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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:
10 March 2017 - 17 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In chemico test system

Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: Ac-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Positive control: cinnamic aldehyde, purity > 95%, was prepared at a concentration of 100 mM in acetonitrile.

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for Cysteine, 100 mM phosphate buffer pH 7.5, for Lysine 100 mM Ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared.
Cysteine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM of test substance, 0.5 mM Cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution. An additional control sample of 5 mM of test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials.
Lysine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in Lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM Lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine.
The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution. An additional control sample of 25 mM of test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Incubation:
The appearance of the substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis:
The concentration of both the cysteine and lysine peptides in the presence of the substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 μm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes
Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Acceptance criteria for analytical measurements are presented in Table 1 in the section "Any other information on materials and methods incl. tables". Information on the interpretation of the results is presented in the same section in Table 2.

Results and discussion

Positive control results:
72.5% depletion (SD 0.11%, n = 3) and 59.7% depletion (SD 1.40%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: cysteine depletion, %
Run / experiment:
1
Value:
0.633
Vehicle controls validity:
valid
Remarks:
stability and precision controls
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: lysine depletion, %
Run / experiment:
1
Value:
0.046
Vehicle controls validity:
valid
Remarks:
stability and precision controls
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable. Acceptance criteria for reference (stability) controls and precision controls of both peptides were met (CV 0.70%, n = 6 and CV 1.17%, n = 6, for cysteine and lysine, respectively, at 0.51 mM).
- Acceptance criteria met for positive control: yes, 72.5% depletion (SD 0.11%, n = 3) and 59.7% depletion (SD 1.40%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.40% and 0.91%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:
Mean depletion of 0.633% and 0.0460% was observed for the test substance with cysteine and lysine peptides, respectively. A small co-elution peak, equivalent to 0.086% of the mean peak area of the Lysine peptide was observed in the Lysine assay. This small co-elution peak in the Lysine assay did not have an impact on the data. No co-elution peak was observed in the Cysteine assay.
Based on these results the reactivity is classified as "no to minimal".

Applicant's summary and conclusion

Interpretation of results:
other: DPRA was negative
Conclusions:
It can be concluded that this DPRA test is valid, and with the mean result of 0.340% depletion the test substance is placed in the reactivity class of “no to minimal reactivity” and thus is predicted to be negative in the DPRA.
Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance (5 mM substance with 0.5 mM Cysteine and 25 mM substance with 0.5 mM Lysine) were successfully analyzed by the validated DPRA analytical method. All analytical acceptance criteria of the test were met. A small co-elution peak, equivalent to 0.086% of the mean peak area of the Lysine peptide was observed in the Lysine assay. This small co-elution peak in the Lysine assay did not have an impact on the data. No co-elution peak was observed in the Cysteine assay. The substance caused 0.633% cysteine peptide depletion and 0.0460% lysine peptide depletion. Based on the mean result of 0.340% depletion the substance is classified as “no to minimal reactivity” based on the DPRA prediction model. The substance was thus considered to be negative in the DPRA.