(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

Endpoint summary

Description of key information

The potential 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 to induce skin and eye irritation/corrosion was evaluated in suitable in vitro tests.

Based on the results from two in vitro skin irritation/corrosion studies conducted according to OECD 431 and OECD 439, the target substance must be considered as irritant to the skin and classification as Skin Irrit. 2, H315 is warranted in accordance with the CLP Regulation 1272/2008.

The potential of the target substance to induce eye irritation was assessed in an in vitro assay conducted according to OECD 437. Based on the results, the target substance can be considered to induce serious damage to the eye. Therefore, classification as Eye Dam.1, H318 is warranted in accordance with the CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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)

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.

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

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

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".

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

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).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-02-20 to 2020-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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.

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

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.: 112719ISA)
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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD570 nm ≥ 0.8 and ≤ 2.8 (1.933)
- Acceptance criteria met for positive control: yes, mean relative tissue viability of the three positive control tissues is ≤ 20% (3.8%)
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%. (0.2-6.5%)

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

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	0.102	0.106	0.111
	1.972	1.720	1.917	0.112	0.114	0.129	0.102	0.107	0.112
Mean Absolute OD570	1.933****			0.118			0.107
OD570(Blank Corrected)	2.018	1.818	2.013	0.073	0.069	0.074	0.056	0.060	0.064
	1.926	1.674	1.871	0.065	0.067	0.083	0.055	0.061	0.065
Mean OD570of the Duplicates (Blank Corrected)	1.972	1.746	1.942	0.069	0.068	0.078	0.055	0.060	0.065
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.887*			0.072			0.060
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.123			0.005			0.005
Relative Tissue Viability [%]	104.5	92.5	102.9	3.7	3.6	4.1	2.9	3.2	3.4
Mean Relative Tissue Viability [%]	100.0			3.8**			3.2
SD of Relative Tissue Viability [%]***	6.5			0.3			0.2
CV [% Viabilities]	6.5			7.5			7.7

*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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this in vitro skin irritation study (OECD 439), the test item is considered to be irritant to the skin. In combination with the results from an OECD 431 test, it can be concluded, that classification as skin irritant (Category 2) is warranted.

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to (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 followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was < 50% (3.2%). The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on this result, the test item

is considered to be irritating to the skin. In combination with the results from a second in vitro test (OECD 431), it can be concluded that classification as Skin Irrit. 2, H315 is warranted.

The study is acceptable and satisfies the guideline requirements for an in vitro skin irritation study (OECD 439).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-09 to 2020-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Storage: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec
Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 °C.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: no data

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): 750 µL

Duration of treatment / exposure:

240 +/- 10 min at 32 ± 1 °C

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Opacity measurements were performed before and directly after exposure. The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (20% imidazole in physiological saline 0.9 NaCl%).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 °C.

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 240 +/- 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM (containing phenol red)

POST-EXPOSURE INCUBATION:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- The optical density was determined at 490 nm (OD490) using a spectrophotometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (triplicates)

Value:

191

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: According to the UN GHS, this IVIS indicates that the test item induces serious eye damage

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: yes, the mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 137 and within two standard deviations of the current historical positive control mean.

For details on the results please refer to box "Any other information on results incl. tables".

Table 2: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score (1), (2)
Negative control	1.3	0.007	1.4
Positive control	117	1.310	137
Test item	134	3.749	191

(1) Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

(2) In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 3: In vitro Irritation Score (IVIS)

Treatment	Final Opacity (2)	Final OD490 (2)	In vitro Irritancy Score (1)
Negative control	0.2	0.005	0.2
	1.1	0.003	1.1
	2.7	0.013	2.9
Positive control	137	1.146	154
	106	1.404	127
	108	1.381	129
Test item	149	5.021	225
	135	2.651	175
	118	3.575	172

(1) In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

(2) Positive control and test item are corrected for the negative control.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, based on the mean in vitro irritation score of 191 obtained in the bovine corneal opacity and permeability assay (OECD 437), it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described above and should be classified category 1 according to the UN GHS criteria.

Executive summary:

The eye irritation potential 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) was investigated in the bovine corneal opacity and permeability assay (OECD 437). The mean in vitro irritation score was determined with 191 after a 4-hour treatment. The positive control induced the appropriate responses, indicating the validity of the assay. In conclusion, since the test item induced an IVIS ≥ 55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described and should be classified as Eye Dam. 1, H318 in accordance with the CLP Regulation 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential 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 to induce skin irritation/corrosion was evaluated in two suitable in vitro tests.

In a primary skin corrosion study conducted according to OECD testing guideline 431, the mean relative tissue viability (% negative control) was greater than 15% (69.2%) after a 60 min treatment and greater than 50% (80.5%) after a 3 min treatment. 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.

Thus, in a primary skin irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to (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 followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was < 50% (3.2%). Based on this result, the test item is considered to be irritating to the skin. In combination with the results from the first in vitro test (OECD 431), it can be concluded that classification as Skin Irrit. 2, H315 is warranted.

The potential of the target substance to induce eye irritation/corrosion was evaluated in an in vitro test conducted according to OECD 437. The bovine corneal opacity and permeability assay (BCOP, OECD 437) allows classification as “not classified” or “Eye Dam 1, H318” based on the In Vitro Irritancy Score (IVIS). Results of an IVIS greater than 55 are identified as “UN GHS Category 1”. Based on the results obtained from the BCOP test (mean IVIS = 191), the target substance must be considered to induce serious damage to the eye and classification as Eye Dam. 1, H318 in accordance with CLP Regulation 1272/2008 is warranted.

Justification for classification or non-classification

Based on the results from suitable in vitro skin and eye irritation/corrosion studies, classification as Skin Irrit. 2, H315 and Eye Dam. 1, H318 is warranted in accordance with CLP Regulation 1272/2008.