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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-17 to 2020-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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).

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

845-262-4

Cas Number:

1809816-36-1

Molecular formula:

C22H17F7N2O2 . C4H6O6

IUPAC Name:

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

Test material form:

solid

Details on test material:

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

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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 mg/mL. The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL (first and second experiments) in the 96-well plate (final concentration DMSO of 0.4%). The test item precipitated at the dose levels of 100 µg/mL and upwards. Test item concentrations were used within 4 hours after preparation. Any residual volumes were discarded.

In vitro test system

Details on the study design:

Test System:
- Test system: U937 human monocytes
- Justification: Inducible CD86 expressing cells
- Source: ATCC (American Type Culture Collection, Virginia, USA), 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 55 – 91%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.2 – 36.8 °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.

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%).

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: Experiments 1 and 2: 1.0, 10, 20, 50, 100 and 200 µg/mL

Results and discussion

Positive control results:

Experiment I: The positive control (TNBS) showed a S.I. ≥ 550% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment II: The positive control (TNBS) showed a S.I. ≥ 594% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment 1

Parameter:

other: highest S.I. of CD86

Value:

552

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Positive indication of skin sensitisation: a biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 1.71 μg/mL.

Results 2

Run / experiment:

other: Experiment 2

Parameter:

other: highest S.I of CD86

Value:

6 013

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 0.1 µg/mL

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'.

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
Test Item
	0.1	-	100	-	150	-	95
	1	100	100	116	176	109	108
	3	-	100	-	326	-	108
	5	-	93	-	607	-	101
	7.5	-	48	-	6013	-	210
	10	57	50	552	5804	113	227
	20	36	23	228	5165	198	247
	50	15	-	139	-	112	-
	100	21	-	465	-	140	-
	200	6	-	-338	-	190	-

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

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 89 34   LA2 100 100 83 40   LA3 100 100 94 34   TNBS1 100 100 577 634   TNBS2 100 100 550 594   TNBS3 100 100 614 771   DMSO (mean) 100 100 141 88     IgG1 value (%) CD86 basal expression (%) CD86-IgG1 expression (%) Experiment Experiment Experiment 1 2 1 2 1 2 RPMI1 0.9 0.7 7.2 2.3 6.3 1.6 RPMI2 0.9 1.3^1 8.7 2.3^1 7.8 1.0^1 RPMI3 1.1 0.8 8.0 2.7 6.9 1.9 DMSO1  -  - -  -  10.0 1.6 DMSO2  - -  -  -  9.3 1.5 DMSO3  - -  -  -  10.3 1.5 RPMI Mean Viability  - 100 100   RPMI Drift  - -2% 46%   LA Drift  - 10% -8%

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

¹ Excluded from analysis, value > 25% from mean

Table 3: Historical Control Data for the U SENS^TMAssay

	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.

Applicant's summary and conclusion

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 an in vitro skin sensitisation study conducted according to OECD 442E with (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) 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. The test item showed toxicity (CV70 values of 7.27 μg/mL and 6.28 μg/mL in experiment 1 and 2, respectively).

A biologically relevant, induction of the CD86 activity (EC150 values of 1.71 μg/mL and 0.1 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. In the first experiment a viability of <70%, test item precipitation and colour interference at concentrations were observed and > 150% increase in CD86 activity was observed at test concentrations with a cell viability of >70% compared to the vehicle control in the second experiment. Based on these results, the test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line).