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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:
weight of evidence
Study period:
From 2019-04-23 to 2019-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl ethylene carbonate
EC Number:
700-261-7
Cas Number:
4427-96-7
Molecular formula:
C5H6O3
IUPAC Name:
Vinyl ethylene carbonate
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%

In chemico test system

Details on the study design:
Test System
- Test System: Synthetic peptides containing cysteine (SPCC) or synthetic peptides containing lysine (SPCL)
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH

Experimental Design
- Preparation of Test Item: For both the cysteine and lysine reactivity assay 17.48 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1532 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of Solutions for Cysteine Reactivity Assay:
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.5 mg of SPCC in 20.96 mL phosphate buffer pH7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
- Preparation of Solutions for Lysine Reactivity Assay:
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.8 mg of SPCL in 20.85 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
- Sample Incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.4 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
- HPLC Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC.

Results and discussion

Positive control results:
Cysteine Reactivity Assay
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 100% ± 0.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Lysine Reactivity Assay
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 55.1% ± 2.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: SPCC depletion%
Value:
0.6
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean of the SPCC depletion
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: SPCL depletion %
Value:
10.5
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean of the SPCL depletion
Other effects / acceptance of results:
- Results Cysteine Reactivity Assay for the Test Item
Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples. For the 210160/A-cys samples, the mean SPCC A220/A258 area ratio was 37.87. Since this was within the 34.06 - 41.63 range, this indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 0.6% ± 0.5%.
- Results Lysine Reactivity Assay for the Test Item
Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 210160/A-lys samples, the mean SPCL A220/A258 area ratio was 31.70. Since this was within the 28.38 – 34.69 range, this again indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 10.5% ± 0.4%.
DPRA Prediction and Reactivity Classification
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 10.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 5.6% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
other: negative
Conclusions:
The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 10.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 5.6%.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.