4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Endpoint summary

Description of key information

No definitive conclusion on skin sensitisation can be drawn on the basis of available data.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

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:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In vitro/chemico assay to be conducted instead of in vivo test.

Details on the study design:

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 °C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Key result

Remarks on result:

not determinable because of methodological limitations

Remarks:

The substance was not soluble in any of the solvents tested and the DPRA study could not be performed.

Interpretation of results:

study cannot be used for classification

Conclusions:

The DPRA study could not be performed as the substance was not soluble in any suitable solvent.

Executive summary:

The objective of the study was to determine the skin sensitisation potential of the substance under GLP to OECD TG 442c, and test the reactivity of the test substance towards synthetic peptides containing cysteine (SPCC) or lysine (SPCL). At a concentration of 100 mM, the test substance was not soluble in acetonitrile, Milli-Q-water or a 1:1 v/v mixture of the two, isopropanol, a mixture of acetone and acetonitrile (1:1, v/v), dimethylsulphoxide and acetonitrile (1:9 v/v), methanol or ethanol. Consequently, the Direct Peptide Reactivity Assay (DPRA) could not be performed with the substance and the study was stopped.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

Two positive controls for cell reactivity check

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: fresh stock solutions were prepared for each test run. A stable dispersion of test substance in DMSO was prepared at the appropriate 8 concentrations applying a dilution factor of 1.2
- Preparation of the test chemical serial dilutions: the stock solution was 250-fold diluted in RPMI-1640 culture medium to obtain the working solutions
- Preparation of the positive controls: 2,4-dinitrochlorobenzene stock solution was prepared at 8 μg/mL in DMSO before treatment by dissolving the substance at a concentration of 2 mg/mL and then diluting 250-fold in cRPMI; nickel sulphate was mixed in 0.9% NaCl solution at a concentration of 12 mg/mL and then 50-fold diluted in cRPMI to obtain a 240 μg/mL stock solution
- Preparation of the solvent, vehicle and negative controls: a DMSO control formulation was included as vehicle control, by dissolving DMSO at 0.2% in cRPMI
- Stable dispersion obtained: yes
- Log Kow of the test chemical: 4.7

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 500 μg/mL
- Solubility in solvents: a stable dispersion in DMSO at 250 mg/mL was obtained
- Solubility in incubation medium: at post-treatment observation, precipitation of test substance was observed at concentrations ≥62.50 μg/mL
- Results of selecting appropriate concentration and determination of cytotoxicity: CV75 was calculated to be 41.47 μg/mL (average of two range finding tests)
- Final concentration range selected on basis of the cyctotoxicity observed in range finder, and on precipitation of test material

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: Run C was invalidated and repeated, because the RFI CD86 of the positive control 2,4-dinitrochlorobenzene was <150
- Test chemical concentrations: 13.89, 16.66, 20, 24, 28.80, 34.56, 41.47 and 49.76 μg/mL
- Application procedure: 500 μL of working solutions was added to the volume of THP-1 cell suspension in each plate (500 μL)
- Exposure time: 24 hours ± 30 minutes
- Study evaluation and decision criteria used: a run is positive if the Relative Fluorescence Intensity (RFI) of CD86 is ≥150 in at least one tested concentration leading to ≥50% viability or the RFI of CD54 is ≥200 in at least one tested concentration leading to ≥50% viability
- Description on study acceptance criteria: for controls the viability of cells treated with cRPMI and DMSO has to be ≥90%, in cRPMI and DMSO control wells MFI ratio of CD86 and CD54 to isotype control should be >105, the RFI values of CD86 and CD54 in the cRPMI and DMSO controls should not exceed the positive criteria (RFI ≥150 for CD86 and RFI ≥200 for CD54), in the positive controls with DNCB and NiSO4, the RFI values of both CD86 and CD54
should meet positive criteria (i.e. CD86 RFI ≥150 and CD54 RFI ≥200) and cell viability should be more than 50%; for test items noted as cytotoxic in the dose range finding study, and resulting in a negative outcome in the main test, cell viability at 1.2 x the CV75 should be <90% in each run and the cell viability of at least 4 out of 8 test concentrations should be >50%.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
- Incubation conditions: humidified incubator at 37 °C and 5% carbon dioxide
- Washing conditions: two washes with 1 mL FACS buffer
- Other: blocked with blocking solution (600 μL in runs A, C, D, 1 mL in run B) and incubated at 4 °C for 15 minutes ± 1 minute. After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μL FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation at 4 °C for 30 minutes ± 2 minutes. Finally, cells were washed with 150 μL FACS buffer twice and resuspended in 200 μL FACS buffer. The plate was positioned inot the plate reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: The non-specific binding of IgG1 and the expression of CD86 and CD54 was analysed by flow cytometry with the acquisition channel B1 to obtain the Mean Fluorescence Intensity (MFI), whereas the viability (PI uptake) was analysed with the acquisition channel B3. A total of 10000 living cells were acquired. Whe the viability was low, a maximum of 100 μL per sample was acquired.
- Plate used: 96-well round bottom plate
- Propidium iodide staining/cytotoxicity measurements: flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations ≥62.50 μg/mL. The corresponding CV75 values were 44.04 μg/mL and 38.91 μg/mL, respectively, in the first and the second range finding study
- Preparation for CD54 and/or CD86 expression measurements/cell staining: A volume of 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4 °C.


DATA EVALUATION
- Cytotoxicity assessment: flow cytometry measurement after Propidium Iodide staining
- Prediction model used: Based on the individual run conclusions, a final prediction is made
• if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted,
• if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) without the need for a third run,
• if however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction is based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered POSITIVE.
An h-CLAT prediction should be considered in the framework of an Integrated Approaches to Testing and Assessment (IATA), considering the AOP on skin sensitization published by OECD, as well as the applicability domain of the h-CLAT method.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The acceptance criteria were fulfilled in each run, except run C, where the RFI value for CD86 of the positive control substance DNCB was <150. The run was invalidated and repeated as run D.

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD54 >200

Value:

41.47 µg/mL

Cell viability:

83% at 41.47 μg/mL, ranging from 82 to 93% at the other concentrations

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD86 >150

Value:

20 µg/mL

Cell viability:

84% at 20 μg/mL, ranging from 79 to 90% at the other concentrations

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

- Slight to strong precipitation was observed in treated wells in run A (at concentrations of ≥20.00 μg/mL) and run B (at concentration ≥34.56 μg/mL), but no precipitation in run D.
- No cell morphology modifications were noted at any tested concentration.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance was found to be negative in this in vitro h-CLAT test.

Executive summary:

The skin sensitisation potential of the substance was studied under GLP to OECD TG 442E in an in vitro human Cell Line Activation Test (h-CLAT), investigating the ability of substances to induce an increase in cell surface markers expression in THP-1 cells. The solubility assessment of the substance revealed that a stable dispersion in DMSO could be obtained at a concentration of 250 mg/mL, and DMSO was therefore selected as the vehicle in the test.In each run of the range finding study and the main study, test substance formulations were applied to THP-1 cells, which were them immediately incubated in a 24-well plate for 24 hours at 37 °C in a humified atmosphere at 5% carbon dioxide. A set of control wells was also added in each plate (2,4-dinitrochlorobenzene and nickel sulphate as positive controls, DMSO as vehicle control) to guarantee the validity of each run. At the end of the incubation period, the treated cells of each well were divided into three aliquots that were distributed to three individual wells of a 96-well plate, and one well was labelled with IgG1-FITC antibodies, one well was labelled with CD86-FITC antibodies and one well was labelled with CD54-FITC antibodies. Finally, all cells were dyed with Propidium Iodide for viability determination just before flow cytometry analysis of CD86 and CD54 expression. The mean fluorescence intensity (MFI) from each test sample was corrected for the isotype control IgG1 MFI value. The corrected MFI value from the vehicle control was set to 100% CD54 and CD86 expression. The MFI values from each test sample treated with the substance or a positive control substance were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) values for CD86 and CD54 expression. The cytotoxicity of the substance was assessed in two dose range finding studies applying test concentrations of 3.91 to 500 μg/mL. The test substance was found to decrease the cell viability to <75% at concentrations >62.5 μg/mL, and the corresponding mean CV75 value was calculated to be 41.47 μg/mL. The maximum test concentrations in the main test was 49.76 μg/mL (i.e. 1.2-fold the mean CV75), and a series of in total 8 test concentrations was obtained by serial dilution to a factor 1.2, giving a test concentration range from 13.89 to 49.76 μg/mL. Under the conditions of the test, the RFI for CD86 was >150 at the test concentration of 20 μg/mL in run A (at a cell viability of 84%), and the RFI for CD54 was >200 at the test concentration of 41.47 μg/mL in run B. As the positive control in run C was invalid, the run was repeated and negative test results were obtained for all test concentrations in run D. The overall conclusion of the test was therefore that the substance was found to be negative in the in vitro h-CLAT test. As the test substance has a log Kow >3.5, the applied method may not be applicable. However, as high cytotoxicity was noted in the range finding tests and a clear dose-response relationship was seen in all tests, exposure of the test system to the substance could be demonstrated. The overall negative result of the test is thus considered as reliable.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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 KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: KeratinoSens

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the basis of solubility results, the test substance was dissolved in DMSO at 200 mM. One stock solution was prepared for each run.
- Preparation of the test chemical serial dilutions: The stock formulation was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to get a total of 12 concentrations in a 96-well plate, which was called the "Master plat 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plated called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within four hours before use, and kept at room temperature and protected from light until use.
- Preparation of the positive controls: For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was further diluted to a final concentration of 6.4 mM. Then, it was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.
- Preparation of the solvent, vehicle and negative controls: This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 μM in culture medium containing 1% DMSO
- Solubility in solvents: the test substance was soluble in DMSO at 100 and 200 mM
- Solubility in incubation medium: no solubilisation of test substance could be obtained at 100 or 200 mM in water for injections and culture medium even after a 40 minute sonication step; once stock solutions in DMSO were 100-fold diluted in the treatment culture medium, a precipitate was immediately observed, and was still noted after the overnight incubation period
- Cytotoxicity assessment performed: for the transparent 96-well cell viability plate the medium was removed by aspiration and replaced with 200 μL of treatment medium; a volume of 27 μL MTT solution at 5 mg/mL in D-PBS was added to each well of the plate, which was then covered with a sealing membrane and incubated at 37 °C in the humidified incubator for 4 hours (± 10 minutes); the medium was then removed and a volume of 200 μL of a 10% SDS solution was added to each well; the plate was covered with a sealing membrane and incubated overnight at 37 °C in the humified atmosphere to extract the formazan from cells; the absorption of each well was determined at 600 nm using the plate reader after the incubation period
- Final concentration range selected on basis of: limited solubility in culture medium

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: two
- Number of repetitions: not applicable
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO
- Application procedure: after the 24-hour growing period, the medium was removed and replaced with 150 μL of treatment medium; from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate; all plates were covered with a sealing membrane and then incubated;
- Exposure time: 48 hours ± 2 hours
- Study evaluation and decision criteria used: The test substance was considered as positive if the following four conditions were all met in two of two or in two of three runs, otherwise the KeratinoSens prediction was considered as negative: the maximal induction factor of luciferase activity compared to the negative control was >1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test), at the lowest concentration with a gene induction >1.5-fold (i.e. at the EC1.5 determining value), the cell viability is ≥70%, the EC1.5 value was <1000 μM (or <200 μg/mL for test item without MW), there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response)
- Description on study acceptance criteria: Each run was considered valid if the following criteria were met: at least 2 consecutive concentrations should have a viability ≥70%, the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations, the average EC1.5 value for the positive control should be within two standard
deviations of the historical mean. In addition, the average induction (Imax) in the 3 replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with
increasing luciferase activity at increasing concentrations for the positive control, the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be <20%.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): cells were grown using general culture procedures up to 80-90% confluence; the day before treatment cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2, and counted using Trypan Blue dye; the cell concentration was adjusted to 80000 cells/mL; cells were distributed into four 96-well plates (three white plates and one tranparent plate) by adding 125 μL (representing 10000 cells) per well taking care to avoid sedimentation of the cells during seeding, after which the cells were grown for 24 hours (± 1 hour) in the 96-well microtiter plates
- Incubation conditions: 37 °C, 5% carbon dioxide, 90% humidity
- Washing conditions: cells were washed with D-PBS
- Precipitation noted: after the 48 hours incubation period, each well was inspected under the microscope for precipitates or emulsions

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: 96-well plate Luminometer with injectors and OD reader (Varioskan Flash)
- Plate used: white assay plates
- Lysate preparation: a volume of 20 μL of passive lysis buffer was added to each well and the cells were
incubated for 20 (± 2) minutes at room temperature and under orbital shaking; the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program: a volume of 50 μL of the luciferase substrate was added to each well, 1 second after this addition, the luciferase signal was integrated for 2 seconds.

DATA EVALUATION
- Cytotoxicity assessment: For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells
- Prediction model used: For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
• Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
• EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
• IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
• indication whether significant 1.5-fold gene induction occurred below the IC30

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

1.83 µM

Cell viability:

109 to 121% at 0.98 to 15.63 μM
73% at 31.25 μM
4 to 26% at 62.5 to 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

31.72 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

39.64 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

Mean Imax = 41.22
A slight to strong precipitate was observed in treated wells at 31.25 μM (first run) or 15.63 μM (second run). The acceptance criteria were met for the test substance and the positive control.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the condition of the study, the substance was positive in the KeratinoSens assay and was considered to activate the Nrf2 transcription factor.

Executive summary:

The skin sensitisation potential of the substance was tested under GLP to OECD TG 442D in an in vitro study using the KeratinoSens cell line, which is an immortalised and genetically modified Human adherent HaCaT keratinocyte cell line used to test the potential to activate the Nrf2 transcription factor. The cells were first plated on 96-well plates and grown for 24 hours at 37 °C. The growth medium was then removed and the cells were exposed to the vehicle control (DMSO) or to 12 different concentrations of the test substance, separated by a factor of 2, in the range from 0.98 to 2000 μM in culture medium containing 1% DMSO. The cells were incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminiscence. In parallel, the cytotoxicity was measured by a MTT reduction test and taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. A positive control with cinnamic aldehyde was also included in the study.

All acceptance criteria were met in both validated runs, and the study was considered as valid. At the tested concentrations, statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison with the negative control at concentrations of ≥1.95 μM in the first validated run and ≥3.91 μM in the second validated run, with an apparent dose-response relationship in both runs. The evaluation criteria for a positive response were met in both runs, and the final outcome of the test was interpreted as positive, i.e. the substance was found to activate the Nrf2 transcription factor in the used KeratinoSens cell line.

Endpoint conclusion

Additional information:

A set of three in chemico/in vitro studies was performed to investigate the skin sensitisation potential of the substance. The direct peptide reactivity assay could not be performed, as the test substance was not soluble in any of the applicable solvents. The human cell line activation test gave a negative result, whereas the ARE-Nrf2 luciferase test method using the KeratinoSens cell line gave a positive result. A definitive conclusion on the skin sensitisation potential of the substance cannot be drawn on the basis of the available data.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available information on the skin sensitisation potential of the substance is not sufficient to draw an unequivocal decision on the need for classification.