Registration Dossier

Administrative data

Description of key information

- In vitro KeratinoSens assay: negative ( OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method) and GLP compliant study)

- In vitro h-CLAT assay: positive ( OECD guideline No. 442E (In vitro skin sensitization: human Cell Line Activation Test (h-CLAT) and GLP compliant study)

- In vivo LLNA: negative (OECD guideline No. 429 and GLP compliant study)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-21 - 2018-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
The test item was first tested in two independent runs using cells from a different passage number. A third run was performed since non-analyzable results were obtained in the first run due to the high cytotoxicity of the test item. The plates were processed as described below in the § Method.

- 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). Magnetic stirring for 10 minutes was used in order to improve the solubility of the test item.

- Method for a run of KeratinoSens assay
* 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.
* 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.
* Endpoint measurements
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:
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.

Acceptance criteria :
Each run was considered valid if the following criteria were met:
. 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 three 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%.

Evaluation criteria of the test item:
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
. the Imax is > 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 is < 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).







Positive control results:
All acceptance criteria were fulfilled for the positive control in each run.
Key result
Parameter:
other: IC50
Run / experiment:
First run
Value:
2.24
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable.
Key result
Parameter:
other: Imax
Run / experiment:
Second and third run
Value:
< 1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assay when tested up to 2.5 μM, highest concentration limited by the high cytotoxicity of the test item.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no data

DEMONSTRATION OF TECHNICAL PROFICIENCY: no data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

SOLUBILITY TEST

In the solubility test, the test item was found not soluble in DMSO at 200µM,whereas it was found soluble in water for injections at 200µM, following a 10-minute of magnetic stirring step. Therefore, water for injections was the vehicle selected for the preparation of the test item formulations.

KERATINOSENS RUN

 First run

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          slight to strongprecipitate were observed in test item-treated wells atconcentrations≥ 62.5 µMat the end of the 48-hour treatment period,

.          a high decrease in cell viability (i.e.cell viability < 70%) was noted from the lowest concentration of 0.98 µM. The corresponding IC30was < 0.98 µM and the IC50was calculated to be 2.24 µM.

 

Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable.

Second run

Due to the high cytotoxicity observed in the first run, a lower range of concentrations was used in the second run in order to determine if a potential gene induction occurred at non cytotoxic concentration. This run was therefore performedusing the following concentrations:0.001, 0.002, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25 and 2.50 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥0.63 µM, andthe corresponding IC30wascalculated to be 0.40 µM.No IC50was calculated since the cell viability was > 50% in this run,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imaxvalue was<1.5 (i.e.1.20).

 

The evaluation criteria for a negative response were met in this second run.

 Third run

Due to results not analyzable obtained in the first run and since two concordant runs are necessary to evaluate the potential of the test itemtoactivate the Nrf2 transcription factor, a third run was performed in the same experimental conditions and using the same concentrations as those tested in the second run.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥1.25 µM, andthe corresponding IC30was 0.62µM. Again, no IC50was calculated since the cell viability was > 50% in this third run,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imaxvalue was again<1.5 (i.e. 1.00).

 

The evaluation criteria for a negative response were also met in this third run.

Thegeometric mean IC30of the two analyzable runs was calculated to be 0.50 µM.

 

The evaluation criteria for a negative response were met in both analyzable 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 sensitization potential.

 

 

Interpretation of results:
other: negative
Conclusions:
The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assay when tested up to 2.5 µM, highest concentration limited by the high cytotoxicity of the test item. Therefore, under the experimental conditions of this study, the test item SOPROMINE 1686 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, SOPROMINE 1686, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment. The design of this study was based on the guideline No. 442D and the study was performedin compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice. The KeratinoSens is an in vitro test which quantifies luciferase gene induction in an immortalized adherent human keratinocyte cell line (HaCAT cell line) as a measure of the activation of the Keap1-Nrf2-ARE pathway.

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 runs were performed as part of the study.

All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered as validated.

 

First run

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          slight to strongprecipitate were observed in test item-treated wells atconcentrations≥62.5 µMat the end of the 48-hour treatment period,

.          a high decrease in cell viability (i.e.cell viability < 70%) was noted from the lowest concentration of 0.98 µM. The corresponding IC30was < 0.98 µM and the IC50was calculated to be 2.24 µM.

 

Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable and two additional runs were necessaryto evaluate the potential of the test item toactivate the Nrf2 transcription factor.

 

Second and third run

Due to the high cytotoxicity observed in the first run, a lower range of concentrations was used in the second and third runs in order to determine if a potential gene induction occurred at non cytotoxic concentration. These runs were therefore performedusing the following concentrations:0.001, 0.002, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25 and 2.50 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations and in either run,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥0.63 µM in the second run and≥1.25 µM in the third run, andthe corresponding IC30values werecalculated to be 0.40 µM and0.62µM, in the second and third runs, respectively.No IC50was calculated since the cell viability was > 50% in these runs,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in any of the runs. Moreover, the Imaxvalue was<1.5 (i.e.1.20 and 1.00 in the second and third runs, respectively).

 

The evaluation criteria for a negative response were met in these two analyzable runs and thegeometric mean IC30was calculated to be 0.50 µM.Therefore, the final outcome is 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 sensitization potential.

The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assaywhen tested up to 2.5 µM, highest concentration limited by the high cytotoxicity of the test item. Therefore, under the experimental conditions of this study, the test item SOPROMINE 1686 was considered to have no potential to activate the Nrf2 transcription factor.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-22 - 2018-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)", 09 October 2017.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
other: THE HUMAN-CELL LINE ACTIVATION TEST (h-CLAT)
Details on study design:
A solubility assessment was first performed in vehicles among 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations.
Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding (DRF) assay in order to select sub-toxic concentrations for testing in the main test.

Dose-range finding assay (DRF)
The DRF consisted in two separated assays. Treatments of DRF assays were performed at the following concentrations: 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 μg/mL.
Each assay was performed as described here below.
Test item stock solutions were prepared at eight different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 50-fold (as 0.9%NaCl 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 0.625 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 tests
The main test consisted in three separated runs being performed as described here below.
Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75 was obtained. The
maximum concentration in the plates was 74.42 μg/mL. All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions. In parallel, the working solutions of 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.
During the main test, treatments were performed at the following concentrations (final concentrations): 20.77, 24.92, 29.91, 35.89, 43.07, 51.68, 62.02 and 74.42 μg/mL.
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-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 (except for run B). 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.

FLOW CYTOMETRY ANALYSIS
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 were acquired (corresponding to an acquisition time of 2 minutes).
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 were 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.

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).
Positive control results:
All acceptance criteria were fulfilled for the positive control in each run.
Key result
Parameter:
other: RFI for CD86
Run / experiment:
Runs A, B, C
Value:
< 200
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54
Run / experiment:
Runs A, B, C
Value:
> 200
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: As the test item led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs, the test item was considered positive in the h-CLAT assay.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no data

DEMONSTRATION OF TECHNICAL PROFICIENCY: no data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Solubility assessment

The test item was found soluble in 0.9% NaCl at the concentration of 100 mg/mL.

Dose-Range Finding

The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 62.02 μg/mL. The highest concentration tested in the main test was therefore 74.42 μg/mL (i.e. 1.2-fold the mean CV75).

Summary results

All controls and test item acceptance criteria were reached in each run.

Run A:

. Post-treatment observations: precipitate was noted in wells treated at concentrations ≥ 51.68 μg/mL,

. Run outcome:

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

- RFI CD54 exceeded the positivity threshold (200) at the concentrations of ≥ 43.07 μg/mL,

- the run was therefore considered positive for RFI CD54.

Run B:

. Post-treatment observations: precipitate was noted in wells treated at concentrations ≥ 51.68 μg/mL,

. Run outcome:

- RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentrations.

- The run was therefore considered negative.

Run C:

. Post-treatment observations: precipitate was noted in wells treated at ≥ 43.07 μg/mL,

. Run outcome:

- RFI CD86 did not exceed the positivity thresholds at any tested concentrations,

- RFI CD54 exceeded the positivity threshold at concentrations ≥ 51.68 μg/mL,

- the run was therefore considered positive for RFI CD54.

As the test item led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs, the test item was considered positive in the h-CLAT assay.

Interpretation of results:
other: positive
Conclusions:
Under the experimental conditions of this study, the test item, SOPROMINE 1686, led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs. The test item SOPROMINE 1686 was therefore considered positive in the h-CLAT assay.
Executive summary:

The objective of the study was to determine the ability of the test item, SOPROMINE 1686, to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method. The design of

this study was based on the guideline No. 442E and the study was performed in compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

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

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding (DRF) assay 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 three successive runs (A, B and C).

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).

Solubility assessment

The test item was found soluble in 0.9% NaCl at the concentration of 100 mg/mL.

Dose-Range Finding

The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 62.02 μg/mL. The highest concentration tested in the main test was therefore 74.42 μg/mL

(i.e. 1.2-fold the mean CV75).

Summary results

All controls and test item acceptance criteria were reached in each run.

Run A:

. Post-treatment observations: precipitate was noted in wells treated at concentrations ≥ 51.68 μg/mL,

. Run outcome:

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

- RFI CD54 exceeded the positivity threshold (200) at the concentrations of ≥ 43.07 μg/mL,

- the run was therefore considered positive for RFI CD54.

Run B:

. Post-treatment observations: precipitate was noted in wells treated at concentrations ≥ 51.68 μg/mL,

. Run outcome:

- RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentrations.

- The run was therefore considered negative.

Run C:

. Post-treatment observations: precipitate was noted in wells treated at ≥ 43.07 μg/mL,

. Run outcome:

- RFI CD86 did not exceed the positivity thresholds at any tested concentrations,

- RFI CD54 exceeded the positivity threshold at concentrations ≥ 51.68 μg/mL,

- the run was therefore considered positive for RFI CD54.

As the test item led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs, the test item was considered positive in the h-CLAT assay.

Under the experimental conditions of this study, the test item, SOPROMINE 1686, led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs. The test item SOPROMINE 1686 was therefore considered positive in the h-CLAT assay.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 May to 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 20.0 – 21.8 grams
- Housing: Group caging / mice were provided with glass tunnel-tubes - Cage type: Type II. polypropylene / polycarbonate
- Diet (e.g. ad libitum): Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable "Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 883 29966, expiry date: 31 October 2018, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel Diet Transport (Batch Number: 60180640040101, Expiry date: 05 March 2019, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 – 24.9°C
- Humidity (%): 32 - 73 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 09 May 2018 (preliminary experiment) To: 12 June 2018
Vehicle:
other: 1% aqueous Pluronic® PE9200 solution
Concentration:
2.5, 5 and 10% (in terms of active ingredient)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test items was examined in a short Preliminary Compatibility Test. The following standard OECD No. 429 vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture (abbreviated as AOO), N,N-dimethylformamide, Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide, and 1% aqueous Pluronic® PE9200 solution (abbreviated as 1% Pluronic). The undiluted creamy test item was not adequate for treatment. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be 1% Pluronic.
- The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 10 and 5 % (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. Based on the observation of the solubility test, the maximum available concentration was 10 % (w/v). The test item was supplied at 25.1%, hence higher concentrations were not practicable. The 25% (w/v) could not be used in the study due to the characteristic of the test item. The next concentration according to the OECD guidelines was 10 % (w/v).
- Irritation: no indications of any irritancy at the site of application
- Systemic toxicity: no systemic toxicity reported
- Ear thickness measurements: The ear thickness values and ear punch weights were within the acceptable range.
- Erythema scores: 0

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The test item is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
- Formulation:
The 10% (w/v) formulation (i.e. 40% (w/v) test item formulation) was the highest concentration which was suitable for the test. No vehicles produced what appeared to be a clear solution at concentrations of 10%.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of Citoxlab Hungary Ltd.

During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics performed.
Positive control results:
The positive control substance was examined at a concentration of 25 % in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 4.4) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Key result
Parameter:
SI
Value:
2.5
Test group / Remarks:
Sopromine 1686 2.5 % (w/v)
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
Sopromine 1686 5 % (w/v)
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
Sopromine 1686 10 % (w/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The appearance of the lymph nodes was normal in the negative control group and in the treatment groups of 10, 5 and 2.5 % (w/v) test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group (see details in table 1)

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index values were 1.8, 1.6 and 2.5 at concentrations of 10, 5 and 2.5 % (w/v) respectively (see details in table 1)
The test item was supplied as creamy white liquid at 25.1 % (w/v) in water, which was formulated in 1% Pluronic. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that Sopromine 1686 is a non-sensitiser. The size of lymph nodes was in good correlation with this conclusion.

EC3 CALCULATION: Not applicable (SI < 3 at all doses)

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. A minimal amount of test item residue was observed on the ears in all 4/4 animals in the 10% (w/v) dose group on Day 3. There were no signs of local irritation observed.
Quantification of the ear thickness was performed by ear punch weight determination. Biopsy weights were within the historical control range.


BODY WEIGHTS
Significant body weight loss (≥5%) was observed only in one animal in the 10% (w/v) group in the main study. Body weight losses were observed in each treatment group, however these felt into the acceptable range (<5%) (see details in table 2).

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM / group

DPM

Number
of lymph nodes

DPN

Stimulation Index

Background

(5 % (w/v) TCA)

35

-

-

-

-

Negative control

(1% Pluronic)

1836

1801.0

8

225.1

1.0

Sopromine 1686

10 % (w/v)
in
1% Pluronic

3306

3271.0

8

408.9

1.8

Sopromine 1686

5 % (w/v)
in
1% Pluronic

2838

2803.0

8

350.4

1.6

Sopromine 1686

2.5 % (w/v)
in
1% Pluronic

4595

4560.0

8

570.0

2.5

Positive control

(25 % (w/v) HCA
in1% Pluronic)

8011

7976.0

8

997.0

4.4

The stimulation index values were 1.8, 1.6 and 2.5 at concentrations of 10, 5 and 2.5 % (w/v) respectively.

The DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range (reported below in table 3).

Table 2:Individual Body Weights for all Animals with Group Means

Animal

Number

Identity

Number

Test Group

Name

Initial Body

Weight (g)

Terminal Body

Weight* (g)

Change#

(%)

6240

1

Negative (vehicle) control

21.2

20.4

-3.8

6244

2

in 1% Pluronic

21.4

20.7

-3.3

6252

3

 

20.3

20.0

-1.5

6249

4

 

20.5

21.2

3.4

 

 

Mean

20.9

20.6

-1.3

6243

5

Sopromine 1686

21.4

20.2

-5.6

6253

6

10 % (w/v)

21.3

20.5

-3.8

6247

7

in 1% Pluronic

20.3

20.6

1.5

6256

8

 

20.5

20.6

0.5

 

 

Mean

20.9

20.5

-1.9

6259

9

Sopromine 1686

21.3

21.8

2.3

6241

10

5 % (w/v)

21.7

20.8

-4.1

6246

11

in 1% Pluronic

20.5

21.0

2.4

6258

12

 

20.4

20.3

-0.5

 

 

Mean

21.0

21.0

0.0

6251

13

Sopromine 1686

21.7

20.7

-4.6

6254

14

2.5 % (w/v)

21.2

22.1

4.2

6242

15

in 1% Pluronic

20.3

20.5

1.0

6257

16

 

20.4

19.9

-2.5

 

 

Mean

20.9

20.8

-0.5

6248

17

Positive control

21.8

21.3

-2.3

6255

18

25 % (w/v) HCA

21.1

20.9

-0.9

6250

19

in 1% Pluronic

20.1

20.3

1.0

6245

20

 

20.0

20.1

0.5

 

 

Mean

20.8

20.7

-0.5

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Table 3:Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2018)

 

Vehicle

 

1% Pluronic PE9200 in water (1%Plu)

 

 

DPN values

SI value

 

 

Control

HCA 25%

HCA 25%

 

Average

198.7

1988.1

11.2

 

Range:min

23.0

154.0

3.0

 

max

680.8

6755.8

33.6

 

Number of cases

234

226

218

 

 

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay, Sopromine 1686, supplied as an aqueous mixture at 25.1 %, tested in a suitable vehicle, was shown to have a no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.
Executive summary:

The objective of this study was to determine the skin sensitisation potential of the test item, Sopromine 1686, following dermal exposure. The study was performed with vertebrate animals as the applied regulatory in vitro alternative tests showed inconclusive/equivocal results (see in vitro tests reported in section 7.4.1). Therefore, an in vivo study was requested to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance. The design of this study was based on the OECD No. 429 guideline and the study was performed in compliance with the OECD Principles of Good Laboratory Practice.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline [1], the best vehicle for the test item was1% aqueous Pluronic® PE9200 solution (abbreviated as 1% Pluronic). Sopromine 1686 being supplied as an aqueous mixture at 25.1 %, a correction factor of 3.98 during the dose formulation was applied. The 10% (w/v) formulation (i.e. 40% (w/v) test item formulation as received) was the highest concentration which was suitable for the test. No vehicles produced what appeared to be a clear solution at concentrations of 10%.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 10 and 5% (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary test, 10% (w/v)was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

-   three groups received Sopromine 1686(formulated in1% Pluronic)at 10, 5,
and 2.5 % (w/v) concentrations respectively,

-   the negative control group received the vehicle (1% Pluronic) only,

-   the positive control group received 25 % (w/v) HCA (dissolved in1% Pluronic).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or signs of systemic toxicity were observed during the study. Minimal amount of test item residue was observed on the ears in all 4/4 animals in the 10% (w/v) dose group on Day 3. Significant body weight loss (≥5%) was observed only in one animal in the 10% (w/v) group in the main study. Body weight losses were observed in each treatment group, however these felt into the acceptable range (<5%).

 

Quantification of the ear thickness was performed by ear punch weight determination. Biopsy weights were within the historical control range.

  

The stimulation index values were 1.8, 1.6 and 2.5 at concentrations of 10, 5 and 2.5 % (w/v), respectively.

 

The result of the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, Sopromine 1686, supplied as an aqueous mixture at 25.1 %, tested in a suitable vehicle, was shown to have a no sensitisation potential (non-sensitiser) in the Local Lymph Node Assay.

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: no category.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

As specified in Annex VII of Reach regulation, paragraph 8.3.1, column 1, in vitro assays were first performed to assess the sensitizing potential of the registered substance. Only KeratinoSens and h-CLAT assays were applicable for the testing of this substance. DPRA could not be performed since it was not applicable to the complexe composition of the substance.

- A GLP-compliant in vitro Skin Sensitisation test (KeratinoSens assay) conducted according to OECD TG 442D (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (CIToxLAB, 2018): The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assay when tested up to 2.5 µM, highest concentration limited by the high cytotoxicity of the test item. Therefore, under the experimental conditions of this study, the test item SOPROMINE 1686 was considered to have no potential to activate the Nrf2 transcription factor.

- A GLP-compliant in vitro Skin Sensitisation test (h-CLAT) conducted according to OECD TG 442E (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (CIToxLAB, 2018): The test item SOPROMINE 1686 led to an increase in CD54 expression above the positivity threshold of 200 in 2 out of 3 consecutive runs. The test item SOPROMINE 1686 was therefore considered positive in the h-CLAT assay.

Based on the results of the KeratinoSens and h-CLAT assays, it was not possible to conclude on the sensitising potential of the registered substance. Consequently, as recommended in Annex VII of Reach regulation, paragraph 8.3.2, columns 1 and 2, an in vivo assay was conducted i.e a LLNA in mice:

A GLP-compliant in vivo LLNA conducted according to OECD TG 429 (of reliability 1 according to Klimisch cotation criteria) is available and was selected as key study (CIToxLAB, 2018): The stimulation index (SI) values were 1.8, 1.6 and 2.5 at concentrations of 10, 5 and 2.5 % (w/v), respectively. Since the SI was below the threshold of 3, it was concluded that the registered substance has no sensitisation potential (non-sensitizer).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Following unconclusive results of two in vitro assays (OECD 442D and OECD 442E), a LLNA (OECD 429) conducted on the registered substance concluded to stimulation index values below 3 at all tested concentrations indicating that the substance has no sensitisation potential.

In conclusion the registered substance is not classified for skin sensitization according to CLP and GHS-UN regulations.