Reaction mass of ammonium(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) hydrogen phosphate and ammonium bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) phosphate

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: in vitro human cell line activation test (h-CLAT)

Version / remarks:

The h-CLAT method is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP) for skin sensitisation, which is the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of ammonium(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) hydrogen phosphate and ammonium bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) phosphate

EC Number:

940-803-1

Molecular formula:

This is a multi-constituent susbstance there is no molecular formula or molecular weight range available

IUPAC Name:

Reaction mass of ammonium(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) hydrogen phosphate and ammonium bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) phosphate

Test material form:

liquid

Specific details on test material used for the study:

Name: Thetawet FS-8250
Batch No.: W17033003
Compounds Substituted alkyl phosphate esters, ammonium salt (EPA accession numbers 278978, 263128, 265259; 25-30 %) Water (70-75 %)
Molecular Weight: 211.55 g/mol
Physical State: liquid
Colour: beige
The test item was freshly prepared immediately prior to use.
The test item was not soluble in 0.9% NaCl at a concentration of 100 mg/mL, it was dissolved in 0.9% NaCl solution at a concentration of 1.00 mg/mL.
Vortex mixing was used to aid solubilisation.
Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.
The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium.
No precipitation, turbidity or phase separation was observed when diluted 1:50 in cell culture medium. Vortex mixing and/or sonication and/or warming to 37 °C were be used to aid solubilisation.

In vitro test system

Details on the study design:

A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 +/- 1°C and 5% CO2.

Results and discussion

Positive control results:

Positiive control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure, resulting in a final DMSO concentration of 0.2% (v/v).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: First experiment

Parameter:

other: Relative cell viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 91.8% (CD86), 91.6% (CD54) and 92.3% (isotype IgG1 control)

Results 2

Key result

Run / experiment:

other: Second experiment

Parameter:

other: Relative cell viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 95.4% (CD86), 94.6% (CD54) and 94.9% (isotype IgG1 control) i

Other effects / acceptance of results:

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiments were performed covering the following concentration steps:
10.00, 8.33, 6.94, 5.79, 4.82, 4.02, 3.35 and 2.79 μg/mL
In all experiments no precipitation or turbidity of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 91.8% (CD86), 91.6% (CD54) and 92.3% (isotype IgG1 control) in the first experiment and to 95.4% (CD86), 94.6% (CD54) and 94.9% (isotype IgG1 control) in the second experiment.
The test meets acceptance criteria if:
 the cell viability of the solvent controls is >90%,
 the cell viability of at least four tested doses of the test item in each run is >50%,
 the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
 the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
 the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 91.8% (CD86), 91.6% (CD54) and 92.3% (isotype IgG1 control) in the first experiment and to 95.4% (CD86), 94.6% (CD54) and 94.9% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be a non-sensitiser.