(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Endpoint summary

Description of key information

The substance was positively tested in the in chemico Direct Peptide Reactivity Assay using synthetic peptides containing cysteine and lysine, the in vitro h-CLAT test using THP-1 cells (human monocytik leukemia cell line) and the in vitro KeratinoSens test using human keratinocytes. Using the results of these studies in a weight-of-evidence approach leads to the conclusion that the substance may have skin sensitising properties warranting a classification under the CLP regulation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

On the basis of solubility results, the test item was solubilized in DMSO at 200 mM. One stock formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 for the first run or using a dilution factor of 1.41 for the second and third runs, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
Test system KeratinoSens cells, Supplier Givaudan, Batch D1, absence of mycoplasm confirmed: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Cell seeding for testing - Cells were grown using general culture procedures up to 80-90% confluence, the day prior to 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. Cell concentration was adjusted to a density of 8 x 10E+04 cells/mL, cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 10E+04 cells) per well taking care to avoid sedimentation of the cells during seeding, after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates
prior to test item addition.
Treatment - After the 24-hour growing period, the medium was removed by aspiration and replaced by 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 for the cytotoxicity evaluation, all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells, the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate: After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
Luminescence flash signal to evaluate induction signal - white plates
• After incubation, the supernatants from the white assay plates were discarded,
• the cells were washed once with D-PBS,
• 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.
Absorbance signal to evaluate the cytotoxicity - transparent plate
• For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
• a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
• the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
• at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
• the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
• after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.
Result analysis - For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted. 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. For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value. 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.
The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose-response curves or large variation may lead to wrong extrapolations. Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

A clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control.

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

1.62

Cell viability:

89% at 2000 μM, ranging from 107 to 143% at all other test concentrations ranging from 45.67 to 1416 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.38

Cell viability:

Ranging from 99 to 141% at all test concentrations in the range from 45.67 to 2000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.62

Cell viability:

72% at 2000 μM, ranging from 113 to 149% at all other test concentrations in the range from 0.98 to 1000 μM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: This parameter has no unit, therefore no unit was provided

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

All acceptance criteria were met in the three validated runs with the exception of the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" which was not fulfilled in the second validated run (i.e. Imax of 8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated.

No geometric mean IC30 or IC50 was calculated since the cell viability was >70% in all runs performed. Since two of the three runs performed were considered as positive, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Interpretation of results:

other: Positive result is considered in a weight-of-evidence approach

Conclusions:

The substance was positively tested in the KeratinoSens assay and considered to activate the Nrf2 transcription factor.

Executive summary:

The potential of the substance to activate the Nrf2 transcription factor was studied under GlP in the KeratinoSens assay to OECD TG 442D. The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent validated runs were performed as part of this study. All acceptance criteria were considered met in the three validated runs, the study was therefore considered as validated. This study was performed at tested concentrations ranging from 0.98 to 2000 μM in the first run, and from 45.67 to 2000 μM in the second and third runs, in culture medium containing 1% DMSO. At these tested concentrations, statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control in two of the three runs performed (at the concentration of 250 μM in the first run and at the two successive concentrations of 128 and 180.5 μM in the third run). The evaluation criteria for a positive response are met in two of the three runs performed, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Solutions for cysteine reactivity assay
Stock solution - A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 20.1 mg of SPCC in 40.12 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
SPCC reference control solutions - 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. In addition, a RCcysCACN:MQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCACN:MQ sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL ACN:MQ (1:1, v/v). A SPCC calibration curve was prepared.
Co-elution control, substance and positive control samples - co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.
Solutions for lysine reactivity assay
Stock solution - A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 20.6 mg of SPCL in 39.77 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
SPCL reference control solutions - Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCACN:MQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCACN:MQ sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN:MQ (1:1. v/v). A SPCL peptide calibration curve was prepared.
Co-elution control, substance and positive control samples - co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.
Sample incubation - 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 times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 22.9 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence 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)

Vehicle / solvent:

1:1 mix, water:acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.7% ± 0.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 65.2% ± 3.5%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

lysine depletion

Value:

0.7 %

At concentration:

0.471 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

lysine depletion

Value:

0 %

At concentration:

0.481 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

0 %

At concentration:

0.482 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

cysteine depletion

Value:

64 %

At concentration:

0.183 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

cysteine depletion

Value:

59.1 %

At concentration:

0.208 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

53.7 %

At concentration:

0.235 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

moderate reactivity [in chemico]

Other effects / acceptance of results:

All acceptability criteria for the cysteine and lysine reactivity assays were met confirming the suitability of the HPLC system and the stability of the HPLC runs over time.

Interpretation of results:

other: Positive result is considered in a weight-of-evidence approach

Conclusions:

The substance was positive in the DPRA and was classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was studied under GLP to OECD TG 442c. After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and nonsensitizers. Acetonitrile (ACN):Milli-Q water (MQ) (1:1, v/v) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and CACN:MQ, 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. In the cysteine reactivity assay the test item showed 58.9% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion. The mean of the SPCC and SPCL depletion was 29.6% and as a result the test item was considered to be positive in the DPRA and classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for SPCL, one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, the percentages of SPCL depletion might be underestimated.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

2018

Deviations:

no

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:

Solubility test - The solubility of the test item was assessed. It was not soluble at the concentration of 100 mg/mL in saline, but a solution was obtained at 250 mg/mL in DMSO after 5 min of vortexing, and this concentration in DMSO was therefore selected. The formulation was then 500-fold diluted in RPMI-1640 culture medium.
Positive control preparation - The positive control DNCB was prepared at the concentration of 8 μg/mL in DMSO as follows:
• on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,
• this solution was then 250-fold diluted in cRPMI in order to obtain a 8 μg/mL DNCB stock solution.
The positive control NiSO4 was prepared at the concentration of 240 μg/mL in 0.9% NaCl as follows:
• on the treatment day, the required quantity of NiSO4 was mixed with 0.9% NaCl at the concentration of 12 mg/mL,
• this solution was then 50-fold diluted in cRPMI in order to obtain a 240 μg/mL NiSO4 stock solution.
Both positive control stock solutions were prepared within 4 hours before use, and kept at
room temperature and protected from light until use.
Vehicle control preparation - As DMSO was the vehicle selected at completion of the solubility assay, DMSO control
formulation was included as vehicle control, and consisted in DMSO dissolved at 0.2% in cRPMI.
Substance preparation - All test item preparations were prepared in glass vials only. Fresh stock formulations of the test item were prepared for each run, using the vehicle DMSO and a stock solution concentration of 250 mg/mL. Test item formulations prepared in DMSO were 500 x concentrated; then 2 x concentrated formulations were prepared by 1:250 dilution in cRPMI. A DMSO vehicle control was also prepared (0.4% DMSO in cRPMI). The above mentioned dilutions of the test item and vehicle controls were performed to insure a constant percentage of the vehicle in the final volume of cell suspension in the well (i.e. 0.2% for DMSO). The aspect of the stock formulations was evaluated and recorded in the study files. The precipitation in the treatment conditions (i.e. when diluted in cRPMI) was checked.
Test system, cells - The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute
monocytic leukemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France). The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37 °C, 5% CO2 and were not allowed to
exceed a cell density of 1 x 106 cells/mL or more than 30 passages. The culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin. During cell culturing, cell viability was checked using trypan blue.
Cell culture - For testing, THP-1 cells were seeded at a density between 0.1 x 10E+06 cells/mL and 0.2 x 10E+06 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. The cells did not exceed density of 1 x 10E+06 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 10E+06 cells/mL. Then, 500 μL of cells suspension were distributed into a 24-well flat-bottom plate (i.e. 1 x 10E+06 cells/well).
Reactivity check - Two weeks after thawing, a reactivity check was performed to qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with Lactic Acid, DNCB and NiSO4 under an internal testing facility reference number. Results from reactivity check tests are compiled in the testing facility internal data, with negative and positive control data obtained during each test.
The study was divided in a dose-range finding assay and a main test with studies of a concentration series in successive runs.
Dose-range finder - Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 250-fold (as DMSO is the selected vehicle) into cRPMI to obtain working solutions. The working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5% CO2. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 μL of FACS buffer. Finally, cells were resuspended in 200 μL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 μL of Propidium Iodide (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.
Main test - Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to the highest achievable non-cytotoxic concentration as no CV75 was obtained. The maximum concentration in the plates was 500 μg/mL. All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions. In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared. All working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5% CO2.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 μL of blocking solution 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 of FITC-labeled 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.
Finally, cells were washed with 150 μL FACS buffer two times and resuspended in 200 μL FACS buffer. The plate was then positioned into 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.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

other: 2,4-dinitrochlorobenze and nickel sulfate

Positive control results:

Positive controls were producing results in the expected acceptability range showing the sensitivity of the test system.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Value:

241.13 µg/mL

Cell viability:

92 to 96%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: RFI CD54 exceeded the positivity threshold at concentrations ≥241.13 μg/mL (with the exception of 347.22 μg/mL)

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Value:

167.45 µg/mL

Cell viability:

94 to 97%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: RFI CD54 exceeded the positivity threshold at concentrations ≥167.45 μg/mL (with the exception of 241.13 and 347.22 μg/mL)

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

Acceptance criteria, dose-range finding study
Viability of control cells treated with cRPMI (and DMSO if applicable) should be ≥90%, viability of control cells treated with 0.2% DMSO should be ≥90%, if applicable.

Acceptance criteria, main test
Controls acceptance criteria
Viability of cells treated with cRPMI and DMSO controls should be ≥90%, in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be >105%, in the DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI >150% and CD54 RFI ≥200%), in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%.
Test item acceptance criteria
For a test item noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 x CV75 should be <90% in each run, cell viability of at least 4 out of 8 concentrations should be >50%.

Main test interpretation
Individual run interpretation
A run conclusion is positive if at least one of the conditions below is met:
RFI of CD86 is ≥ 150 in at least one tested concentration leading to ≥50% viability, RFI of CD54 is ≥200 in at least one tested concentration leading to ≥50% viability. In other circumstances, the run is considered as negative.
Prediction model
Based on the individual run conclusions, a final prediction is made as follows:
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 will be 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 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered positive. An h-CLAT prediction should be considered in the framework of an IATA, considering the AOP on skin sensitization published by OECD, as well as the applicability domain of the h-CLAT method.

Interpretation of results:

other: Positive result is considered in a weight-of-evidence approach

Conclusions:

The substance was tested positively in the h-CLAT assay.

Executive summary:

The potential of the substance to induce an increase in cell surface markers expression in THP-1 cells was studied under GLP to OECD TG 442E using the h-CLAT test method. A solubility assessment was performed in vehicles among 0.9% NaCl and DMSO and the substance was found soluble in DMSO at the concentration of 250 mg/mL. Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay. As no decrease in cell viability (i.e. cell viability > 75%) was noted in test item treated wells, no mean CV75 value was calculated and the highest tested concentration in the two assays of the main test was 500 μg/mL. In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37 °C, 5% CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination. For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54). The threshold RFI for CD54 was exceeded at five test concentrations in experiment A and four concentrations in experiment B, whereas the threshold RFI for CD86 was exceeded only for the lowest test concentration in the second experiment B. Under the conditions of the study, the substance was positively tested in the h-CLAT assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitising potential of the substance was assessed in chemico under GLP in a Direct Peptide Reactivity Assay to OECD TG 442C. The substance showed a mean depletion of 58.9% in the cysteine reactivity assay in three independent assays, while the mean depletion of three experiments in with lysine reactivity assay was only 0.2%. However, the percentage of lysine depletion may be underestimated due to precipitation after the incubation period. Overall, the mean depletion was 29.6%, and as a result the substance was considered to be positive in the DPRA and classified in the "moderate" reactivity class when using the appropriate prediction model.

The skin sensitisation potential was also investigated under GLP in an in vitro KeratinoSens test to OECD TG 442D and in an in vitro h-CLAT test to OECD TG 442E. Statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison with the negative control in two of the three runs performed in the KerationSens test. The evaluation criteria for a positive response were met and the final study outcome was therefore considered to be positive. Two independent runs were performed with the solvent DMSO in the h-CLAT assay. In both assays the Relative Fluorescence Index for CD54 expression was exceeding the threshold at several test concentrations, whereas the RFI for CD86 expression was only exceeded at one test concentration in the second experiment. Based on the results, the substances was considered to be positive in the h-CLAT test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

On the basis of the available in chemico and in vitro test data, the substance is considered to have a skin sensitising potential. It is therefore proposed to classify the substance for skin sensitisation, Category 1, under Regulation (EC) No. 1272/2008.