(2R)-2-(2,4-difluorophenyl)-1,1-difluoro-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}-3-(1H-tetrazol-1-yl)propan-2-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-07 to 2020-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

adopted 18 Jun 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,3,4-tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol

EC Number:

841-499-2

Cas Number:

1340593-59-0

Molecular formula:

C23H16F7N5O2

IUPAC Name:

2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-1,2,3,4-tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol

Test material form:

solid

Details on test material:

- CAS No.: 1340593-59-0
- Purity: 99.9 % w/w
- Appearance: white solid
- Storage conditions: ambient temperature (10 °C to 30 °C)
- Expiry date: 2021-07-06

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Approximately 50 mg (83.3 mg/cm²) of the test item were applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

6 ± 0.25 h

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 2 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 2 °C, 5.0% CO2 / 95% air. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS.
Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 5 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 2°C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings. For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 30644 for main experiment, 30640 for killed tissue controls),
1x bottle EpiOcularTM assay medium (Lot No.: 021020ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 112719ISE),

- Doses of test chemical and control substances used:
1. Negative Control: 50 µL Aqua dest.
2. Positive Control: 50 µL methyl acetate (CAS No. 79-20-9 (Merck, Lot No.: S7816211)
3. Test Item: 50 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 2 °C, 5.0% CO2/95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 2 °C, 5.0% CO2/95% air

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): no prediction can be made
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”

Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8
- mean relative tissue viability of the positve control is < 50%
- relative tissue viability difference of replicate tissues is < 20%

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative Tissue Viability (%)

Run / experiment:

Mean of replicates

Value:

104.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed no irritating effects. The mean relative tissue viability (% negative control) was >60% (104.9%). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Main Results
Name	NC		PC		TM
Tissue	1	2	1	2	1	2
OD570values (blank-corrected)	1.379	1.390	0.621	0.536	1.480	1.410
	1.327	1.391	0.619	0.540	1.474	1.412
Mean of the Duplicates	1.353	1.391	0.620	0.538	1.477	1.411
Mean OD	1.372*		0.579		1.444
SD of mean OD	0.027		0.058		0.046
Relative Tissue viability [%]	98.6	101.4	45.2	39.2	107.7	102.9
Relative Tissue Viability Difference [%]^***	2.8		6.0		4.8
Mean Tissue Viability [%]	100.0		42.2**		104.9 (NSMTT corrected)

NC: negative control; PC: positive control; TM: test material

*corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**mean relative tissue viability of the positive control is < 50%

***relative tissue viability difference of replicate tissues is < 20%

****mean absolute OD570 of the negative control is >0.8 and < 2.8 (1.422)

Table 2: Acceptance Criteria
		Value	Cut-off	pass/fail
Mean absolute OD570 nm NC		1.422	0.8 < NC < 2.8	pass
Mean relative viability PC [%]		42.2	< 50%	pass
Max. difference of % viability [%]		6.0	< 20%	pass

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions, the test item showed no irritating effects. The test item is classified as non-irritant in accordance with UN GHS “No Category”.

Executive summary:

In this study conducted according to OECD 492, the potential of (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol (99.9% purity) to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6-hour exposure and 18-hour post-incubation period and compared to those of the concurrent negative controls. The test item showed reduction of MTT as compared to the control after mixture of 50 mg test item with 1mL MTT medium. Therefore, NSMTT was determined to be 0.3% and the killed control corrected viability (KCCV) was determined to 104.9%. Then, the test item showed non-specific reduction of MTT, and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 60% (104.9% NSMTT-corrected).

Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.