[No public or meaningful name is available]

Endpoint summary

Description of key information

REACH_sensitising | KeratinoSens | OECD 442D | #key study#
REACH_sensitising | hCLAT | OECD 442E | #key study#

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

Study period:

August - October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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.

Positive control results:

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

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%

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%

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

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.

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25.06.2018

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

09.03.2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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 KeratinoSens™ assay 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

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 the test item was dissolved in DMSO. Based on a molecular weight of 248.32 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 in experiments 1 and 2 and a constant dilution factor of 1:1.333 in experiment 3. These master solutions were diluted 1:100 in cell culture medium. 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.

Positive control results:

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

Key result

Run / experiment:

other: Experiment 1 (62.50 µM)

Parameter:

other: I max

Value:

14.49

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Cell viability: 10.4%

Run / experiment:

other: Experiment 2 (31.25 µM)

Parameter:

other: I max

Value:

4.09

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cell viability: 35.5%

Key result

Run / experiment:

other: Experiment 3 (60 µM)

Parameter:

other: I max

Value:

15.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Cell viability: 86.2%

Other effects / acceptance of results:

The controls confirmed the validity of the study.

In the first experiment, a max luciferase activity (Imax) induction of 14.49 was determined at a test item concentration of 62.50 μM. The corresponding cell viability was 10.4%. The lowest tested concentration with a significant luciferase induction >1.5 (2.66) was found to be 31.25 μM. The corresponding cell viability was >70% (109.8%). The calculated EC1.5 was <1000 μM (18.05 μM). Furthermore, a slight but apparent overall dose-response for luciferase induction was observable.

Therefore, the test item is considered as sensitiser in this experiment.

In the second experiment, a max luciferase activity (Imax) induction of 4.09 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 35.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.58) was found to be 15.63 μM. The corresponding cell viability was <70% (54.5%). The calculated EC1.5 was <1000 μM (14.34 μM).

Therefore, the test item is considered as non-sensitiser in this experiment.

As the first and second experiment showed different outcomes, a third experiment with an adapted concentration range was performed.

In the third experiment, a max luciferase activity (Imax) induction of 15.34 was determined at a test item concentration of 60 μM. The corresponding cell viability was 86.2%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 25.33 μM. The corresponding cell viability was >70% (93.2%). The calculated EC1.5 was <1000 μM (22.65 μM). Furthermore, an overall dose-response for luciferase induction was observable.

Therefore, the test item is considered as sensitiser in this experiment.

The test item showed sensitizing potential in two out of three independent experiments. Under the condition of this study the test item is therefore considered as sensitiser.

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 induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiments. Therefore, the test item can be considered as 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 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 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. Based on a molecular weight of 248.32 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 in experiments 1 and 2 and a constant dilution factor of 1:1.333 in experiment 3. 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 (Experiment 1 and 2)

60, 45.01, 33.77, 25.33, 19, 14.26, 10.69, 8.02, 6.02, 4.52, 3.39, 2.54 μM (Experiment 3)

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, a max luciferase activity (Imax) induction of 14.49 was determined at a test item concentration of 62.50 μM. The corresponding cell viability was 10.4%. The lowest tested concentration with a significant luciferase induction >1.5 (2.66) was found to be 31.25 μM. The corresponding cell viability was >70% (109.8%). The calculated EC1.5 was <1000 μM (18.05 μM). Furthermore, a slight but apparent overall dose-response for luciferase induction was observable.

Therefore, the test item is considered as sensitiser in this experiment.

In the second experiment, a max luciferase activity (Imax) induction of 4.09 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 35.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.58) was found to be 15.63 μM. The corresponding cell viability was <70% (54.5%). The calculated EC1.5 was <1000 μM (14.34 μM). Therefore, the test item is considered as non-sensitiser in this experiment.

As the first and second experiment showed different outcomes, a third experiment with an adapted concentration range was performed.

In the third experiment, a max luciferase activity (Imax) induction of 15.34 was determined at a test item concentration of 60 μM. The corresponding cell viability was 86.2%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 25.33 μM. The corresponding cell viability was >70% (93.2%). The calculated EC1.5 was <1000 μM (22.65 μM). Furthermore, an overall dose-response for luciferase induction was observable. Therefore, the test item is considered as sensitiser in this experiment.

The test item showed sensitizing potential in two out of three independent experiments. Under the condition of this study the test item is therefore considered as sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

For assessment of the skin sensitization potential a binary testing strategy using Keratinosens (OECD 442D) and h-CLAT (OECD 442E) has been chosen. In the KeratinoSens assay the test item did induce the luciferase activity in the transgenic KeratinoSens cell line in two independent experiments and was therefore considered as sensitizer. In the h-CLAT the test item did upregulate the cell surface marker CD86 in two independent experiments. The test item was therefore considered to be a skin sensitizer.

 

Based on the available data 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 is classified as skin sensitizer.

 

(Skin Sens. 1 – no subcategorization)