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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September 2020 - 11 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
June 18, 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4,19,19-tetraethoxy-3,20-dioxa-8,15-dithia-4,19-disiladocosane and S-(6-{[3-(triethoxysilyl)propyl]thio}hexyl) ethanethioate
EC Number:
951-696-6
Molecular formula:
C17H36O4S2Si and C24H54O6S2Si2
IUPAC Name:
4,4,19,19-tetraethoxy-3,20-dioxa-8,15-dithia-4,19-disiladocosane and S-(6-{[3-(triethoxysilyl)propyl]thio}hexyl) ethanethioate
Test material form:
liquid

In chemico test system

Details on the study design:
Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
The molecular weight and purity of all components (excluding water) was used to calculate a final purity and to derive a molecular weight of the test item. Based on the derived molecular weight a 100 mM stock solution was prepared. Additionally, the neat test item was tested without any prior dilutions. All test item preparations were pre-weighed into glass vials and were dissolved in an appropriate solvent previously determined in a pre-experiment. A factor of 1.05 was used to correct the purity of the test item.

Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run .
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples

Peptides
21.56 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (41.8 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.92 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.889 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100-400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using a HPLC procedure.

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 63.51%.

In vitro / in chemico

Results
Key result
Run / experiment:
other: 1
Parameter:
other: peptide depletion (%)
Value:
3.54
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Precipitation was observed for the three test item samples and for the sample of the coelution control. Therefore, samples were centrifuged and pipetted into new vials prior to the HPLC analysis.
Other effects / acceptance of results:
For the 100 mM stock solution of the test item turbidity, but no 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 three test item samples and for the sample of the coelution control. Therefore, samples were centrifuged and pipetted into new vials prior to the HPLC analysis.
For the 100 mM solution of the test item turbidity but no 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 three test item samples and for the sample of the coelution control. Therefore, samples were centrifuged and pipetted into new vials prior to the HPLC analysis.
The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (3.54%). Since a precipitation was observed, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.
Therefore, no prediction can be made.
According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered. Due to precipitations in both peptides no prediction can be made and the borderliner experiment is not necessary.
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 63.51%.
The controls confirmed the validity of the study for both, the cysteine and lysine run.

Conclusion
In this study under the given conditions the test item showed minimal reactivity towards both peptides. 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 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.

Any other information on results incl. tables

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm [mAU]

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

5.3370

0.1608

68.09

68.36

0.26

0.37

5.2520

0.1582

68.60

5.2850

0.1592

68.40

Test Item

14.6970

0.4419

5.48

6.70

1.08

16.09

14.4510

0.4345

7.07

14.3770

0.4323

7.54

 

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm [mAU]

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

5.8980

0.2018

59.55

58.65

0.78

1.33

6.0880

0.2083

58.25

6.1000

0.2087

58.16

Test Item

14.4950

0.4953

0.24

0.38

0.21

54.25

14.4900

0.4951

0.28

14.4410

0.4934

0.61

Categorization of the Test Item

Prediction Model

Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)

Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

3.54

Minimal Reactivity

 --*

6.70

Minimal Reactivity

 --*

Positive Control

63.51

High Reactivity

positive

68.36

Moderate Reactivity

positive

*due to the observed precipitation, no prediction can be made

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.