2,2,3,3,8,8,9,9-octafluorotricyclo[8.2.2.2⁴,⁷]hexadeca-1(12),4,6,10,13,15-hexaene

Endpoint summary

Description of key information

Based on the OECD 497 (Defined Approaches on Skin Sensitisation), the following in chemico and in vitro methods were performed:

- direct peptide reactivity assay (DPRA; OECD TG 442C)
- keratinocyte activation (KeratinoSens™; OECD TG 442D)

- dendritic cell activation (human cell line activation test (h-CLAT; OECD TG
442E)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Qualifier:

equivalent or similar to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 23rd, 2015

GLP compliance:

yes

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

Batch No.: 340421
Purity: 98.5%

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.
In the present study PARYLENE HT® was dissolved in acetone. For the dose finding assay stock solutions with concentrations ranging from 140.63 mg/mL to 1.10 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.
Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:
281.26, 234.38, 195.32, 162.77, 135.64, 113.03, 94.19, 78.49 µg/mL
Turbidity of the test item was observed in experiment 2 for all 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.

Vehicle / solvent control:

other: acetone

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

170 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 92.2% (CD86), 90.9% (CD54) and 91.6% (isotype IgG1 control) in the first experiment and to 90.3% (CD86), 88.0% (CD54) and 90.0% (isotype IgG1 control) in the second experiment

Vehicle controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

167 %

Cell viability:

Relative cell viability at the highest test item concentration was reduced to 92.2% (CD86), 90.9% (CD54) and 91.6% (isotype IgG1 control) in the first experiment and to 90.3% (CD86), 88.0% (CD54) and 90.0% (isotype IgG1 control) in the second experiment.

Vehicle controls validity:

valid

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study PARYLENE HT® was dissolved in acetone. For the dose finding assay stock solutions with concentrations ranging from 140.63 mg/mL to 1.10 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

281.26, 234.38, 195.32, 162.77, 135.64, 113.03, 94.19, 78.49 µg/mL

Turbidity of the test item was observed in experiment 2 for all 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.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item can be considered as a skin sensitiser

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:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Batch No.: 340421
Purity: 98.5%

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Based on a molecular weight of 352.23 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.

Vehicle / solvent control:

other: acetone

Negative control:

other: DMSO

Positive control:

cinnamic aldehyde [442D]

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no relevant increase

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no relevant increase

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.
No dose response for luciferase activity induction was observed for each individual run as well as for
an overall luciferase activity induction.

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 be not 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.

Executive summary:

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 PARYLENE HT® was dissolved in aceton.
Based on a molecular weight of 352.23 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.
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.

Reference 3

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 Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Qualifier:

equivalent or similar to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013 [5].

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch No.: 340421
Purity: 98.5%

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

In the present study PARYLENE HT® was dissolved in acetonitrile, based on the results of the pre experiments.
Based on a molecular weight of 352.23 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 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. Precipitation was observed for the samples of test item (including co-elution control). Samples were not centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Remarks on result:

not determinable

Interpretation of results:

study cannot be used for classification

Conclusions:

Due to the observed precipitation the prediction model does not apply and a prediction cannot be
made.
The data generated with this test should be considered in the context of an integrated approache such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in chemico and in vitro methods performed were DPRA, KeratinoSensTM and h-CLAT. Based on the OECD 497(Defined Approaches on Skin Sensitisation), the data was compared. 

Two concordant results were not obtained from methods. One test resulted inconclusive (DPRA), one test resulted negative (keratinosens) and one test resulted positive (h-CLAT).

These predictions were nevertheless considered in a weight-of evidence approach with other information, considering the classification of other similar substances and the worst case.

The substance is classified a skin sensitiser, cat. 1.