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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:
28 Jun - 06 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
Adopted: 4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH
- Synthetic cysteine-containing peptide: Ac-RFAACAA-COOH
- Synthetic lysine-containing peptide: Ac-RFAAKAA-COOH

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile (ACN)
- For both the cysteine and lysine reactivity assay: 40.92 mg of test item was solved in 1628 μL ACN (100 mM solution). Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Concentration: 100 mM in ACN

CONTROLS
- Reference control, calibration solution and co-elution control were prepared.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: at least 22.5 ± 0.5 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: HPLC-PDA Method
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Milli-Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Cysteine %B: 10, 25, 90, 90, 10, 10
Lysine %B: 10, 20, 90, 90, 10, 10
Time (min): 0, 10, 11, 13, 13.5, 20
- Detector Wavelength: Photodiode array detection, monitoring at 220 and 258 nm
- Calibration standard concentrations of both peptides: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 and 0.000 mM
- Column temperature: 30 °C
- Injection volume: 10 µL

Results and discussion

Positive control results:
- The mean percent cysteine-containing peptide depletion for the positive control cinnamic aldehyde was 71.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
- The mean percent lysine-containing peptide depletion for the positive control cinnamic aldehyde was 48.5% ± 1.2%. 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

Results
Key result
Parameter:
other: % depletion
Remarks:
mean of cysteine- and lysine-containg peptide
Run / experiment:
Cysteine 1:10 / Lysine 1:50 Prediction Model
Value:
27.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
moderate reactivity
Other effects / acceptance of results:
OTHER EFFECTS:
A phase separation was observed after the incubation period for lysine-containing peptide, one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, it might be possible that the reactivity class for lysine-containing peptide is underestimated. No precipitate or phase separation was observed after the incubation of the synthetic peptide containing cysteine with the test item sample.

ACCEPTANCE OF RESULTS:
Acceptability of the Cysteine Reactivity Assay
- The correlation coefficient (r2) of the cysteine-containing peptide standard calibration curve was 0.995. Since the r2 was >0.99, the standard calibration curve was accepted.
- The mean peptide concentration of reference controls A was 0.533 ± 0.009 mM while the mean peptide concentration of reference controls C was 0.536 ± 0.011 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the percent cysteine-containing peptide depletion.
- The coefficient of variation (CV) of the peptide areas for the nine reference controls B and C was 3.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the reference control samples was 16.90. The mean A220/A258 ratio ± 10% range was 15.21-18.59. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The percent cysteine-containing peptide depletion was calculated versus the mean cysteine-containing peptide peak area of reference controls C. The mean percent cysteine-containing peptide depletion for the positive control cinnamic aldehyde was 71.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay
- The correlation coefficient (r2) of the lysine-containing peptide standard calibration curve was 0.995. Since the r2 was >0.99, the lysine-containing peptide standard calibration curve was accepted.
- The mean peptide concentration of reference controls A was 0.501 ± 0.005 mM while the mean peptide concentration of reference controls C was 0.465 ± 0.008 mM. The means of reference control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent lysine-containing peptide depletion.
- The CV of the peptide areas for the nine reference controls B and C was 3.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the reference control samples was 14.51. The mean A220/A258 ratio ± 10% range was 13.06-15.96. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The percent lysine-containing peptide depletion was calculated versus the mean lysine-containing peptide peak area of reference controls C. The mean percent lysine-containing peptide depletion for the positive control cinnamic aldehyde was 48.5% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).



Any other information on results incl. tables

Table : DPRA Prediction and Reactivity Classification for the Test Item

 

% Cysteine-containing peptide depletion

% Lysine-containing peptide depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

 

Mean

SD

Mean

SD

 

Cysteine 1:10 / Lysine 1:50 prediction model

Test item

51.3

1.7

2.9

4.6

27.1

Positive: Moderate reactivity

SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: skin sensitising potential based on the key event “protein reactivity”.
Conclusions:
Under the conditions of the Direct Peptide Reactivity Assay the test substance showed moderate peptide reactivity. Skin sensitising potential was observed based on the key event “protein reactivity”.
Executive summary:

The test substance gave a positive outcome in the DPRA and the KeratinoSensTM assay. These results indicate that it is likely that the test substance will interact with protein moieties in vivo and that the substance is able to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. Both properties can potentially lead to skin sensitization in humans. This conclusion is supported by the positive DEREK assessment, which concludes on strong sensitizing properties related to the presence of an alpha,beta-unsaturated ester. Taken all data together it is concluded that it is sufficiently demonstrated that the test substance has skin sensitizing properties. The test substance should therefore be classified as a skin sensitizer (cat. 1). Currently, no conclusion on sub-category can be made based on in vitro studies. Please also refer to attachment in Section 13: Weight of Evidence for Skin Sensitisation (statement 2018).