(R)-2-(2,4-Difluorphenyl)-1,1-difluor-3-(tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluorethoxy)-phenyl]-2-pyridyl}-2-propanol-(R,R)-tartrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-03-20 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 18th June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2R)-3-amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol; (2R,3R)-2,3-dihydroxybutanedioic acid

EC Number:

845-262-4

Cas Number:

1809816-36-1

Molecular formula:

C22H17F7N2O2 . C4H6O6

IUPAC Name:

(2R)-3-amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol; (2R,3R)-2,3-dihydroxybutanedioic acid

Test material form:

solid

Details on test material:

- CAS: 1809816-36-1
- Content (q-NMR): 99.9% w/w
- Content (HPLC): 99.5% area
- Appearance: White solid
- Storage conditions: Ambient temperature (10 °C to 30 °C)
- Expiry date: December 2020

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 25 mg of the test item were dispensed directly atop the EpiDerm tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model EpiDerm™ (EPI-200, MatTek)

EpiDerm Kit:
The EpiDerm tissues were provided as kits (MatTek), consisting of the following components relevant for this study:
- 1x sealed 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 (Lot No.: 30854F, 30858 [repetition 60 min])
- 2x 24-well plates
- 4x 6-well plates
- 1x bottle of assay medium (DMEM-based medium; Lot: 032620MJC, 041620MJD)
- 1x bottle of DPBS Rinse Solution (Lot: 012120MJA)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C


REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
- 3 min experiment: After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- 60 min experiment: After 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper
- 3 min and 60 min experiment: After the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: final concentration 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
See Table 1 in box "Any other information on materials and methods incl. tables".

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 plus 25 µL H2O

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL potassium hydroxide
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure, mean of replicates

Value:

80.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure, mean of replicates

Value:

69.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%.
- Colour interference with MTT: The mixture of 25 mg test item per 300 μL Aqua dest. and 300 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC is determined to be 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative & positive control: The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.798, 1.942). The mean relative tissue viability (% negative control) of the positive control was < 15% (6.3%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (0.6% - 6.6%).

For details of the results please refer to Tables 2 and 3 in section "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2: Results of the 3-min experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.776	1.676	0.182	0.426	1.381	1.340
	1.852	1.768	0.189	0.384	1.518	1.493
	1.904	1.811	0.190	0.469	1.525	1.484
Mean Absolute OD570	1.798****		0.307		1.457
OD570- Blank Corrected	1.727	1.627	0.133	0.377	1.333	1.291
	1.803	1.720	0.141	0.336	1.469	1.445
	1.856	1.763	0.142	0.420	1.476	1.435
Mean OD570of 3 Aliquots (Blank Corrected)	1.795	1.703	0.139	0.378	1.426	1.390
SD OD570 of 3 Aliquots	0.065	0.069	0.005	0.042	0.081	0.086
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.749*		0.258		1.408
SD OD570 of 2 Replicate Tissues	0.065		0.169		0.025
Mean Relative Tissue Viability [%]	100.0		14.8		80.5
Coefficient Of Variation [%]^***	3.7		65.5		1.8

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

***  coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is 30%.

^****  The mean absolute OD₅₇₀of the negative control is ≥ 0.8 and ≤ 2.8

Table 3: Results of the 60-min experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.842	1.995	0.175	0.153	1.370	1.370
	1.855	2.053	0.174	0.160	1.341	1.352
	1.862	2.042	0.172	0.161	1.344	1.369
Mean Absolute OD570	1.942****		0.166		1.358
OD570- Blank Corrected	1.795	1.948	0.128	0.106	1.323	1.322
	1.808	2.006	0.126	0.113	1.294	1.305
	1.815	1.995	0.125	0.114	1.297	1.322
Mean OD570of 3 Aliquots (Blank Corrected)	1.806	1.983	0.126	0.111	1.305	1.316
SD OD570 of 3 Aliquots	0.010	0.031	0.002	0.005	0.016	0.010
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.895*		0.118		1.311
SD OD570 of 2 Replicate Tissues	0.125		0.011		0.008
Mean Relative Tissue Viability [%]	100.0		6.3**		69.2
Coefficient Of Variation [%]^***	6.6		9.2		0.6

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

** mean relative tissue viability of the 60 min positive control < 15%

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is 30%.

**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was above 15% after 60 min treatment and above 50% after 3 min treatment. Therefore, the test item is classified as “non-corrosive“.

Executive summary:

In a primary skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 25 mg of (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol L-tartrate (99.5% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was greater than 15% (69.2%) after 60 min treatment and greater than 50% (80.5%) after a 3 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on the results from this study, the test item can be classified as non-corrosive. To specify the classification in accordance to CLP regulation 1272/2008 the results from an OECD 439 study must be additionally consulted.

The study is acceptable and satisfies the guideline requirements for an in vitro skin corrosion study (OECD 431).