[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 09 November 2017 to 12 January 2018. Report issue: 21 August 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Multiple adult human donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 09 January 2018. On the day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium / the pre-incubation phase of the EpiSkin™ tissues started.

TEMPERATURE AND OTHER TEST CONDITIONS
- Pre-warming: Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for 22.5 hours.

- Temperature and other test conditions used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2 for 15 minutes
- Temperature and other test conditions post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5% CO2 for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Once - the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.


MTT ASSAY
- Method: A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plate. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 2.5 hours at room temperature with gentle agitation. For each tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate for optical density reading..
- Spectrophotometer: Versamax (Molecualr Devices, 85737 Isaminng, Germany)
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three tissue samples per endpoint

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
10 µL (26.3 µL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
10 µL


POSITIVE CONTROL
- Amount(s) applied (volume or weight):
10 µL
- Concentration (if solution): 5% w/w

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

Number of replicates:

Negative control = 3
Negative Control Freeze Killed = 3
Positive control = 3
Test Item = 3
Test Item Freeze Killed Tissue = 3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 1

Value:

70.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 2

Value:

62.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 3

Value:

55.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (of the 3 tissue samples tested)

Value:

60.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item passed the colour interference pre-test.

The test item reduced MTT in the pre-test for direct MTT reduction. Consequently, additional tests with freeze-killed tissues were necessary to show the amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue. The relative viabilities were correct for MTT reduction in killed tissue.

Any other information on results incl. tables

Data Evaluation

For the test item and the positive control, the mean relative viability ±rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:

In Vitro Result	in vivo Prediction	EU Classification	UN GHS Classifciation
mean tissue viability <50%	Irritant	H315	Category 2
mean tissue Viability > 50%	Non-irritant

 

For the current test, an irritation potential leading to H315 classification of EU according to regulation (EC) 1272/2008, and GHS category 2 according to UN GHS (published 2003, last (7^th revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced to<50% of the negative control. After treatment with the test item the mean relative absorbance value was reduced to 60.13%.This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the test, the test substance is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

Introduction

The in vitro skin irritation potential of the test substance as assessed a Human Skin Model Test design to meet the following guideline:

• OECD Guideline for the testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 28, 2015)

Method

Three tissues of the human skin model EpiSkin™ were treated with the test item, a negative control (PBS) or a positive control (5% sodium lauryl sulfate) for 15 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. Tissues were then washed to remove test material and following a further 42 hours post exposure incubation an MTT assay was run on the tissue samples to assess tissue viability were included.

The test item reduced MTT (pre-test for direct MTT reduction); however, it did not dye water, when mixed with it (pre-test for colour interference). Due to MTT reduction additional tests with freeze-killed tissues were necessary.

Result

The negative and positive control absorbance values were well within the required acceptability criterion thus showing the quality and validity of the test system.

Following treatment with the test item the mean relative absorbance value decreased to 60.13%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Conclusion

In conclusion, it can be stated that in this study and under the experimental conditions reported the test substance is not irritant to skin according to UN GHS and the EU CLP regulation.