1-Butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Apr 25, 2017 - June 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

EC Number:

833-005-9

Cas Number:

1003218-33-4

Molecular formula:

C24H34F2O

IUPAC Name:

1-butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied undiluted.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier:SkinEthic Laboratories (Nice, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch No: 17-RHE-050
- Expiration date: May 8, 2017

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

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL PBS using a pipette. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

87.9

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
After treatment with the negative control the absorbance values reached a mean OD of 1.871 (SD 9.0%). Therefore, the negative control fulfilled the validity criteria.
Treatment with the positive control revealed a mean relative viability of 1.8% (SD 16.7%), thus the positive control reached the validity criteria.

Any other information on results incl. tables

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.871	100
Positive Control	42	0.034	1.8
Test Material	42	1.645	87.9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439.Under the experimental conditions reported, the test material is not irritating to the skin.

Executive summary:

Objective

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test.

Study Design

The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.Triplicates of the human skin model RHEä(Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue.

Results

After treatment with the negative control (DPBS-buffer) the mean OD was 1.871. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.8%. Thus, the acceptance criteria stated in part 5.9 were met.

Following treatment with the test item, the tissue viability was 87.9% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Conclusion

Under the experimental conditions reported, the test material is not irritating to the skin.