[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

KERATINOSENS TEST

The objective of this study was to evaluatethe potential of the test item toactivate the Nrf2 transcription factor. according to the OECD guideline 442D.

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.

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.

For each run, the test itemwas solubilised in water for injections at 200 mM, after at least 45 minutes of magnetic stirring.

With one exception in the first run (i.e.%CV of negative control at 20.4% instead of being < 20%) which was considered not to have any impact on the validity of the results, all acceptance criteria were fulfilled and both runs were therefore considered to be valid.

Both runs were 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 wells at the end of the 48-hour treatment period up to the highest tested concentration of 2000 µM,

.            a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations ≥250 µM corresponding to geometric meansIC₃₀and IC₅₀of the two runs of 406.54 and 577.28µM, respectively,

.            statistically gene-fold inductions above the threshold of 1.5 were noted at non-cytotoxic concentrations ≥ 1.95 µM, with an apparent dose‑related relationship. The I_maxwere 5.06 and 7.15 in the first and second runs, respectively and the calculated EC_1.5were 1.18 and 1.99 µM in the first and second runs, respectively.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

HUMAN-CELL LINE ACTIVATION TEST (h-CLAT)

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 assessmentwas 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).

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.

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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the Guidance on the application of CLP criteria (ECHA, 2017): ''Validated in vitro/in chemico methods exist with the aim to identify a sensitising potential of a chemical. These include OECD TG442C (Peptide/protein binding), TG442D (keratinocyte response) and TG 442E (monocytic/dendritic cell response). The in vitro/in chemico tests are not regarded as stand alone tests and the result from such a test should be used together with other data in an overall assessment. Further, at present there is no agreed strategy on how to use in vitro/in chemico methods for direct estimation of sensitising potency, but data from such tests can be used together with other data in order to assess skin sensitisation potency. See also the Guidance on IR&CSA, especially Section R.7.3.4.1.''

OECD guideline 442C was however not conducted as the test item is a metal complex. Regarding the h-CLAT and Keratinosens tests carried out on the test substance, discordant results were obtained. Using a conservative approach the test substance has been classified as skin sensitizer Categoy 1, according to the CLP criteria (ECHA 2017).