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Administrative data

Description of key information

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 skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered as non-irritant to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 (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
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".

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 (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TODTT= ODTM– (ODKT– ODKU) = 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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
2020-02-07 to 2020-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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.

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.
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%
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".

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
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”.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered as non-irritant to the skin and to the eye and no classification in accordance with CLP regulation 1272/2008 is warranted.

Justification for classification or non-classification

Based on the results from suitable in vitro test methods (OECD 439, OECD 492), the target substance can be considered as non-irritant to the skin and eye. Thus, no classification in accordance with CLP regulation 1272/2008 is warranted.