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

Endpoint summary

Description of key information

This end point has been assessed from three newly performed in-vitro and in-chemico test procedures which have confirmed that the original assessment from reliable published data relating to other short chain alkyl perfluorinated substances, which are very similar in structure to this substance, was accurate in concluding that this test substance is not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Not possible to isolate from water without decomposition

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

This publication is a general assessment of various human health and environmental effects of these substances. The result detailed gives data from DuPont 2009

Deviations:

not specified

GLP compliance:

not specified

Remarks:

The results within the report do not specify if GLP was used.

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CA/JHsd

Sex:

not specified

Vehicle:

not specified

Concentration:

25 μl per ear (5%,25%, 50%, or 100% v/v)

Parameter:

SI

Value:

< 3

Remarks on result:

other: Not sensitising. The EC3 value was not calculable because the SI for all test concentrations was <3

No clinical signs of toxicity were observed. A statistically significant ↑in cell proliferation measurements compared to the vehicle control group at 100%. However, stimulation indices of less than 3.0 were observed at all test concentrations. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable

Interpretation of results:

GHS criteria not met

Reference 2

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

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

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.

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.

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).

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)

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%.

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.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
In the present study Thetawet FS-8250 was dissolved in DMSO.
Based on a molecular weight of 211.55 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

Positive control results:

A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001055932) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
The controls confirmed the validity of the study

Key result

Run / experiment:

other: First

Parameter:

other: luciferase induction

Remarks:

The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Value:

1.5 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Second

Parameter:

other: luciferase induction

Remarks:

The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Value:

1.5 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non sensitiser.
The controls confirmed the validity of the study

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

Preparation of the Test Item, study plan, p. 10.This deviation did not influence the quality or integrity of the present study

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC). This is an in-chemico methodology and not in-vivo.

Specific details on test material used for the study:

Name: Thetawet FS-8250
Batch No.: W17033003
Calculated Molecular Weight: 211.55 g/mol
Compounds: Substituted alkyl phosphate esters, ammonium salt (EPA accession numbers 278978, 263128, 265259; 25-30 %)
Water (70-75 %)
Physical State: liquid
Colour: beige
Stability: stable under standard conditions
Storage Conditions: room temperature
Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water. All test item solutions were freshly prepared immediately prior to use

Details on the study design:

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP. In the case of Thetawet, this approach has been taken and other in-vitro test methods have been used to confirm the non- lassification

Positive control results:

Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Key result

Run / experiment:

other: Results of the Cysteine Peptide Depletion

Parameter:

other: % depletion value of cysteine and lysine peptide

Remarks:

The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

Value:

0.76

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Cysteine peptide depletion % for positive control was 70.42

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Results of the Lysine Peptide Depletion

Parameter:

other: % depletion value of cysteine and lysine peptide

Remarks:

The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

Value:

1.25

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Lysine peptide depletion % for positive control was 66.09

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Categorization of the Test Item: Based on the results of the peptide depletion, categorization according to the prediction model might be performed.
Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Thetawet FS-8250 was dissolved in dist. water, based on the results of the pre-experiments.

Based on a molecular weight of 211.55 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation was regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cwater).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (1.00%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 68.26%.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach, which has been done.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)