using a sterile cotton swab. The insert was then transferred to clean 6-well plate containing fresh medium (0.9 mL/well). Medium was refreshed after 18 ±2 hours. Following an additional 22.5 hours incubation period at ca. 37 °C and ca. 5 % CO₂ in a humidified incubator, viability was determined using the MTT assay.

MTT ASSAY
An MTT solution of 1 mg/mL was prepared by diluting MTT concentrate 5 times in MTT diluent (both provided with the kit). The bottom of the inserts was blotted dry and the inserts placed in a 24-well plate containing 300 µL of MTT medium/well. The plates were placed in an incubator at ca. 37 °C and ca. 5 % CO₂. After 180 ± 1 minutes, the tissues were rinsed three times with PBS. The formazan product was extracted from the tissue using 2 mL MTT extractant (provided with the kit). Extraction was performed in the dark for 2 days at 2 to 10 °C.

After completion of the extraction period, the skin membrane was pierced with a needle to allow the extract to run into the well from which the membrane was taken. The optical density was measured in triplicate in 200 µL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control.

INTERPRETATION OF THE RESULTS
The test is considered valid if the optical density of the negative control is ≥1.0 and ≤2.5 and the optical density of the extractant solvent alone is <0.1. Tissues treated with the positive control should reflect their ability to respond to an irritant chemical.

The irritation potential of the test material was determined from the relative mean tissue viability compared to the negative control tissues, using the following prediction model:

Mean tissue viability in vitro In vivo prediction
(% of negative control)

Mean tissue viability ≤50 % Irritant
Mean tissue viability >50 % Non-irritant

Results and discussion

In vivo

Irritant / corrosive response data:

The results are summarised in Table 1.
The mean optical density of the test material was 94 % of the negative control.

The optical densities of the negative and positive control were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. This demonstrated validity of the model.

Any other information on results incl. tables

Table 1 Summary of Control and Test Material Results

Material			MTT Conversion (OD₅₇₀)
			Measurement 1	Measurement 2	Measurement 3	Replicate Mean
Negative Control	Replicate 1		1.924	1.921	1.983	1.943
	Replicate 2		1.980	1.978	1.995	1.984
	Replicate 3		1.933	1.975	1.970	1.959
	Mean	1.962
	SD	0.021
	% CV	1.1
	% of control	100
Test Material	Replicate 1		2.060	2.090	2.059	2.070
	Replicate 2		1.611	1.643	1.630	1.628
	Replicate 3		1.828	1.861	1.837	1.842
	Mean	1.847
	SD	0.221
	% CV	12.0
	% of control	94
Positive Control	Replicate 1		0.127	0.136	0.138	0.134
	Replicate 2		0.137	0.137	0.135	0.136
	Replicate 3		0.133	0.132	0.133	0.133
	Mean	0.134
	SD	0.002
	% CV	1.4
	% of control	7

 Data shown in the table were corrected for background absorbance values (extractant solvent alone), which were on average 0.040.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material was determined to be a non irritant in accordance with EU criteria.

Executive summary:

The potential of the test material to cause skin irritation was assessed in an in vitro study conducted under GLP conditions in accordance with the draft guideline “OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009”.

The study used the EpiDerm reconstituted skin membranes. The undiluted test material was applied topically to the skin membranes for 60 minutes. After a 42 hour incubation period, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate. 5 % Sodium Dodecyl Sulphate (SDS) was used as the positive control and Phosphate Buffered Saline (PBS) as the negative.

The mean optical density of the test material was 94 % of the negative control.

The optical densities of the negative and positive control were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. This demonstrated validity of the model.

Under the conditions of this study, the test material was determined to be a non irritant and requires no classification in accordance with EU criteria.