Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

Endpoint summary

Description of key information

The test item was found positive in the DPRA and the h-CLAT assays and negative in the KeratinoSens assay. Since 2 out of 3 in vitro/in chemico assays were positive, the registered substance is considered sensitizing to skin and classified Skin Sens. Cat. 1 according to CLP and GHS classification criteria.

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

Study period:

From 16 April to 20 August 2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

- Appearance: Clear paste
- Storage condition: At room temperature
- Purity: 99.7%
- Correction factor: No correction factor

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

1. TEST AND CONTROL ITEMS
1.1) Vehicle
Based on solubility results, the retained vehicle was milli-Q water. A sonication of 1 minute was used to improve solubilisation.

1.2) Positive control
The positive control was cinnamaldehyde (CAS No. 104-55-2), batch No. MKBX8146V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 99.1%.
As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was a colorless liquid and was used just after its preparation.

1.3) Co-elution control samples
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were p
repared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

1.4) Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent specified in § Reference control samples preparation.
These samples were used to:
- reference control A: check the accuracy of the calibration curve for peptide quantification,
- reference control B: check the stability of the peptide during analysis,
- reference control C: check that the solvent did not impact the percentage of peptide depletion.

1.5) Test item formulation preparation
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (milli-Q water) at 100 mM. The formulation was sonicated for 1 minute. This formulation was an opaque white solution and was used just after its preparation.

1.7) Additional samples to test the neat test item
Since the test item is a mixture, the reactivity of neat test item was evaluated. Neat test item was dissolved to its maximum soluble concentration in the same vehicle used to prepare the apparent 100 mM solution. Then, it was tested as such without any further dilution by incubating it at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively.


2. DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.

2.1) Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

2.1.1) Co-elution control samples preparation
- For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
- For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

2.1.2) Reference control samples preparation
2.1.2.1) Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

2.1.2.2) Reference control C samples
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
- For the reference control C prepared with cysteine peptide: 50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- For the reference control C prepared with lysine peptide: In parallel, 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

2.1.2.3) Cinnamaldehyde (positive control) depletion control samples preparation
- For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- For the reactivity of cinnamaldehyde with lysine peptide:- In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.1.3) Test item samples preparation
- For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
- For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated

2.1.4) Preparation of additional test item neat samples
- For reactivity of the test item with cysteine peptide: 50 µL of test item formulation prepared at the maximum soluble concentration was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- For reactivity of the test item with lysine peptide: In parallel, 250 µL of test item formulation prepared at the maximum soluble concentration was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.2) Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation (see § Results).
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system


2.3) Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also in cluded in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

2.4) HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
- one blank sample (peptide dilution buffer),
- one calibration curve injected at the beginning of the analytical batch,
- three reference control A samples,
- the co-elution control sample,
- three reference control B samples,
- reference control C sample (replicate 1),
- positive control sample (replicate 1),
- test item study sample (replicate 1),
The injection order of the reference control C, positive control and all test item study samples were reproduced identically for replicate 2 and then replicate 3:
- three reference control B samples.
- additional neat co-elution control sample for the neat test item,
- additional neat test item samples (3 replicates),
- three reference control B samples.

The HPLC/UV method used for the samples analysis is described in CiToxLAB France internal analytical method.

Positive control results:

The mean of the percent cysteine and percent lysine depletions of Cinnamaldehyde was 75.60% which corresponds to Hight reactivity.

Key result

Run / experiment:

other: at 100 mM in milli-Q water with sonication.

Parameter:

other: mean of the percent cysteine and percent lysine depletions

Value:

13.79

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: test item neat

Parameter:

other: mean of the percent cysteine and percent lysine depletions

Value:

21.07

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

SOLUBILITY RESULTS

Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile and in 1:1 mixture of acetonitrile:milli-Q water even after 1 minute of sonication. A solution was obtained at 100 mM with milli-Q water after 1 minute of sonication. Therefore, the vehicle retained was milli-Q water.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis. As precipitate was observed in the positive samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Only supernatants were then injected into the HPLC/UV system. For the other samples, the vials were directly transferred into the HPLC/UV system.

EVALUATION OF THE RESULTS

1) at 100 mM in milli-Q water with sonication.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 23.59%,

- for the lysine peptide, the mean depletion value was 3.98%.

The mean of the percent cysteine and percent lysine depletions was equal to 13.79%. Accordingly, the test item was considered to have low peptide reactivity.

2) test item neat

The test item was also tested neat . The mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 35.61%,

- for the lysine peptide, the mean depletion value was 6.53%.

The mean of the percent cysteine and percent lysine depletions with the test item neat was equal to 21.07%.

These results confirm those obtained with test item prepared at 100 mM (low peptide reactivity). Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

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 sensitization potential of the test item

Interpretation of results:

other: the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Conclusions:

Under the experimental conditions of this study, the test item Miranol C2M AA, was considered to have low peptide reactivity. The test item is considered positive in the DPRA assay.

Executive summary:

The reactivity of the test item (Miranol C2M AA) to synthetic cysteine and lysine peptides was evaluated was based on the OECD Guideline 442C and the study was performed in compliance with the OECD Principles of Good Laboratory Practice.

This test is part of a tiered strategy for skin sensitization assessment.

 

Method:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nM. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent)

 

Results:

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

The test item was dissolved at 100 mM in milli-Q water with sonication.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide

- for the cysteine peptide, the mean depletion value was 23.59%,

- for the lysine peptide, the mean depletion value was 3.98%.

The mean of the percent cysteine and percent lysine depletions was equal to 13.79%. Accordingly, the test item was considered to have low peptide reactivity.

The test item was also tested neat. The mean percent depletion values were calculated for each peptide

- for the cysteine peptide, the mean depletion value was 35.61%,

- for the lysine peptide, the mean depletion value was 6.53%.

The mean of the percent cysteine and percent lysine depletions with the test item neat was equal to 21.07%. These results confirm those obtained with test item prepared at 100 mM (low peptide reactivity). Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

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 sensitization potential of the test item.

 

Conclusion:

Under the experimental conditions of this study, the test item, Miranol C2M AA, was considered to have a low peptide reactivity. The test item is considered positive in the DPRA assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 March to 01 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In vitro skin sensitization assays addressing the Key Event on activation of dendritic cells on the adverse outcome pathway for Skin sensitization)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Quantification of the changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical.

Specific details on test material used for the study:

- Appearance: Clear paste
- Storage condition: At room temperature
- Purity: 99.7%
- Correction factor: No correction factor

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
1. SOLUBILITY ASSESSMENT
The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc) and recorded in the study files. A preparation was deemed appropriate for cell treatment as long as it was qualified as a solution or stable dispersion (homogenous emulsion/suspension). As the test item was a surfactant, saline solution (0.9% NaCl) was the only possible vehicle to be used. The test item was first dissolved at 100 mg/mL. If the test item was not soluble at 100 mg/mL in 0.9% NaCl, the Highest Soluble Concentration (HSC) was determined by testing lower concentrations such as 50 mg/mL, 25 mg/mL, 10 mg/mL and 1 mg/mL (i.e. minimal possible concentration). If the test item was soluble or gave a stable dispersion (homogenous emulsion/suspension) the HSC was determined by testing the concentration of 500 mg/mL (and 300 mg/mL if no solution/stable dispersion obtained at 500 mg/mL). Vortexing, sonication or heating were used to help solubilize the test item in vehicle, as the Sponsor did not mention that such process can affect the test item stability. Once a solution was obtained in 0.9% NaCl, the formulation was then 100-fold diluted in RPMI-1640 culture medium. Then a visual inspection was performed immediately after dilution as well as after an overnight period. The presence or absence of precipitate/emulsion was recorded in the study files.

2. TEST ITEM AND CONTROLS PREPARATION
2.1 Positive controls 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 200 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 10 mg/mL,
- this solution was then 50-fold diluted in cRPMI in order to obtain a 200 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.

2.2 Vehicle control
Since 0.9%NaCl was the retained vehicle (i.e. aqueous vehicle), test item formulations data was compared to data obtained from cells treated with culture media and therefore cRPMI was considered as the vehicle control.

2.3 Test item preparation
All test item preparations were prepared in glass vials only. Test concentrations prepared and vehicle used were indicated by the Study Director in the study files, and no study plan amendment was issued for these purposes. Fresh stock formulations of the test item were prepared for each run, using the vehicle and concentration identified in the § Solubility assessment. These concentrations were the same for all runs.
Test item stock formulations prepared in 0.9% NaCl were 100 x concentrated, and then 2 x concentrated formulations were prepared by 1:50 dilution in cRPMI. 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 and any observation was reported in the study files. The test item formulations were kept at room temperature and protected from light until use, i.e. within 4 hours after preparation of the stock formulations. No control of concentration was performed during the study.

3 TEST SYSTEM
3.1 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 (100 units/mL) and streptomycin (100 µg/mL). During cell culturing, cell viability was checked using trypan blue.

3.2 Cell culture for testing
For testing, THP-1 cells were seeded at a density between 0.1 x 106 cells/mL and 0.2 x 106 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cell did not exceed density of 1 x 106 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. Then, 500 µL of cells suspension were distributed into a 24 well flat bottom plate (i.e. 1 x 106 cells/well).

3.3 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 Citoxlab France reference number. Results from reactivity check tests are compiled in Citoxlab France internal data, with negative and positive control data obtained during each test.

4. STUDY DESIGN
The study was divided in two successive phases. First, Dose-Range Finding assays (DRF) were performed to assess test item toxicity and, determine the CV75 (i.e. the test item concentration that result in 75% cell viability compared to the vehicle control). Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in two successive runs in the main test in order to evaluate the CD86 and CD54 expression.

4.1 Dose-Range Finding assay (DRF)
The DRF consisted in two separated assays, for which the treatments were performed at the following concentrations (final concentrations in wells): 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.
Each assay was performed as described here below. Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. Since this selected vehicle was 0.9%NaCl, the stock formulations were then diluted 50-fold 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, an inspection under microscope was performed to each well and the presence or absence of precipitate/emulsion was recorded in the study files. Cells were then 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.

4.2 Main test
The main test consisted in two separated runs. During these two runs, treatments were performed at the following concentrations (final concentrations in wells): 8.61, 10.33, 12.40, 14.88, 17.85, 21.42, 25.71 and 30.85 µg/mL.
Each run was performed as described here below. Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle (i.e. NaCl 0.9%). The highest concentration corresponded to 1.2-fold the mean CV75 (i.e. a maximal concentration in the wells of 30.85 µg/mL). All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of both positive controls (DNCB and NiSO4) were prepared as noted in § Test item and controls preparation.
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, an inspection under microscope was performed to each well and the presence or absence of precipitate/emulsion was recorded in the study files. Cells were then 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-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.
Finally, cells were washed with 150 µL FACS buffer 2 times and re-suspended 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.

5. FLOW CYTOMETRY ANALYSIS
5.1 DRF assays
The PI uptake is analyzed using flow cytometry with the acquisition channel B3. A total of 10 000 living cells (PI negative) are acquired. In case of low viability which does not allow obtaining 10 000 living cells, a maximum of 100 µL per sample was acquired (corresponding to an acquisition time of 2 minutes).

5.2 Main test
The non-specific binding of IgG1 and the expression CD86 and CD54 was analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10 000 living cells, a maximum of 100 µL per sample was acquired (corresponding to an acquisition time of 2 minutes). In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

Positive control results:

All acceptance criteria were reached in both runs (see summary results in attachement). Tstudy was therefore considered to be valid.

Key result

Run / experiment:

other: A

Parameter:

other: RFI for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative

Key result

Run / experiment:

other: A

Parameter:

other: RFI for CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive

Key result

Run / experiment:

other: B

Parameter:

other: RFI for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative

Key result

Run / experiment:

other: B

Parameter:

other: CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive

Other effects / acceptance of results:

All acceptance criteria were reached in both runs (see summary results in attachement. Tstudy was therefore considered to be valid.

SOLUBILITY RESULTS:

Since a homogeneous suspension was observed in the test item formulation at 25 mg/mL in 0.9% NaCl after 5 minutes of sonication, it was then decided to evaluate the effect of sonication on a test item formulation at 100 mg/mL. As a result, an additional solubility assessment was performed

After vortexing and 5 minutes of sonication a yellowish homogenous solution was obtained. Therefore, 100 mg/mL in 0.9% NaCl was retained as the highest stock concentration to be used (after vortex and 5 minutes of sonication) and the following test item concentrations were tested in the DRF phases:7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL (final concentrations).

 DRF RESULTS:

The following results were obtained in both DRF assays:

.            at post-treatment observation, no precipitate/emulsion were observed at any tested concentrations. However a low cell density was noted at concentrations≥250 µg/mL and≥125 µg/mL in the DRF 1 and DRF2, respectively,

.            test item interacted with Propidium Iodide (PI), making concentrations of 500 and 1000 µg/mL unusable. These concentrations were therefore excluded from the analysis,

.            flow cytometry measurement after PI staining revealed a cell viability decrease below 75% at concentrations≥31.25 µg/mL. The corresponding CV75 values were 25.70 µg/mL and 25.71 µg/mL in the DRF1 and DRF2, respectively.

 

Based on the results from both DRF runs, the mean CV75 was 25.71 µg/mL, and the highest concentration tested in the main test was therefore 30.85 µg/mL (i.e.1.2-fold the mean CV75).

MAIN TEST (see detailed results and summary in attachement):

Both runs were performed using the followingconcentrations (final concentrations in wells): 8.61, 10.33, 12.40, 14.88, 17.85, 21.42, 25.71 and 30.85 µg/mL.

 At these tested concentrations and for both runs:

.            at post-treatment observation,no precipitate/emulsion andno cell morphology modificationwere noted in treated wells,at any tested concentrations,

.            RFI CD86 did not exceed the positivity thresholdat any tested concentrations,

.            RFI CD54 reached or exceeded the positivity threshold at the concentrations≥25.71µg/mLand≥ 12.4µg/mLin the runs A and B, respectively.

Both runs were therefore considered positive.

Interpretation of results:

other: the test item Miranol C2M AA was found to be positive in the h-CLAT test method thus it may have potential to cause skin sensitization.

Conclusions:

Under the experimental conditions of this study, the test item Miranol C2M AA was found to be positive in the h-CLAT test method.

Executive summary:

The objective of the study was to determine the ability of the test item, Miranol C2M AA, to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

Methods:

A solubility assessment was first performed in 0.9% NaCl to select the highest concentration to be used for test item formulation preparations.

Following the solubility assays, the cytotoxic potential was assessed in Dose-Range Finding assays in order to select sub-toxic concentrations for testing in the main test.

The skin sensitizing potential of the test item was then tested in the main test in two successive runs.

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 Fluorescence 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).

Results:

Solubility assessment: The test item was found soluble at 100 mg/mL in 0.9% NaCl, after vortexing and sonication.

Dose-Range Finding: During both DRF assays, the test item induced a decrease in cell viability < 75% and the calculated mean CV75 value was 25.71 μg/mL. The highest concentration tested in the main test was therefore 30.85 μg/mL (i.e. 1.2-fold the mean CV75).

All acceptance criteria were reached in both runs. The study was therefore considered to be valid.

For both runs:

- RFI CD86 did not exceed the positivity threshold (≥ 150) at any tested concentrations,

- RFI CD54 reached or exceeded the positivity threshold (≥ 200) with cell viability ≥ 50% at the concentrations ≥ 25.71 μg/mL and ≥ 12.4 μg/mL in the runs A and B, respectively.

Both runs were therefore considered positive.

 

Conclusion:

Under the experimental conditions of this study, the test item Miranol C2M AA was found to be positive in the h-CLAT test method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 May 2018l to 12 April 2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

- Appearance: Clear paste
- Storage condition: At room temperature
- Purity: 99.7%
- Correction factor: No correction factor

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
1) TEST SYSTEM
. KeratinoSens cells: 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. (Supplier: this cell line was provided by Givaudan; Batch: D1; Storage condition: at -80°C; Absence of mycoplasm was confirmed)

2) VEHICLE, CONTROLS, FORMULATION
2.1) Vehicle
Based on solubility results, the vehicle was water for injections.

2.2) Controls
2.2.1) Negative control
DMSO was selected as negative control was applied to cells at a concentration of 1% in culture medium. Since several test items were assayed concurrently, the results of the negative control were shared.
2.2.2) Positive control
. Name : Cinnamic Aldehyde (CA)
. Synonym : trans-Cinnamaldehyde
. CAS number : 14371-10-9
. Storage conditions : at +4°C and under nitrogen gas
Since several test items were assayed concurrently, the results of the positive control were shared.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. 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.

2.3) Test item formulations
On the basis of solubility results, the test item was suspended in water for injections at 200 mM. After preparation, the formulations were sterilized by filtration through a filter prior treatment. One formulation was prepared for each run. It was then diluted in water for injections serial dilutions, using a dilution factor of 2 for the first run and a dilution factor of 1.41 for the second run, 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.

3) STUDY DESIGN
The test item was tested in two independent runs using cells from a different passage number.
3.1) Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).

3.2) Method for a run of KeratinoSens assay
3.2.1) 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 104 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 104 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.
3.2.2) 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.
3.2.3) Test itemp concentrations
. The first run was performed using the following 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 and 1% water.
. the second run was performed using the following concentrations: 5.71, 8.05, 11.35, 16.00, 22.56, 31.81, 44.90, 63, 89, 126, 177 and 250 µM in culture medium containing 1% DMSO and 1% water.

3.2.3) Endpoint measurements
3.2.3.1) 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.
3.2.3.2) 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:
− 50 μL of the luciferase substrate was added to each well,
− 1 second after this addition, the luciferase signal was integrated for 2 seconds.
3.2.3.3) 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.

Positive control results:

First run:
The criterion “the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8” was not fulfilled (i.e. Imax of 9.65). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
Second run:
The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" was not fulfilled (i.e. Imax of 12.01). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.

Key result

Run / experiment:

other: Run 1

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations.

Key result

Run / experiment:

other: Run 2

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations.

Key result

Run / experiment:

other: Run 1

Parameter:

other: IC50

Remarks:

(µM)

Value:

103.56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Run 2

Parameter:

other: IC50

Remarks:

(µM)

Value:

28.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were fulfilled for the positive and negative controls in both runs (see details above in the field "Positive control results"). The runs were therefore considered to be valid.

SOLUBILITY RESULTS

In the solubility test, the test item was not found soluble in DMSO at 200 mM, whereas it was found soluble in water for injections at 200 mM. Therefore, this vehicle (water for injections) was selected for the preparation of the test item stock formulations

KERATINOSENS RUN

First run (see Figure 1):

This run was performed using the following 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 and 1% water. At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM,

. the corresponding IC30 and IC50 were calculated to be 55.80 and 103.56 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5.

Second run (see Figure 2):

This run was performed using the following concentrations: 5.71, 8.05, 11.35, 16.00, 22.56, 31.81, 44.90, 63, 89, 126, 177 and 250 μM in culture medium containing 1% DMSO and 1% water. At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 22.56 μM,

. the corresponding IC30 and IC50 were calculated to be 18.04 and 28.90 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5.

The geometric mean IC30 and IC50 of the two validated runs was calculated to be 31.72 and 54.71 μM, respectively.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. This negative 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.

Interpretation of results:

other: negative

Conclusions:

Under the experimental conditions of this study, the test item, Miranol C2M AA, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Miranol C2M AA, to activate the Nrf2 transcription factor. This study was based on the OECD Guideline 442D and was performed in compliance with the OECD Principles of Good Laboratory Practice.

This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Methods:

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. Two independent runs were performed.

Results:

For each run, the test item was suspended in water for injections at 200 mM.

- The first run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM. At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM,

. the corresponding IC30 and IC50 were calculated to be 55.80 and 103.56 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5.

- The second run was performed using the following concentrations: 5.71, 8.05, 11.35, 16.00, 22.56, 31.81, 44.90, 63, 89, 126, 177 and 250 μM. At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 22.56 μM,

. the corresponding IC30 and IC50 were calculated to be 18.04 and 28.90 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5.

The geometric mean IC30 and IC50 of the two validated runs was calculated to be 31.72 and 54.71 μM, respectively.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative.

In conclusion, under the experimental conditions of this study, the test item, Miranol C2M AA, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Three Klimish score 1 in vitro/in chemico studies are available: A Direct Peptide Reactivity Assay (DPRA) performed according to OECD Test guideline 442C, a KeratinoSens assay performed according to OECD Test guideline 442D and a human Cell Line Activation Test (h-CLAT)

performed according to OECD Test guideline 442E. The registered substance was found positive in the DPRA and the h-CLAT assays and negative in the KeratinoSens assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item was found positive in the DPRA and the h-CLAT assays and negative in the KeratinoSens assay. Since 2 out of 3 in vitro/in chemico assays were positive, the registered substance is considered sensitizing to skin and classified Skin Sens. Cat. 1 according to CLP and GHS classification criteria.