1-fluoro-3-(trans-4-propylcyclohexyl)benzene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 FEB 2015 - 26 MAR 2015

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, 65189 Wiesbaden (18 November 2014)

Test material

Test material information

Constituent 1

Reference substance name:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

EC Number:

443-100-3

EC Name:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

Cas Number:

138679-81-9

Molecular formula:

Hill formula: C15H21F CAS formula: C15H21F

IUPAC Name:

1-fluoro-3-[(1s,4r)-4-propylcyclohexyl]benzene

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The RHE™-model was obtained by culturing adult human keratinocytes on a polycarbonate filter under conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer.

Justification for test system used:

To reduce animal testing, this alternative in vitro method was used. The human skin RHE™-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: RHE™-model (SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 15-RHE-029
- Expiration date: March 9, 2015
- Date of initiation of testing: March 4, 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL DPBS to remove any residual test item. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.454 (±0.542) (Acceptance criterion: OD > 0.7)
- Barrier function: 9.2 h (Acceptance criterion: 4.0 h <= ET50 <= 10.0 h)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. 7 viable cell layers present. Absence of significant histological abnormalities.

NUMBER OF REPLICATE TISSUES:
3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

40.25

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The visual evaluation in the pretest for MTT-reducing capacity of the test item after 3 hours incubation with MTT-reagent did not show blue color, i.e. MTT was not reduced by the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values were 1.792, 1.650 and 1.719 and, thus, in the range of ≥ 0.8 and ≤ 3.0. After treatment with the negative control (DPBS-buffer) the mean OD was 1.720 (standard deviation: 4.13%) and, thus, higher than the historically established boundary of 1.400.
- Acceptance criteria met for positive control: After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.36% (standard deviation: 5.75%) and, thus, lower than the historically established boundary of 3.55%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the three tissues treated with the test item was 11.28% and, thus, ≤ 18%. The standard deviation of the negative control and the positive control was <= 18%, respectively.

Any other information on results incl. tables

A table of the results is attached under 'Attached background material'.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS Category 2 or Category 1

Conclusions:

The tissue viability after treatment with the test item was lower than 50% (mean viability: 40.25%). Therefore, the test item is considered to possess an irritant potential to skin.

Executive summary:

This in vitro study was performed according to OECD Guideline 439 (Reconstructed Human Epidermis Test) following GLP. The test consisted of a topical exposure of the test item to a human reconstructed skin 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 the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 1.720. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.36 %. Therefore, the study fulfilled the validity criteria. The tissue viability after treatment with the test item was 40.25 % and, thus, lower than 50 %, i.e. according to UN GHS classification the test item is considered as irritant to skin.