Vinyl 4-(1,1-dimethylethyl)benzoate

Endpoint summary

Description of key information

Skin Sensitisation

REACH_sensitising | DPRA | OECD 442C | #key study#

REACH_not sensitising | KeratinoSens | OECD 442D | #key study#

REACH_sensitising | h-CLAT | OECD 442E | #key study#

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-Dec. 2021

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. 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.
In the present study SAT 200028 was dissolved in acetonitrile, based on the results of the pre-experiments.
Based on a molecular weight of 204.26 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.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

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 67.59% with cysteine experiment 1 and 67.27% with cysteine experiment 2.

Key result

Group:

test chemical

Run / experiment:

other: Cysteine, Experiment 1

Parameter:

mean cystein depletion

Value:

13.43 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

73.63 %

Remarks on result:

other: Precipitation observed, no prediction can be made

Key result

Group:

test chemical

Run / experiment:

other: Cysteine Depletion, Experiment 2

Parameter:

mean cystein depletion

Value:

14.78 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

72.98 %

Remarks on result:

other: Precipitation observed, positive with low reactivity

Group:

test chemical

Run / experiment:

other: Lysine

Parameter:

mean lysine depletion

Value:

0.85 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

61.56 %

Remarks on result:

other: Co-elution observed

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The controls confirmed the validity of the study for both, the cysteine and lysine run.

Cysteine Experiments:
Experiment 1:
For the 100 mM stock solution of the test item turbidity and phase separation 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 the test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.
Experiment 2:
For the 100 mM stock solution of the test item turbidity 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 the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

 

Lysine Experiment:
For the 100 mM stock solution of the test item turbidity 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. Slight turbidity was observed for the samples of the positive control (excluding 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 turbidity was regarded as not relevant.

 

A minor co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.
Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).

 

In the cysteine experiment 1, precipitation was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.
In the cysteine experiment 1, the 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (13.43%). According to the evaluation criteria in the guideline, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

 

Furthermore, according to the evaluation criteria in the guideline, for test items with a cysteine peptide depletion between 9% and 17% a second run should be considered. If the mean depletion of the cysteine peptide would then be >13.89%, the test item would be classified as positive. If the mean depletion of the cysteine peptide would then be ≤13.89%, no prediction could be made due to the observed precipitation in the cysteine experiment.
In the cysteine experiment 2, precipitation was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.
In the cysteine experiment 2, the 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (14.78%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as sensitiser.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Consideration in the context of integrated approach such as IATA.

Conclusions:

In this study under the given conditions the test item showed low reactivity towards the cysteine peptide. The test item might be considered as “sensitiser”.
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.

Executive summary:

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 SAT 200028 was dissolved in acetonitrile, based on the results of the pre-experiments.
Based on a molecular weight of 204.26 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.
Cysteine Experiments:
Experiment 1:
For the 100 mM stock solution of the test item turbidity and phase separation 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 the test item (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.
Experiment 2:
For the 100 mM stock solution of the test item turbidity 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 the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.
Lysine Experiment:
For the 100 mM stock solution of the test item turbidity 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. Slight turbidity was observed for the samples of the positive control (excluding 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 turbidity was regarded as not relevant.
A minor co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.
Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).
In the cysteine experiment 1, precipitation was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.
In the cysteine experiment 1, the 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (13.43%). According to the evaluation criteria in the guideline, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.
Furthermore, according to the evaluation criteria in the guideline, for test items with a cysteine peptide depletion between 9% and 17% a second run should be considered. If the mean depletion of the cysteine peptide would then be >13.89%, the test item would be classified as positive. If the mean depletion of the cysteine peptide would then be ≤13.89%, no prediction could be made due to the observed precipitation in the cysteine experiment.
In the cysteine experiment 2, precipitation was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.
In the cysteine experiment 2, the 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (14.78%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as 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 67.59% with cysteine experiment 1 and 67.27% with cysteine experiment 2.

 

Conclusion
In this study under the given conditions the test item showed low reactivity towards the cysteine peptide. The test item might be considered as “sensitiser”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-Oct. 2021

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)

Version / remarks:

23rd July 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

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 sensitiser and non-sensitisers.
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 SAT 200028 was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 to 3.91 mg/mLwere 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 248.51 ± 81.32 μg/mL was derived in the dose finding assay
Based on the CV75, the main experiment was performed covering the following concentration steps:
298.21; 248.51; 207.09; 172.58; 143.81; 119.84; 99.87; 83.22 μg/mL
In all experiments a phase separation was observed after mixing the test item stock solutions with cell culture medium. Ultrasonication was used to aid solubilisation.
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:

DMSO

Negative control:

other: medium control

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

RFI of positive control of CD86: 192 / 281 / 338 (> 150; pass);
RFI of positive control of CD54: 297 / 263 / 523 (> 200; pass)

Key result

Group:

test chemical

Run / experiment:

other:

Parameter:

EC200, CD54 [442E]

Value:

92.11 µg/mL

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other:

Remarks:

RFI CD86 [442E]

Value:

127 %

Cell viability:

50.1 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD86 [442E]

Value:

139 %

Cell viability:

80.0 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

other: RFI CD86 [442E]

Value:

132 %

Cell viability:

90.7 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other:

Remarks:

RFI CD54 [442E]

Value:

297 %

Cell viability:

75.7 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD54 [442E]

Value:

194 %

Cell viability:

73.4 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

other: RFI CD54 [442E]

Value:

484 %

Cell viability:

45.3 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

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

Moderate or severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 62.2% (CD86), 64.5% (CD54) and 66.5% (isotype IgG1 control) in the first experiment and to 74.1% (CD86), 73.0% (CD54) and 74.2% (isotype IgG1 control) in the second experiment and to 24.4% (CD86), 23.0% (CD54) and 23.2% (isotype IgG1 control) in the third experiment.

 

The expression of the cell surface marker CD54 was upregulated to 297% in experiment 1 and to 484% in experiment 3. The upregulation above the threshold of 200% was observed at several concentrations, starting at 83.22 μg/mL in experiment 1 and at 99.87 μg/mL in experiment 3. In experiment 2 no upregulation of the surface marker CD54 was observed.

 

In contrast, the expression of cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments.

 

The EC200 value was calculated with 92.11 µg/mL. Since only two independent experiments showed an upregulation above the threshold, the higher EC200 of the two calculated values was adopted. The EC values could potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.

 

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

 

The controls confirmed the validity of the study for all experiments

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Consideration in the context of integrated approach such as IATA.

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 is considered as skin 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 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.
In the present study SAT 200028 was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 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 248.51 ± 81.32 μg/mL was derived in the dose finding assay
Based on the CV75, the main experiment was performed covering the following concentration steps:
298.21; 248.51; 207.09; 172.58; 143.81; 119.84; 99.87; 83.22 μg/mL
In all experiments a phase separation was observed after mixing the test item stock solutions with cell culture medium. Ultrasonication was used to aid solubilisation.
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.

 

Moderate or severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 62.2% (CD86), 64.5% (CD54) and 66.5% (isotype IgG1 control) in the first experiment and to 74.1% (CD86), 73.0% (CD54) and 74.2% (isotype IgG1 control) in the second experiment and to 24.4% (CD86), 23.0% (CD54) and 23.2% (isotype IgG1 control) in the third experiment.

 

The expression of the cell surface marker CD54 was upregulated to 297% in experiment 1 and to 484% in experiment 3. The upregulation above the threshold of 200% was observed at several concentrations, starting at 83.22 μg/mL in experiment 1 and at 99.87 μg/mL in experiment 3. In experiment 2 no upregulation of the surface marker CD54 was observed.

 

In contrast, the expression of cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments.

 

The EC200 value was calculated with 92.11 µg/mL. Since only two independent experiments showed an upregulation above the threshold, the higher EC200 of the two calculated values was adopted. The EC values could potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.

 

Since one of the cell surface markers clearly exceeded the threshold in two out of three independent experiments the test item is considered to be a skin sensitiser.
The controls confirmed the validity of the study for all experiments.

 

Conclusion
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 is considered as skin 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July-Oct. 2021

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 09, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

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 SAT 200028 was dissolved in DMSO. Based on a molecular weight of 204.26 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 and a stable suspension was formed. 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:

DMSO

Negative control:

other: blank well (background)

Positive control:

cinnamic aldehyde [442D]

Positive control results:

No. of positive control conc. steps with significant luciferase activity induction >1.5: 3.0 (> 1; pass)

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Induction of Luciferase Activity / Fold Induction (mean max. value)

Value:

1.14

Cell viability:

108.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

The controls confirmed the validity of the study.

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.

Interpretation of results:

GHS criteria not met

Remarks:

Consideration in the context of integrated approach such as IATA.

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.

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 SAT 200028 was dissolved in DMSO. Based on a molecular weight of 204.26 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 and a stable suspension was formed. 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.

The controls confirmed the validity of the study.

 

Conclusion
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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The three in chemico/in vitro tests DPRA, KeratinoSens and h-CLAT were performed according to OECD principles (OECD 442c,d,e). One test was considered negative (KeratinoSens), two tests considered the substance to be a skin sensitiser (DPRA, h-CLAT). According to the results of the OECD QSAR toolbox (V 4.5), no alerts for skin sensitisation were found, but the result was out of applicability domain. Overall and according to Integrated Testing Strategy 2 described in the OECD Guideline No. 497: Defined Approaches on Skin Sensitisation, the substance is considered to be a weak skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance was considered a skin sensitiser in the in chemico/in vitro Skin Sensitisation studies OECD 442C and 442E. According to Integrated Testing Strategy 2 described in the OECD Guideline No. 497: Defined Approaches on Skin Sensitisation, the substance is considered to be a weak skin sensitiser. According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as a weak skin sensitiser (Category 1B).