Dipotassium titanium oxide dioxalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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 (In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation (h-CLAT))

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as a surrogate for human myeloid dendritic cells, since these cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of Oxalate de Titanyl Potassium in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of Oxalate de Titanyl Potassium are determined by cytotoxicity tests.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium titanium oxide dioxalate

EC Number:

238-475-3

EC Name:

Dipotassium titanium oxide dioxalate

Cas Number:

14481-26-6

Molecular formula:

C4K2O9Ti

IUPAC Name:

dipotassium oxotitaniumbis(ylium) dioxalate

In vitro test system

Details on the study design:

Reasons for the Choice of THP-1 Cells:
THP-1 cells (Human monocytic leukemia cell line) are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

Experimental Design and Procedures of h-CLAT:
The test item was tested in three independent runs. The tests were performed on different days. The test item was prepared separately for each run.
The following concentrations of the test item were tested in the main experiments (h-CLAT):
85, 102, 122, 147, 176, 211, 253 and 304 µg/mL (in the first main experiment)
87, 105, 126, 151, 181, 217, 261 and 313 µg/mL (in the second and third main experiment)


-Treatment of the Cells
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. The two highest test item concentrations of the three h-CLAT runs could not be microscopically evaluated for morphological alterations, due to observed precipitation.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

-Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (289  g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

-Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

-Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.

Preparation of the acquisition
The following acquisition plots were prepared:
•2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
•Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) is calculated, but excluded from the evaluation, if the cell viability is less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

-Data Analysis and Interpretation
Flow Cytometry Analysis
The RFI is used as an indicator of CD86 and CD54 expression.
The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation: Cell Viability [%]= (Number of living cells)/(Total Number of acquired cells) ×100
Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

-Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
•Cell viability of medium control and DMSO control should be more than 90%.
•In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
•For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
•In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
•For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is > 90% (OECD 442E guideline).

-Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.7.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

Results and discussion

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: all runs

Parameter:

other: RFI of CD86 (%)

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: see Remarks

Remarks:

The RFI of CD86 was equal or greater than 150% in at least one concentration of the first run and the RFI of CD86 was equal or greater than 150% in at least one concentration of the second run. Due to fluctuating CD86 values observed in the first h-CLAT run, a confirmatory third run was conducted. The RFI of CD86 was equal or greater than 150% in at least one concentration of the third run

Results 2

Key result

Run / experiment:

other: second run

Parameter:

other: RFI of CD54 (%)

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The RFI of CD54 was equal or greater than 200% in at least one concentration of the second run.

Applicant's summary and conclusion

Interpretation of results:

other: The test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Conclusions:

In conclusion, the test item Oxalate de Titanyl Potassium with a log Pow <0 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).