[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: 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)

Version / remarks:

23.07.2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

01.07.2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the human cell line activation test (h-CLAT) showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Molecular formula:

C15 H20 O3

IUPAC Name:

Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Test material form:

liquid

Details on test material:

- Name: 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester
- CAS No.: 68169-12-0 (outside EU)
- EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7ahexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate
- EC Number: 951-956-9
- Batch No.: OX8J0799
- Molecular Weight: 248.32 g/mol
- Physical State: liquid
- Colour: clear
- Active Components: 98.52% active components (different isomers), 1.48% 2-Cyclopenta-1,3-dienyloxyethan-1-ol
- Purity: 98.52%
- Expiry Date: 17 September 2023
- Storage Conditions: room temperature
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Results and discussion

Positive control results:

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: CD86 Experiment 1 (158.75 µg/mL)

Parameter:

other: RFI

Remarks:

[%]

Value:

187

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Cell viability: 55.7%

Results 2

Key result

Run / experiment:

other: CD86 Experiment 2 (110.24 µg/mL)

Parameter:

other: RFI

Remarks:

[%]

Value:

426

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Cell viability: 25.8%

Results 3

Run / experiment:

other: CD54

Parameter:

other: RFI

Remarks:

[%]

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

In the present study 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester; EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 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.

A CV75 of 132.29 ± 4.06 μg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

158.75, 132.29, 110.24, 91.87, 76.56, 63.80, 53.17, 44.30 μg/mL

In the dose finding assay 1 precipitation of the test item was observed in the three highest working solutions when mixing the test item stock solutions with cell culture medium. In the other experiments, no precipitation or turbidity of the test item was observed 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 55.7% (CD86), 54.6% (CD54) and 50.4% (isotype IgG1 control) in the first experiment and to 18.0% (CD86), 23.7% (CD54) and 19.3% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated up to 187% (158.75 μg/mL) in the first experiment and up to 426% (110.24 μg/mL) in the second experiment.

In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

The controls confirmed the validity of the study for all experiments.

The data generated with this test should be considered in the context of integrated approached 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.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

in the context of an integrated approach only

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in two independent experiments. Therefore, the test item is considered to be a skin sensitiser.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

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

In the present study 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester; EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 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.

A CV75 of 132.29 ± 4.06 μg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

158.75, 132.29, 110.24, 91.87, 76.56, 63.80, 53.17, 44.30 μg/mL

In the dose finding assay 1 precipitation of the test item was observed in the three highest working solutions when mixing the test item stock solutions with cell culture medium. In the other experiments, no precipitation or turbidity of the test item was observed 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 55.7% (CD86), 54.6% (CD54) and 50.4% (isotype IgG1 control) in the first experiment and to 18.0% (CD86), 23.7% (CD54) and 19.3% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated up to 187% (158.75 μg/mL) in the first experiment and up to 426% (110.24 μg/mL) in the second experiment.

In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Conclusion

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in two independent experiments. Therefore, the test item is considered to be a skin sensitiser.

The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.