3-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 22, 2017 - November 7, 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:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 28, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,

Version / remarks:

September 14, 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

EC Number:

936-120-3

Cas Number:

97476-05-6

Molecular formula:

C21 H25 F

IUPAC Name:

3-fluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre-treatment, the test item was applied neat to the tissues.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30°C

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

in vitro: duplicate design

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular Tissues (OCL-200, OCL-212) (Lot No: 27005) was obtained from MatTek In Vitro Life Science Laboratories
- Doses of test chemical and control substances used: undiluted
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours at 37°C (exposure), 25 min at room temperature (post-exposure immersion), 18 hours at 37°C (post-exposure incubation)
- Number of tissue replicates used per test chemical and controls: two tissues (test item, negative and positive control)
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm wavelength
- Description of the method used to quantify MTT formazan:

After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2. The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 ml isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Viabililty (%) = (test item OD/mean negative control OD) x 100
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category).
If the test item-treated tissue viability is <=60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
1. The negative control OD is > 0.8 and < 2.5 (0.945 and 1.025).
2. The mean relative viability of the positive control is below 50% of the negative control viability (33.9%).
3. The difference of viability between the two relating tissues of a single chemical is < 20% (values between 1.1% to 8.2%) in the same run (for positive and negative control tissues and tissues of single chemicals).
- Reference to historical data of the RhCE tissue construct
1. Tissue viability: OD (540- 570 nm) [1.1 – 3.0] (acceptance criteria); 1.419 +/- 0.105 (result)
2. Barrier function: ET-50 [12.2 – 37.5 min] (acceptance criteria); 15.97 min (result)
3. Sterility: No contamination (acceptance criteria); sterile (result)
- Positive and negative control means and acceptance ranges based on historical data
Negative control: OD >0.8 and <2.5
The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
- Acceptable variability between tissue replicates for positive and negative controls: The difference of viability between the two relating tissues of a single chemical is <20% in the same run.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single chemical is <20% in the same run.

Results and discussion

In vitro

Results

Irritation parameter:

other: Viability %

Run / experiment:

Run 1 / Experiment 1

Value:

111.4

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:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (0.945 and 1.025).
2. TThe mean relative viability of the positive control is below 50% of the negative control viability (33.9%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 1.1% to 8.2%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

Any other information on results incl. tables

	Mean OD	Mean Viability
Negative Control	0.985	100.0%
Positive Control	0.334	33.9%
Test Item	1.098	111.4%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (UN GHS: No Category).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 0.985 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.9% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 111.4% and, thus, higher than 60%, i.e. according to OECD 492 the test item is labeled non-irritant (UN GHS: No Category).

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (UN GHS: No Category).