1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-22 and 2015-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

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:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin SOP, Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

Cas Number:

1154521-93-3

Molecular formula:

C40H68N2O4

IUPAC Name:

1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France,
- Supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 15-EKIN-018
- Expiry date: 11 May 2015
- Date of initiation of testing: 6 May 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The EpiSkinTMSM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source.
- Observable damage in the tissue due to washing: care was taken to avoid damaging to the epidermis.
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours at 37 °C in an incubator with 5 % CO2, ≥95% humidified atmosphere and protected from light
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

IN CASE OF MTT DIRECT INTERFERENCE:
Optical properties of the test item or its chemical action on MTT may interfere with the assay and lead to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test item acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls are used to detect and correct for test item interference with the viability measurement.

Check-method for possible direct MTT reduction with test item:
Approximately 10 μL test item was added to 2 mL MTT 0.3 mg/mL (diluted with assay medium) solution and mixed. The mixture was incubated for three hours at 37 °C protected from light. Subsequently, any observed colour changes of the solution was recorded:
- Test items which do not interact with MTT: yellow
- Test items interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test item interacts with the MTT. It is then
necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
The test item showed no direct interaction with MTT. Using of additional control was not necessary.

Check-method to detect the colouring potential of test item:
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in
contact with water (simulating a tissue humid environment). Approximately 10 μL test item was added to 90 μL of water. The mixture was shaken for 15 minutes at room temperature and any development of colouring was monitored (unaided eye assessment). The test item showed no ability to become coloured in contact with water. Using of additional control was not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irrtating to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: unchanged

NEGATIVE ONTROL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: SDS 5% aq.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test animals

Details on test animals or test system and environmental conditions:

not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

82

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:

OTHER EFFECTS:
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be 1.473 %. As the NSMTT was below 30 % the true MTT metabolic conversion and the correction of viability percentages were undertaken.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is brownish orange, however in low amount (such as treatment amount) it was observed as a very light coloured liquid and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not
necessary. A false estimation of viability can be precluded.


ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues was 0.836. The mean OD value obtained for the positive control was 0.033 and this result corresponds to 4 % viability when compared to the results
obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

According to the current OECD guideline No. 439, the test item is considered as non-irritant to skin and is therefore not classified.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and untreated killed tissues) were used to detect and correct for test substance interference with the viability measurement. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 81 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item is considered as non-irritant to skin and is therefore not classified.