(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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-07 to 2020-06-24

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

Version / remarks:

adopted 18 June 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
- Treatment of test material prior to testing: Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 30849 (main experiment), 30838 (killed tissue controls)

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport.
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 022020MJD)
1x bottle of DPBS Rinse Solution (Lot No.: 112719ISE)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow at room temperature and for the remaining time of 35 ± 1 min transferred to the incubator at 37 °C.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by 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 ± 5 min at 37 °C
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-incubation 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): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 2124835)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution (TC-SDS 5%, MatTek, CAS No.: 151-21-3, Lot No: 110519MSA).

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

100

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:

For detailed results see Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Results of the Pre-Experiments:

- The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The medium as well as the test item turned blue/purple. The test material sedimented or suspended. NSMTT was≤30% (4.3%) relative to the negative control of livingepidermis, the true MTT metabolic conversion (TOD_TT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TOD_TT= OD_TM– (OD_KT– OD_KU) = 1.968 – (0.740 – 0.659) = 1.887

- The mixture of 25 mg of the test item per 300 µL aqua dest. or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC was determined to be 0%.

The test item showed non-specific reduction of MTT, but 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.

Results of the main experiment:

Table 1: Result of the Test Item

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570****	2.064	1.864	2.060	0.120	0.116	0.120	2.155	1.899	2.042
	1.972	1.720	1.917	0.112	0.114	0.129	2.075	1.978	1.936
OD570(Blank Corrected)	2.018	1.818	2.013	0.073	0.069	0.074	2.108	1.853	1.995
	1.926	1.674	1.871	0.065	0.067	0.083	2.029	1.931	1.889
Mean OD570of the Duplicates (Blank Corrected)	1.972	1.746	1.942	0.069	0.068	0.078	2.069	1.892	1.942
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.887*			0.072			1.968
SD of mean OD570	0.123			0.005			0.091
Relative Tissue Viability [%]	104.5	92.5	102.9	3.7	3.6	4.1	109.7	100.3	103.0
Mean Relative Tissue Viability [%]	100.0			3.8**			104.3 100 (NSMTT corrected)
SD of Relative Tissue Viability [%]***	6.5			0.3			4.8
CV [% Viabilities]	6.5			7.5			4.6

*Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is ≤ 20%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is≤ 18%.

****The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8 (1.933).

Table 2: Quality criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNC	1.933	0.8 ≤ NC ≤ 2.8	pass
Relative Viability [%] PC	3.8	≤ 20%	pass
SD Viability [%]	0.3 -6.5	≤ 18%	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 irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to (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) for 60 min followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained from three tissues compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was > 50% (100%). Based on this result, the test item is classified as a non-irritant according to the UN GHS.