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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:
09 March 2018 to 29 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline" for further information
Principles of method if other than guideline:
Deviations from the study plan included:
- the test material was directly added to the selected vehicle to reach the concentration of 100 mM in order to avoid the any loss of test material
- all the lysine samples (co-elution controls, reference controls, test material and positive control samples) were incubated up to 29 hours and 38 minutes instead of 24 (± 2) hours. Since the incubation was homogeneous on this lysine analytical sequence and since the acceptance criteria were met, this slight deviation was considered not to have any significant impact on the validity or integrity of the study.

These deviations were considered not to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This test is part of a tiered strategy for skin sensitisation assessment, approved by regulatory authorities in the EU.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: colourless limpid liquid
- Storage conditions: in darkness at room temperature
Specific details on test material used for the study:
As the test material was found to be volatile, it was sampled with a pipette and the respective weight of the sampling was determined using the density of the test material. The test material was directly added to the selected vehicle (acetonitrile) to reach the concentration of 100 mM. This formulation had the aspect of a colorless limpid solution and was used just after its preparation.

In chemico test system

Details on study design:
CONTROL ITEMS
- Positive control: Cinnamaldehyde

- Co-elution control samples
In order to detect possible co-elution of the test material with a peptide, co-elution control samples were prepared by incubating the test material formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

- Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorised in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM. These samples were used to:
reference control A: check the accuracy of the calibration curve for peptide quantification,
reference control B: check the stability of the peptide during analysis,
reference control C: check that the solvent did not impact the percentage of peptide depletion.

TEST SYSTEMS
- Cysteine peptide
Peptide sequence: AC-RFAACAA-COOH
Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
Molecular weight: 750.88 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 111016HS_MHeW1017
Storage condition: At -20 °C
Description: White powder

The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

- Lysine peptide
Peptide sequence: AC-RFAAKAA-COOH
Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
Molecular weight: 775.91 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 120514HSDWW1017
Storage condition: At -20 °C
Description: White powder
Specific handling conditions : None

The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

METHOD
The test material was tested in one run. The run was processed as described below.

- Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

- Co-elution control samples preparation
For the co-elution control with cysteine peptide:
50 µL of test material formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile.
For the co-elution control with lysine peptide:
In parallel, 250 µL of test material formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

- Reference control samples preparation
Reference control A and B samples:
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples:
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide:
50 µL of vehicle (acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
For the reference control C prepared with lysine peptide:
In parallel, 250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

- Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

- Test material samples preparation
For the reactivity of test material with cysteine peptide:
50 µL of test material formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
For the reactivity of test material with lysine peptide:
In parallel, 250 µL of test material formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

- Incubation of the samples
All samples (co-elution controls, reference controls, test material and positive control samples) were then incubated during 24 (± 2) hours for the cysteine-peptide analytical sequence or up to 29 hours and 35 minutes for the lysine-peptide analytical sequence at 25 °C and protected from light before injection into the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.

HPLC/UV Conditions:
- Analytical column: Zorbax SB C18, 100 x 2.1 mm, 3.5 µm, (Waters). In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
- Mobile phase: Mobile phase A: acetonitrile + 0.085 % TFA; Mobile phase B: milli-Q water + 0.1 % TFA
- Flow: 350 µL/minute
- Gradient:
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90
- UV wavelength: 220 nm
- Rinse solution: acetonitrile
- Oven temperature: 30 °C
- Autosampler temperature: nominal temperature +25 °C
- Injection volumes: 7 µL for the Cysteine-peptide analytical sequence and 3 µL for the Lysine-peptide analytical sequence
- Retention times: Cysteine-peptide: approx. 9.5 minutes; Lysine-peptide: approx. 6.8 minutes
- Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test material samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test material replicate, the percent depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:

% depletion = [1 - (peptide peak area in replicate injection / mean peptide peak area in relevant reference control C samples)] x 100

Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

- Evaluation of the possible co-elution of the test item with the lysine or cysteine peptides
In order to detect possible co elution of the test materials with a peptide, chromatograms of the co-elution control samples were analysed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r²) ≥ 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10 % of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100 % with a SD < 14.9 %,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6 %,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0 %,
- the test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test material replicates should be < 14.9 % for the percent cysteine depletion value and < 11.6 % for the percent lysine depletion value.

DATA INTERPRETATION AND CLASSIFICATION
A test material was considered to have the following classification for peptide reactivity according to the mean peptide depletion results for cysteine and lysine:
Mean cysteine and lysine % depletions No co-elution Co-elution with Cys Co-elution with Lys Co-elution with cystein & lysine
0 % ≤ mean %depletion ≤ 6.38 % no/ minimal reactivity Inconclusive apply Cys
6.38 % < mean %depletion ≤ 22.62 % low reactivity ≥ low reactivity 1:10 only inconclusive
22.62 % < mean %depletion ≤ 42.47 % moderate reactivity ≥ moderate reactivity prediction
42.47 % < mean %depletion ≤ 100% high reactivity high reactivity model

Cysteine 1:10-only prediction model:
Mean of cysteine depletion Reactivity class
0 % ≤ Mean % depletion ≤ 13.89 % No or minimal reactivity
13.89 % < Mean % depletion ≤ 23.09 % Low reactivity
23.09 % < Mean % depletion ≤ 98.24 % Moderate reactivity
98.24 % < Mean % depletion ≤ 100 % High reactivity

The result of the DPRA is considered as positive as soon as low reactivity is detected.

Results and discussion

In vitro / in chemico

Results
Key result
Parameter:
other: percent cysteine and percent lysine depletions
Run / experiment:
mean
Value:
0.58
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
SOLUBILITY RESULTS
The test material was found soluble at 100 mM in acetonitrile without sonication. Acetonitrile was therefore chosen to be vehicle.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test material and positive control samples) was performed prior to HPLC analysis.
As precipitate was observed in the positive control samples incubated with the cysteine and lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.

EVALUATION OF THE RESULTS
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides.
- for the cysteine peptide, the mean depletion value was 0.20 %,
- for the lysine peptide, the mean depletion value was 0.95 %.


Applicant's summary and conclusion

Interpretation of results:
other: Not sensitising
Conclusions:
The mean of the percent cysteine and percent lysine depletions was equal to 0.58%. Accordingly, the test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.
Executive summary:

The potential for the test material to be sensitising to human skin was investigated in chemico, in a study which was conducted in accordance with the standardised guideline OECD 442C, under GLP conditons.

The reactivity of the test material was evaluated by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides.

-  for the cysteine peptide, the mean depletion value was 0.20 %,

-  for the lysine peptide, the mean depletion value was 0.95 %.

The mean of the percent cysteine and percent lysine depletions was equal to 0.58 %. Accordingly, the test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.