Vinyl 4-(1,1-dimethylethyl)benzoate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Vinyl 4-(1,1-dimethylethyl)benzoate

EC Number:

239-510-5

EC Name:

Vinyl 4-(1,1-dimethylethyl)benzoate

Cas Number:

15484-80-7

Molecular formula:

C13H16O2

IUPAC Name:

ethenyl 4-tert-butylbenzoate

Test material form:

liquid

Details on test material:

- Name: SAT 200028
- Batch No.: 19K2505
- Physical State: liquid
- Purity: 99.0%
- Expiry Date: 30 June 2021
- Storage Conditions: at room temperature, protected from light
- Safety Precautions: The routine hygienic procedures were sufficient to assure
personnel health and safety.

In chemico test system

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

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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”.