[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 06 to October 04, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

IUPAC Name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

Test material form:

solid

In vitro test system

Details on the study design:

The study was divided in two successive phases. First, a Dose-Range Finding assay (DRF) was 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 negative control). Secondly, based on cytotoxicity data obtained from the DRF assay, a concentration series was tested in two successive validated runs in the main test in order to evaluate the expression of CD86 and CD54. An additional run was performed in order to have a minimum of two concordant runs reaching significant cytotoxicity.

Dose-Range Finding assay (DRF)
The DRF consisted of two separated assays (i.e. DRF1 and DRF2), for which the treatments were performed at the following final concentrations: 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500 and 5000 µg/mL.
Each assay was performed as described below.
Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 50-fold into cRPMI to obtain working solutions.
The working solutions were finally used for treatment after 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 a light microscope was performed to each well. Then, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 µL of FACS buffer. Finally, cells were resuspended in 200 µL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 µL of Propidium Iodide (PI) solution at 12.5 µg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 µg/mL per well.

Main test
The main test consisted of seven separated runs, for which four were invalidated and three were validated.
Test item stock solutions were prepared at 8 concentrations by 1.2-fold dilutions (except for Runs B and C, for which a 2-fold dilution factor was applied) using the selected vehicle. The highest final concentration used in the Runs A, B and C corresponded to 1.2-fold the mean CV75, i.e. 968.17 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.
All working solutions were finally used for treatment after 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 final test item concentrations:
- Run A: 270.20, 324.24, 389.09, 466.90, 560.28, 672.34, 806.81 and 968.17 µg/mL,
- Runs B and C*: 7.56, 15.13, 30.26, 60.51, 121.02, 242.04, 484.09 and 968.17 µg/mL,
- Run D**: 70.03, 84.03, 100.84, 121.01, 145.21, 174.25, 209.10 and 250.92 µg/mL,
- Runs E, F and G***: 111.63, 133.96, 160.75, 192.90, 231.48, 277.78, 333.33 and 400 µg/mL.

*: Due to unexpected cytotoxicity obtained in the run A, a dilution factor of 2 was selected in order to achieve both cytotoxic and non-cytotoxic concentrations and therefore reach interpretable concentrations in further runs,
**: In order to fully comply with the OECD Test Guideline, the dilution factor of 1.2 has been used again. Based on previous runs B and C, a second CV75 value was calculated (i.e. 209.10 µg/mL) and used for the selection of concentrations to be used in the Run D (i.e. 250.92 µg/mL).
***: Since significant cytotoxicity was not obtained in the Run D compared to what obtained in the previous runs, a highest tested concentration of 400 µg/mL was selected to be used as the highest tested concentration. Then the dilution factor of 1.2 was still used.

At the end of the treatment phase, an inspection under a light microscope was performed to each well. Then, 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.
Finally, cells were washed with 150 µL FACS buffer twice 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
1.DRF assay
The PI uptake is analyzed by flow cytometry using 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).

2.Main test
The non-specific binding of IgG1 and the expression CD86 and CD54 was analyzed by flow cytometry using 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).
Where viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration is considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: C

Parameter:

other: RFI for CD86

Value:

105

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 95 %

Results 2

Run / experiment:

other: C

Parameter:

other: RFI for CD54

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 97 %

Results 3

Run / experiment:

other: D

Parameter:

other: RFI for CD86

Value:

106

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 93 %

Results 4

Run / experiment:

other: D

Parameter:

other: RFI for CD54

Value:

129

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 93 %

Results 5

Run / experiment:

other: G

Parameter:

other: RFI for CD86

Value:

101

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 82 %

Results 6

Run / experiment:

other: G

Parameter:

other: RFI for CD54

Value:

63

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability = 82 %

Any other information on results incl. tables

DRF RESULTS

The following test item concentrations were tested in the DRF assay (final concentrations in wells): 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.

At these tested concentrations, and in both DRF 1 and DRF2:

.           at post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification was observed at any tested concentrations,

.           flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations≥1250 µg/mL. The corresponding CV75 values were 926.29 µg/mL and 687.33 µg/mL in the DRF1 and DRF2, respectively.

Note: In the DRF1, due to unusual and atypical profile observed during flow cytometry analysis at 312.50 µg/mL, the corresponding viability result was considered not biologically relevant and was therefore not taken into account in the results analysis. 

Based on the results from both DRF runs, the mean CV75 was 806.81 µg/mL, and the highest concentration tested in the first main test was therefore 968.17 µg/mL (i.e.1.2-fold the mean CV75).

For the dose selection of the run D and since viability results obtained in runs A, B and C were not in line with those obtained in the DRF assay, a second CV75 value was therefore calculated based on viability results obtained in Runs B and C (i.e.209.10 µg/mL).

 

MAIN TEST: individual run results

All acceptance criteria regarding negative and positive controls were reached in runs A, C, D, E and G. However due to a strong cytotoxicity of the test item observed in all tested concentrations of both runs A and E, values were considered out of the acceptance criteria on the test item and these runs were considered invalidated. Therefore only Runs C, D and G were considered validated for the interpretation.

 

These three validated runs were performed using the following concentrations (final concentrations in wells):

.           run C: 7.56, 15.13, 30.26, 60.51, 121.02, 242.04, 484.09 and 968.17 µg/mL,

.           run D: 70.03, 84.03, 100.84, 121.01, 145.21, 174.25, 209.10 and 250.92 µg/mL,

.           run G: 111.63, 133.96, 160.75, 192.90, 231.48, 277.78, 333.33 and 400 µg/mL.

 

At these tested concentrations,the following results were obtained:

.           At post-treatment observations, no precipitate/emulsion was noted in treated wells,

.           run outcomes:

o  In the Run C, cytotoxicity (i.e.cell viability <50%) was observed at concentrations≥ 484.09 µg/mL,

o  In all runs: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any non‑cytotoxic tested concentrations. These runs were therefore considered negative.

Applicant's summary and conclusion

Interpretation of results:

other: Not skin senitizer

Remarks:

Based on the criteria described on the OECD guideline 442E

Conclusions:

Not skin sensitizer

Executive summary:

Method

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

The design of this study was based on the OECD Guideline 442E.

A solubility assessment was first performed using vehicles among saline solution (0.9% NaCl) and Dimethylsulfoxide (DMSO) to select an appropriate vehicle and the highest concentration to be used for test item formulations.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay 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 7 successive runs (i.e. four invalidated runs and three validated runs).

In each run, test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO₂in a humidified incubator. A set of control wells was also added in each plate to assure the validity of each run. At the end of the incubation period, cells from each well were distributed into three wells of a 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. 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) from each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. The corrected MFI value from the corresponding negative control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding negative 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 in saline solution (0.9% NaCl) at concentrations ≤ 500 mg/mL, after 30 minutes of magnetic stirring and 12 minutes of heating at 45°C.

Dose-Range Finding

The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 806.81 µg/mL. The highest concentration tested in the first main test was therefore968.17 µg/mL (i.e.1.2-fold the mean CV75).

Summary results

All acceptance criteria regarding negative and positive controls were reached in runs A, C, D, E and G. However due to a strong cytotoxicity of the test item observed in all tested concentrations of both runs A and E, values were considered out of the acceptance criteria on the test item and these runs were considered invalidated. Therefore only Runs C, D and G were considered validated for the interpretation.

These three validated runs were performed using the following concentrations (final concentrations in wells):

-         run C: 7.56, 15.13, 30.26, 60.51, 121.02, 242.04, 484.09 and 968.17 µg/mL,

-         run D: 70.03, 84.03, 100.84, 121.01, 145.21, 174.25, 209.10 and 250.92 µg/mL,

-         run G: 111.63, 133.96, 160.75, 192.90, 231.48, 277.78, 333.33 and 400 µg/mL.

At these tested concentrations,the following results were obtained:

-         At post-treatment observations, no precipitate/emulsion was noted in treated wells,

-         run outcome:

o  In the Run C, cytotoxicity (i.e.cell viability <50%) was observed at concentrations≥ 484.09 µg/mL,

o  In all runs: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any non‑cytotoxic tested concentrations. These runs were therefore considered negative.

Conclusion 

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