Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 April to 25 July, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other: study plan
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
yes
Remarks:
See "Any information on materials and methods"
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

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

In chemico test system

Details on study design:
ANALYTICAL INSTRUMENTS
1. HPLC System
Designation: HPLC_4
Components: Degasser G1322A; Quaternary pump G1311A; Autosampler G1313A; -Column compartment G1316A; UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
Usage and calibration followed the corresponding SOP 114 00 526 in the current edition.

2. Column
An ACE Excel SuperC18 150 x 3 mm column with 3 μm particles and pre-column Phenomenex SecurityGuard C18, 4 x 3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline.

3. HPLC Program
Eluent A: H2O + 0.1 % TFA
Eluent B: Acetonitrile + 0.085 % TFA
Gradient: % over time as follows:
- 0 minutes: 90 % A; 10 % B
- 10 minutes: 75% A; 25 % B
- 10.5 minutes: 10 % A; 90 % B
- 12 minutes: 10 % A; 90 % B
- 13 minutes: 90 % A; 10 % B
- 20 minutes: 90 % A; 10 % B
Flow rate: 0.55 mL/min
Injection volume: 7 μL
Column temperature: 30 °C
Wavelength 1: 220 nm
Wavelength 2: 258 nm

TEST SYSTEM
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
- Cys-Peptide (Cysteine): Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol); Batch no.:P170415-2-LR569638; Purity: 98.10%
- Lys-Peptide (Lysine): Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol); Batch no.: P170415-2-LR569640; Purity: 98.85%

INSTRUMENTS AND DEVICES
- Analytical scales Mettler Toledo XS205DU
- Adjustable pipettes with one-way tips Rainin ®
- Repeater pipette AutoRep E, Rainin ®-
- Fridge
- Carbon analyser multi N/C 2100 S Analytik Jena AG
- Conductometer 538, wtw
- Radio controlled watch
- Ultrasonic batch SONOREX RK510H, Bandelin
- pH-meter wtw 540 GLP
- Incubation chamber Memmert ICP-600
- Temperature Data logger ebro
- Standard laboratory glassware
- Centrifuge EBA 20

CHEMICALS
- Water for chromatography H2O, Honeywell, HPLC grade H2O, J.T. Baker, LC/MS grade
- Demineralised water H2O, from ion exchange cartridge. Total organic carbon (TOC) < 1 ppm, conductivity < 0.1 S/cm
- Acetonitrile for chromatography CH3CN, ACN, Honeywell, HPLC grade
- Trifluoroacetic acid TFA, for spectroscopy, Merck
- Phosphoric acid: H3PO4, 85%, p.a.
- Ammonia solution: NH3, 25%, p.a.
- Sodium hydroxide solution: NaOH, 45%, p.a
- Ammonium acetate, CH3COONH4, p.a, Sigma Aldrich
- Sodium dihydrogen phosphate, Na2HPO3 * 2H2O, p.a., Roth

BUFFERS
- 25 mM Phosphate buffer (batch no. 20180711): 1.1109 g disodium hydrogen phosphate dihydrate were dissolved in demineralised water, pH was adjusted to 7.50 with 85 % H3PO4 (final volume 250 mL).
- 25 mM Ammonium acetate (batch no. 20180412): 482.0 mg ammonium acetate (anhydrous) were dissolved in demineralised water, pH was adjusted to 10.19 with 25 % ammonia (final volume 250 mL).

CONTROLS
Positive controls:
- Cinnamaldehyde (CAS 104-55-2, food grade 99.1 %, batch no. MKBT8955V) was used as 100 mM solution in acetonitrile for the Cys-peptide.
- 2,3-Butanedione (CAS 431-03-8, 99.4 %, batch no. BCBS3560V) was used as 100 mM solution in acetonitrile for the Lys-peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls:
- For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion of positive controls.

Co-elution control:
Sample prepared from the respective peptide buffer and the test item, but without peptide.

CONDUCT OF THE STUDY
The test was performed two times for Cys-peptide and one time for Lys-peptide. The determination was invalid for one experiment:
- Cys-peptide assay performed on 25. Apr. 2018 (Standard derivation of the test item is out of range)
The results of these experiment are not reported but saved together with all other raw data of the study in the test facility’s GLP document archive.

DISSOLUTION OF THE TEST ITEM
As the test item is a mixture with unknown composition, it was used neat for testing and treated as 100 mM solution for evaluation.

TEST SOLUTIONS
Dilution buffers:
- 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.5 (Peptide dilution buffer C)
- 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.2 (Peptide dilution buffer K)

Peptide stock solutions:
- 0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2. (batch no. 20180426)
- 0.667 mM Cys-Peptide solution was prepared by dissolving 17.5 mg of the peptide in 35.0 mL phosphate buffer, pH 7.5. (batch no. 20180724)

Peptide calibration standards:
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer (see chapter 7.2.1): 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM Peptide.
Calibration samples were analysed before the samples containing the test item. Blank dilu-tion buffer was also measured.

Test item samples:
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.

INCUBATION
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22 h for Cys-peptide and 24 h for Lys-peptide.
All three Replicates of test item incubated with Cys-peptide (both experiments) and all three Replicates of positive control Cinnamaldehyde (batch 20180425) were turbid after incubation.

MEASUREMENTS
Measurements were performed using the HPLC method. Results are shown in Table 7.4–a and Table 7.4–b of "Illustrations" in "Overall remarks, attachments".

CALCULATION OF CALIBRATION CURVE
Determination:
From the peak areas of the peptide calibration standards detected at 220 nm, linear calibration curves in the form y = b*x + a were calculated for both peptides using validated Microsoft Excel® spreadsheets, where:
y = Measured peak area [mAU*min]
b = Slope [mAU*min / mM]
a = Intercept [mAU*min]
x = Standard concentration [mM]
See Table 8.1-a and FIgures 8.1-a and 8.1-b in "Illustrations" of "Overall remarks, attachments".

Acceptance Criteria: the r² value of linear calibration should be > 0.99.
Assessment: the calibration curve was linear with acceptable coefficient of determination 0.99988 and 0.99999 for the Cys-peptide and the Lys-peptide, respectively.

DETERMINATION OF SOLVENT CONTROLS
The peptide concentrations in the solvent controls were calculated using the linear regression (a = intercept, b = slope).
Peptide concentration [mM] = (Peak area [mAU*min]-a) / b
See Table 8.2-a in "Illustrations" of "Overall remarks, attachments".

Acceptance criterion
- The mean peptide concentration of solvent control samples of sets A and C should be 0.50 ± 0.05 mM
- The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
Assessment: the peptide concentrations of all solvent controls (Reference A and Reference C) were in the acceptable range of 0.50 ± 0.05 mM for both peptides. The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 0.5 % for the Lys-peptide and 4.3 % for the Cys-Peptide, respectively.

CALCULATION OF PEPTIDE DEPLETION
The peptide depletion was calculated for each individual sample using the following equa-tions (shown for Cys-Peptide, Lys-peptide is calculated analogously):
Peptide depletion C,i = (1 - (measured peptide peak area in sample / mean peptide peak area in solvent controls C)) * 100

The mean peptide depletion of the Cys-peptide was calculated as follows:
Peptide depletion cys = (sum of (cys,i=1,2,3 Peptide depletion i)) / 3
The mean peptide depletion of the test item was calculated using the following equation:
Mean peptide depletion (%) = (% Peptide depletion of cysteine + % Peptide depletion of lysine) / 2


EVALUATION OF RESULTS

Results and discussion

Positive control results:
The calculated peptide depletion value for the lysine peptide assay were 33.60 %, 39.36 % and 35.75 % (mean = 36.24 %).
The calculated peptide depletion value for the cysteine peptide assay were 77.39 %, 79.15 % and 80.26 % (mean = 78.93 %).

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: mean Lysine peptide depletion (%)
Value:
3.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: mean Cysteine peptide depletion (%)
Value:
98.29
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Mean depletion of both peptides after incubation with the test item: 50.84 %.

Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion

Assessment
a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.
c) The maximum standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item. The maximum standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Evaluation of results
According to the test guideline, the reactivity is classified as “high”, “moderate”, “low” or “minimal” using the Cysteine 1:10/Lysine 1:50 prediction model shown in Table 8.4–a in "Illustrations" of "Overall remarks, attachments".
The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 50.84 %, therefore the test item was classified with:
- DPRA Prediction: Positive
- Reactivity class: High

Proficiency
The ten proficiency chemicals listed in the guideline were tested using the analysis method. Eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline (in-house study).

Historical data
Historical data of Lys-peptide depletion values of valid tests support the acceptance criteria for the proficiency chemical 2,3-butanedione defined in the guideline (mean depletion of the Lys peptide of 10 – 45 %). Therefore, the usage as positive control with the same acceptance criteria is reasonable. See Table 8.6-a in "Illustrations" of "Overall remarks, attachments".

DISCUSSION
All acceptance criteria were fulfilled, therefore the test was considered valid. The DPRA prediction for the test item was positive with "reactivity class: high" according to the Cysteine 1:10/Lysine 1:50 prediction model.
No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Applicant's summary and conclusion

Interpretation of results:
other: induces molecular interactions via skin proteins
Conclusions:
The test item reacted positive with "reactivity class: high" in the Cysteine 1:10/Lysine 1:50 prediction model, therefore, the test item induces molecular interactions via skin proteins.
Executive summary:

The sensitising potential of the test item was evaluated using the Cysteine 1:10/Lysine 1:50 prediction model of the direct peptide reactivity assay (DPRA), according to the OECD Guideline 442C (2015). The test item was incubated for 24 ± 2 hours at 25 °C together with cysteine and lysine peptides, and the subsequent peptide concentration was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cysteine and Lysine peptides, respectively. Triplicate samples of a solvent control, peptide buffer controls and positive controls (Cinnamaldehyde, 100 mM in acetonitrile for cysteine; 2,3 -Butanedione, 100 mM in acetonitrile for lysine) were incubated and measured in parallel.

Mean cystein depletion was 98.29 %; mean lysine depletion was 3.40 %. Mean peptide depletion was therefore 50.84 %. The test item was therefore found to be positive with high reactivity to the Cysteine 1:10/Lysine 1:50 prediction model, indicating that the test item induces a molecular interaction via skin proteins, via peptide/protein bonding.