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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 to induce skin sensitisation was evaluated in three suitable in vitro test methods conducted according to OECD 442C, OECD 442D and OECD 442E and further supported by the results of an OECD QSAR Toolbox v4.4.1 prediction.

Based on the results, the target substance can be considered as non-sensitiser. Therefore, classification is not warranted in accordance with the CLP regulation 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-02-13 to 2020-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 442E
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitization to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the U-SENS^TM assay, which is recommended in an international guideline (e.g. OECD 442E).
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the composition/purity of the test item. A solubility test was performed. The test item was either dissolved or suspended in complete medium to a final concentration of 0.4 and 50 mg/mL and in DMSO (Hybrimax, Sigma, Zwijndrecht, The Netherlands) to a final concentration of 50 mg/mL. In DMSO the test item formed a clear colourless solution at 50 mg/mL. DMSO was selected as solvent for the main assay. In the main experiments the test item was dissolved in DMSO at 50 and 12.5 mg/mL in the first and second experiment, respectively. The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL (first experiment) and 50, 20, 10, 7.5, 5 and 1 µg/mL (second experiment) in the 96-well plate (final concentration DMSO of 0.4%). The test item precipitated at the dose levels of 50 µg/mL and upwards. Test item concentrations were used within 3 hours after preparation. Any residual volumes were discarded.

Details on the study design:
Test System:
- Test system: U937 human monocytes
- Justification: Inducible CD86 expressing cells
- Source: ATCC no. CRL-1593.2TM
Stock cultures of these cells are stored in the freezer (-150 °C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test. Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Cell Culture:
- Cell culture medium: Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).
- Environmental conditions: All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 48 – 93%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.7 – 36.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door.

EXPERIMENTAL DESIGN:
Plating of cells:
- Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 10^5 viable cells/mL. Cell viability was >90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.

Number of experiments:
- Two valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated.

Treatment of cells:
- Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.

Precipitate evaluation:
- After 45 ± 3 hours of exposure, wells were checked for precipitate.

Cell antibodies staining for IgG1 and CD86:
- Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded, and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

Flow cytometry method:
- Acquisition: Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
- Analysis: All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary, in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analysed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
- Color Interferences - On IgG1 analysis: There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).7.8 µM, 16 µM, 31 µM, 63 µM, 125 µM, 250 µM

Vehicle:
- The vehicle of the test item, i.e. 0.4 % dimethyl sulfoxide (DMSO, Sigma, Zwijndrecht, The Netherlands) in complete medium (RPMI-1640, Life Technologies, Bleiswijk, The Netherlands). The vehicle control formulation was shared with parallel studies.

Dose Groups:
- Negative Control: Lactic Acid (LA; Sigma, Zwijndrecht, the Netherlands) : 200 µg/mL
- Positive Control: 2,4,6-Trinitrobenzenesulfonic acid (TNBS; Sigma, Zwijndrecht, the Netherlands): 50 µg/mL
- Test Item:
Experiment 1: 1.0, 10, 20, 50, 100 and 200 µg/mL
Experiment 2: 1.0, 5.0, 7.5, 10, 20 and 50 µg/mL.


Positive control results:
The positive control (TNBS) showed in both experiments a S.I. higher than 150 % in all wells (S.I. ≥ 535% and S.I. ≥ 257% in Experiments 1 and 2, respectively) and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Run / experiment:
other: Experiment 1
Parameter:
other: CD86 upregulation S.I at highest tested concentration with a viability of 70% or higher
Remarks:
10 µg/mL
Value:
525
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: Experiment 2
Parameter:
other: CD86 upregulation S.I at highest tested concentration with a viability of 70% or higher
Remarks:
50 µg/mL
Value:
555
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442E were met in this test.

For individual results please refer to Table 1, Table 2 and Table 3 in box 'Any other information on results incl. tables'.

Table 1: Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the test item

Test items

Dose
(µg/mL)

% Viability (Mean)*

CD86-IgG1 S.I.*

Colour Interference S.I.*

Experiment

Experiment

Experiment

1

2

1

2

1

2

 

 

 

 

 

 

 

1

100

100

132

90

98

109

 

5

-

100

-

233

-

115

 

7.5

-

100

-

315

-

115

 

10

99

99

525

311

78

91

 

20

65

71

813

728

268

290

 

50

53

72

1152

555

265

286

 

100

48

-

1146

-

270

-

 

200

48

-

1023

-

281

-

* Red values are either below 70% viability or above 150 S.I..

- Not Applicable

Table 2: Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls

% Viability (Mean)*

CD86-IgG1 S.I.*

Experiment

Experiment

1

2

1

2

LA1

100

100

107

67

LA2

100

100

112

76

LA3

100

100

98

76

TNBS1

100

100

740

581

TNBS2

100

100

535

257

TNBS3

100

100

730

452

DMSO (mean)

100

100

155

127

* Red values are either below 70% viability, above 150 S.I.

Table 3: Historical Control Data for the USENSTMAssay

Positive control

Negative control

S.I. (%)

Viability (%)

S.I. (%)

Viability (%)

Range
(Mean ± 2 * SD)

79 – 962

92 – 103

51 – 132

95 – 101

Mean

520

98

92

99

SD

221

2.9

20

1.3

n

495

495

496

496

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Nov 2017 to Dec 2019.

Interpretation of results:
other: positive indication of a sensitising potential
Conclusions:
In this study under the given conditions the test item did trigger an upregulation of the expression of the cell surface marker CD86 in two independent experimental runs. Based on these results, the test item can be considered to have a sensitising potential.
Executive summary:

In a dermal sensitisation study conducted according to OECD 442E with (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) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the U937 cell line activation Test (U-Sens™) assay in two independent experimental runs. Cells were incubated with the test item for 45 ± 3 hours and later checked for cell viability and expression of CD86 cell surface markers. Both experiments passed the acceptance criteria. In both experiments the positive (TNBS) and negative (LA) control were considered valid.

The test item showed toxicity in the first experiment (CV70 value of 18 µg/mL). The test item showed no toxicity in the second experiment. A biologically relevant induction of the CD86 activity (EC150 values of 1.4 µg/mL and 2.7 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test item is classified as positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. Therefore, the test item can be considered to have a sensitising potential.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-02-13 to 2020-05-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted June 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSens^TM assay, which is recommended in international guidelines (e.g. OECD 442D).
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 composition/purity of the test item. A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (colourless solution). The 100-fold dilutions in DMEM glutamax of 6.3 to 200 mM formed a non-homogeneous solution (moderate or heavy precipitate) and were therefore not suitable to test. The 100-fold dilution of the 3.1 mM DMSO stock formed a homogeneous solution (microscopic slight precipitation). The 100-fold dilution of the 1.6 mM DMSO stock in DMEM glutamax formed also a homogeneous solution (no precipitation). The concentration of 3.1 mM was selected as highest concentration for the main assay (limit of solubility). In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 3.1 mM (colourless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03 and 0.02 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. The test item precipitated at the highest two dose levels tested in the second experiment at the end of the incubation period. Test item concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.
Details on the study design:
Test System:
- A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell Culture:
- Basic medium : Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
- Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum and geneticin (500 µg/mL).
- Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56 °C; 30 min) fetalfoetal calf serum.
- Environmental consitions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 37 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.3 - 37.3 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
- Subculturing: Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25).

Plating of cells:
- For testing, cells were 80-90% confluent. One day prior to testing cells were harvested and distributed into 96-well plates (10,000 cells/well) in basic medium.
For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 5 in experiment 1 and 7 in experiment 2.

Treatment of cells:
- The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ± 1.0 °C in the presence of 5% CO2. In total 2 valid experiments were performed.

Luciferase activity measurement:
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity assessment:
- For the KeratinoSens^TM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37 °C ± 1.0 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Dose groups:
- Negative Control: DMSO in test item exposure medium
- Positive Control: Ethylene dimethacrylate glycol (EDMG) : 7.8 to 250 µM
- Test Item: 12 concentrations of the test item: Experiments 1 and 2: 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24, 0.12, 0.06, 0.03 and 0.02 µM


Positive control results:
- Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.21 and the EC1.5 21 µM.
- Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.62 and the EC1.5 50 µM.
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: max. luciferase activity (Imax) induction
Value:
1.22
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.22 and therefore no EC1.5 could be calculated.
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: max. luciferase activity (Imax) induction
Value:
1.31
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.31 and therefore no EC1.5 could be calculated.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
Both experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (21 µM and 50 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.21-fold and 2.62-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (3.3% and 7.8 % in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

For individual results see Table 1, Table 2 and Table 3 in box 'Any other information on results incl. tables'.

Table 1: Mean Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)

0.02

0.03

0.06

0.12

0.24

0.49

0.98

2.0

3.9

7.8

16

31

Exp 1 luminescence

1.09

1.17

1.17

1.18

1.20

1.16

1.22

1.15

1.14

1.04

0.01

0.00

Exp 1 viability (%)

100.0

98.2

94.5

92.9

90.3

93.1

93.5

97.3

110.4

127.0

0.0

-0.1

Exp 2 luminescence

1.05

1.03

1.18

1.16

1.25

1.31

1.28

1.12

1.03

0.68

0.01

0.01

Exp 2 viability (%)

97.1

95.8

90.3

87.6

85.3

82.2

82.4

81.8

85.0

62.8

-0.2

-0.3

Table 2: Mean Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.24

1.44

1.59***

1.87***

2.45***

3.21***

Exp 1 viability (%)

103.3

99.2

98.5

100.1

99.0

105.8

Exp 2 luminescence

1.04

1.26

1.36

1.60***

1.90***

2.62***

Exp 2 viability (%)

108.4

102.4

98.3

94.3

90.5

89.2

***p< 0.001 Student’s t test

Table 3: Overview EC1.5, Imax, IC30 and IC50Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.22

11

12

Test item Experiment 2

NA

1.31

6.5

9.3

Pos Control Experiment 1

21

3.21

NA

NA

Pos Control Experiment 2

50

2.62

NA

NA

NA = Not applicable

Table 4: Historical Control Data for the KeratinoSensTM Studies

Positive control

EC1.5(µM)

Imax

95% control Range

-4.3 – 123

-7.34 – 14.22

Mean

59.4

3.44

SD

31.8

5.39

n

484

484

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to December 2019.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, test item can be considered to be a non-sensitizer.
Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with (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) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. The test item showed toxicity (IC30 values of 11 µM and 6.5 µM and IC50 values of 12 µM and 9.3 µM in experiment 1 and 2, respectively). Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5.

For both experiments 1 and 2 no significant luciferase induction was observed within the tested concentration range. Therefore, no EC1.5 value could be calculated. Based on the results, the test item is considered to be a non-sensitizer.

Therefore, in this study, the test item is considered to be non-sensitiser to skin.

Endpoint:
skin sensitisation, other
Remarks:
in silico, OECD QSAR Toolbox, profiling of structural alerts for skin sensitisation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2020-03-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source
Justification for type of information:
1. SOFTWARE
OECD QSAR Toolbox

2. MODEL (incl. version number)
version 4.4.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O[C@@](Cn1cnnn1)(c1ccc(F)cc1F)C(F)(F)c1ccc(cn1)-c1ccc(OCC(F)(F)F)cc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Name of profilers:
- Protein binding potency Lys (DPRA 13%)
- Protein binding by OASIS
- Protein binding by OECD
- Protein binding potency Cys (DPRA 13%)
- Protein binding potency GSH
- Respiratory sensitisation
- Protein binding alerts for skin sensitization according to GHS
- Protein Binding Potency h-CLAT
- Keratinocyte gene expression
- Protein binding alerts for skin sensitization by OASIS

5. APPLICABILITY DOMAIN
Target chemical within the applicability domain.

6. ADEQUACY OF THE RESULT
These results are used in a weight-of-evidence approach to assess the skin sensitising potential of the target chemical. The selected profilers detect structural alerts, which are associated with the potential to bind to or interact with proteins. The binding to skin proteins is the first step of the Adverse Outcome Pathway for skin sensitisation i.e the molecular initiating event leading to the skin sensitisation.
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Profiling of structural alerts for skin sensitisation was performed with the QSAR Toolbox Version 4.4.1.
Specific details on test material used for the study:
- other information: Smiles code O[C@@](Cn1cnnn1)(c1ccc(F)cc1F)C(F)(F)c1ccc(cn1)-c1ccc(OCC(F)(F)F)cc1
Parameter:
other: OECD QSAR Toolbox_alerts for skin sensitisation
Remarks on result:
no indication of skin sensitisation
Remarks:
no alerts found by profilers

Table 1: (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 profiling for structural alerts for skin sensitisation sensitisation:

PROFILERS

RESULTS

 
Protein binding potency Lys (DPRA 13%) DPRA less than 9% (DPRA 13%)
DPRA less than 9% (DPRA 13%) >> No protein binding alert
Protein binding by OASIS No alert found
Protein binding by OECD No alert found
Protein binding potency Cys (DPRA 13%) DPRA less than 9% (DPRA 13%)
DPRA less than 9% (DPRA 13%) >> No protein binding alert
Protein binding potency GSH Not possible to classify according to these rules (GSH)
Endpoint Specific  
Respiratory sensitisation No alert found
Protein binding alerts for skin sensitization according to GHS No alert found
Protein Binding Potency h-CLAT No alert found
Keratinocyte gene expression Not possible to classify according to these rules
Protein binding alerts for skin sensitization by OASIS No alert found
Interpretation of results:
GHS criteria not met
Conclusions:
The test item has been profiled for structural alerts for skin sensitisation by using the OECD QSAR Toolbox, version 4.4.1. A negative indication of the skin sensitisation of the test item is given by ten different profilers. For example, no protein-binding alerts were found, which indicates that the (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 can be predicted to be negative in the direct peptide reactivity assay (DPRA).
Executive summary:

The test item has been profiled for structural alerts for skin sensitisation by using the OECD QSAR Toolbox, version 4.4.1. The target substance was within the applicability domain of the profilers used. The profiling results showed a negative indication for skin sensitisation of the test item based on ten different profilers. For example, no protein-binding alerts were found, which indicates that (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 can be predicted to be negative in the direct peptide reactivity assay (DPRA).

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-05-18 to 2020-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted 18th June 2019
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of European Centre for the Validation of Alternative Methods (ECVAM) and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction for the purity/composition of the test item was performed. For both the cysteine and lysine reactivity assay 90.42 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1714 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the aa clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Details on the study design:
Test system:
Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Experimental Design:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

Sample Incubations:
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.3 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:

System 1 (used for Cysteine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)

System 2 (used for Lysine Reactivity Assay):
Alliance separations module 2695 (Waters, Milford, MA, USA)
Dual λ absorbance detector 2487 (Waters)
Positive control results:
SPCL mean deplition for Cinnamic Aldehyde is 62.8% ± 7.7%. SPCC mean deplition for Cinnamic Aldehyde is 71.4% ± 0.2%.
Key result
Run / experiment:
other: 1
Parameter:
other: SPCC mean depletion (%)
Value:
1.5
Vehicle controls validity:
not examined
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Precipitation observed following the incubation
Key result
Run / experiment:
other: 1
Parameter:
other: SPCL mean depletion (%)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Precipitation observed following the incubation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes

The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.

Table 2: Acceptability of the direct peptide reactivity assay (DPRA)

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.998

>0.99

0.9999

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.519 ± 0.002

0.50 ± 0.05

0.499 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.506 ± 0.004

0.50 ± 0.05

0.478 ± 0.006

CV (%) for RC samples

B and C

<15.0

1.2

<15.0

0.8

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

71.4

40.2-69.0

62.8

SD of peptide depletion cinnamic aldehyde (%)

<14.9

0.2

<11.6

7.7

SD of peptide depletion for the test item (%)

<14.9

0.6

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

(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

1.5%

±0.6%

0.0%

±0.0%

0.8%

Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:
other: negative ("no or minimal reactivity class")
Conclusions:
In the in chemico skin sensitisation assay conducted according to OECD guideline 442C (DPRA), the substance was tested negative and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

In this in chemico sensitisation study conducted in accordance with OECD guideline 442C, the test item (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 (99.1% purity) was dissolved in acetonitrile.

The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24.3 h at 25 ± 2.5 °C. Subsequently samples were analyzed by high-performance liquid chromatography (HPLC).

Sensitizing potential of the test item was predicted from the mean peptide depletion of both analyzed peptides (cysteine and lysine) by comparing the peptide concentration of the test item-incubated samples to the corresponding reference controls. All acceptability criteria were met, and the test was considered to be valid.

The test item showed minimal reactivity towards the synthetic peptides. In the cysteine reactivity assay the test item showed 1.5% peptide depletion while in the lysine reactivity assay the test item showed 0.0% peptide depletion. The mean of both values was 0.8% and as a result, the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

However, since precipitation or phase separation was observed after the incubation period for both synthetic peptides containing cysteine (SPCC) or lysine (SPCL), one cannot be sure how much test item remained in the solution to react with the peptides.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
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 to induce skin sensitisation was evaluated in three suitable in vitro test methods conducted according to OECD 442C, OECD 442D and OECD 442E and additionally by an OECD QSAR Toolbox v4.4.1 prediction.

In the first study conducted according to OECD 442D, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the KeratinoSens™ cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, test item can be considered to be a non-sensitizer.

In the second study, conducted according to OECD 442E, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells in vitro using the human cell line activation test (U937 human monocytes). Cells were incubated with the test item for 45 ± 3 hours and later checked for cell viability and expression of CD86 cell surface markers. Under the given conditions the test item did trigger an upregulation of the expression of the cell surface marker CD86 in two independent experimental runs. Based on these results, the test item can be considered to have a sensitising potential.

In the third study, conducted according to OECD 442C, the sensitisation potential of the test item was assessed on the basis of the reactivity of the test item with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24.3 h at 25 ± 2.5 °C. Subsequently samples were analyzed by high-performance liquid chromatography (HPLC).

Sensitizing potential of the test item was predicted from the mean peptide depletion of both analyzed peptides (cysteine and lysine) by comparing the peptide concentration of the test item-incubated samples to the corresponding reference controls. All acceptability criteria were met, and the test was considered to be valid.

The test item showed minimal reactivity towards the synthetic peptides. In the cysteine reactivity assay the test item showed 1.5% peptide depletion while in the lysine reactivity assay the test item showed 0.0% peptide depletion. The mean of both values was 0.8% and as a result, the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

As in the DPRA assay precipitation was observed after the incubation period for both synthetic peptides containing cysteine and lysine, it can be assumed that if the test item precipitates in aqueous solution and/or is highly lipophilic, it also precipitate in body fluids the protein binding in the skin is also be less likely.

In addition, based on physicochemical properties of the test item a low dermal absorption can be assumed due to a log P > 4 and a molecular weight of >500.

Based on assessing the results from the in vitro studies in a weight of-evidence approach, the target substance can be considered as non-sensitiser for the skin. The non-sensitizing potential of the test item is further supported by OECD QSAR Toolbox profiling module which did not indicate protein binding alerts for skin sensitization with the target chemical.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

By assessing the available data in a weight-of-evidence approach, the target substance can be considered as non-sensitizer. Thus, in accordance with CLP regulation 1272/2008 no classification for skin sensitization is warranted.