4-Ethoxy-2,3-difluorphenylboronic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 18, 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 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:

October 9, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

EC Number:

606-729-6

Cas Number:

212386-71-5

Molecular formula:

C8 H9 B F2 O3

IUPAC Name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability :
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Analysis for tissue functionality and quality: please refer to "any other information on methods and materials"

- Description of the cell system used
Desigantion: EpiOcular Tissue (OCL-200, OCL-212)
Keratinocyte strain: 4F1188

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied neat to the tissues.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

6 hours +/- 15 min

Duration of post- treatment incubation (in vitro):

18 hours +/- 15 min

Number of animals or in vitro replicates:

2 tissues

Details on study design:

- Details of the test procedure used

- RhCE tissue construct used, including batch number: EpiOcular Tissue (OCL-200, OCL-212), Lot No. 27033

- Doses of test chemical and control substances used: 50 mg test material, 50 µL negative control, 50 µL positive control

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 6 hours (+/- 15 min) at 37°C
Post-exposure immersion: 25 min (+/- 2 min) at room temperature
Post-exposure incubation: 18 hours (+/- 15 min) at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Pre-experiments to assess direct MTT reduction and colored or staining test item were performed.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 (test item), 2 (negative control), 2 (positive control)
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): wavelength = 570 nm
- Description of the method used to quantify MTT formazan
After the post-treatment incubation period, the treated tissues were incubated with 300 µL MTT solution (1.0 mg/mL MTT) and incubated for 180 min (+/- 10 min) at 37°C. The inserts were removed from the 24-well plate after 180 min (+/- 10 min). 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 MTT, the plates were placed on an orbital shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extracts 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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
No categeory: Mean tissue viability > 60%
No prediction can be made: Mean tissue viability: <= 60%

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Negative control: OD > 0.8 and < 2.5
Positive control: mean relative viability is a) 30 min exposure: below 50% of control viability and b) 6 hour exposure: below 50% of control viability
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
- Positive and negative control means and acceptance ranges based on historical data
1. Negative control is OD > 0.8 and < 2.5 (1.577 and 1.795)
2. The mean relative viability of the positive control is below 50% of the negative control viability (17.8%)
- Acceptable variability between tissue replicates for positive and negative controls: < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: viability (%)

Run / experiment:

1

Value:

13.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: viability (%)

Run / experiment:

2

Value:

13.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (OD > 0.8 and < 2.5 (1.577 and 1.795))
- Acceptance criteria met for positive control: Yes (below 50% of the negative control (17.8%))

Any other information on results incl. tables

The pre-test for direct MMT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Following treatment with the test item, the tissue viability was 13.8% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

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 min). 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 1.686 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.8% (study acceptance criterion: < 50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 13.8% and, thus. lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.