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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:
3 Dezember 2018 - 6 December 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04. Feb. 2015
Deviations:
yes
Remarks:
Please refer to "Other effects/acceptance of results"
Qualifier:
according to
Guideline:
other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal test-ing): “DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensi-tisation Testing.”
Version / remarks:
29. Jun. 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: SOP 118 00 875 edition 1, valid from 29. Oct. 2018, „Durchführung des DPRA-Tests nach OECD 442C
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

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

In chemico test system

Details on study design:
Synthetic peptides:
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Instruments and Devices:
HPLC system
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenom-enex SecurityGuard C18, 4x3 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.

Heating chamber, Centrifuge, Fridge, Repeater pipette, pH-meter, Analytical scale, Precision scale, Pipette 100 – 1000 µL, Vortexer, Glass thermometer, Conductometer, Carbon analyser

Chemicals:
Water for chromatography
H2O, Honeywell, HPLC 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
CH3CN, ACN, AppliChem, analysis grade
Trifluoroacetic acid
TFA, Merck, for spectroscopy
Ammonium hydroxide
NH3,25 %, p.a.
Ammonium acetate,
CH3COONH4, p.a, Sigma Aldrich
Sodium dihydrogen phosphate
NaH2PO4 * 1 H2O, p.a.
Disodium hydrogen phosphate
Na2HPO4 * 7 H2O, p.a.

Buffers:
100 mM Phosphate buffer (mix out of solution A + B)
Solution A: 1.38 g sodium dihydrogen phosphate monohydrate (monobasic) are dissolved in 100 mL demineralized water.
Solution B: 6.70 g disodium hydrogen phosphate heptahydrate (dibasic) are dissolved in 250 mL demineralized water.
Final 100 mM phosphate buffer is mixed out of 18 mL of solution A and 82 mL of solution B. The pH is adjusted to 7.503 with solution B.
100 mM Ammonium acetate buffer (batch no. 20181204)us) are dissolved in 200 mL demineralised water, pH is adjusted to 10.199 with 25 % ammonium hydroxide solution.

Positive control:
Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %),100 mM solution in acetonitrile for the cysteine peptide
2,3-Butanedione (CAS 431-03-8, >97 %), 100 mM solution in ace-tonitrile for the lysine 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 are prepared in triplicate (Sets A, B1, B2 and C, total 12 samples per peptide). Set A is analysed together with the peptide calibration standards, sets B1 and B2 are analysed at the start and end of the analysis sequence and are used as stability control for the peptide over the total analysis time. Set C is incubated and analysed together with the samples and is used for calculation of the peptide depletion.

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

Peptide stock solutions
The peptide stock solutions are freshly prepared for each assay.
0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45 mL phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45 mL ammonium acetate buffer, pH 10.2.

Test item stock solution:
The test item stock solution is freshly prepared for each assay.
100 mM test item solution was prepared by dissolving 154.4 mg test item in 3 mL of the solvent acetonitrile for the Cys-peptide and 154.5 mg test item for the Lys-peptide, respectively. The solution was vortexed until the test item was dissolved.

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

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 solution), the Lys-peptide sam-ples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item solution) using the stock solutions described. A final volume of 1 mL per sample was prepared for each sample.

Incubation:
The positive control, solvent control and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h. Samples appearing turbid or where precipitation is visible by the unaided eye are centrifuged (benchtop centrifuge, 10 min at 4500 rpm) and only the clear supernatant is used for measurement.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Evaluation criteria of results according to the cysteine 1:10 prediction model.
mean Cys peptide depletion [%] Reactivity Evaluation
> 98.24 - ≤ 100 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
0 - ≤ 13.89 minimal or no reactivity negative

Acceptance criteria
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3- Butane-dione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion.

Results and discussion

Positive control results:
The mean Cys-Peptide depletion of the positive control was 80.01%.

In vitro / in chemico

Results
Key result
Parameter:
other: mean peptide depletion Cys peptide (%)
Run / experiment:
1st run
Value:
78.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline were tested using the analysis method described. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Deviations from the Guideline
The mean percent area ratio 220 nm/258 nm of the positive control 2,3 Butanedione in experiment 1 is 120 %. This is marginal out of the given range 90-110 % and shows a peak impurity and co-elution. This was considered uncritical, because the value of peptide-depletion was reported as “co-elution – percent depletion estimated”, it is not an acceptance criterium for the study and the results of the Lys-peptide were not considered for the evaluation with the Cysteine 1:10 prediction model.


Any other information on results incl. tables

Table 1: Historical Data

Parameter

Depletion [%]

Depletion [%]

Peptide

Cys- Peptide

Lys- Peptide

Mean

82.34

35.35

Standard
Deviation

8.28

6.80

Range 2σ

65.77 – 98.91

21.76-48.95

Study

80.01

13.58

Table 2: Calculated peptide depletion values for the Cys-Peptide

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

77.46

80.01

2.28

Positive control Rep. 2

80.72

Positive control Rep. 3

81.85

Test item Rep. 1

74.29

78.11

3.77

Test item Rep. 2

78.21

Test item Rep. 3

81.83

Table 3: Calculated peptide depletion values for the Lys-Peptide

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

12.48

13.58 **

1.25

Positive control Rep. 2

13.32

Positive control Rep. 3

14.95

Test item Rep. 1

0 (-39.12) *

0.00 ***

0.00

Test item Rep. 2

0 (-38.23) *

Test item Rep. 3

0 (-34.79) *

* Note: Negative depletion values were considered as “zero” when calculating the mean.
** Note: The area ratio 220 nm/258 nm was marginal out of range. The value is reported as “co-elution – percent depletion estimated”
*** Note: Due to co-elution the results of the Lys-peptide were not considered when using the Cysteine 1:10 prediction model for the evaluation.

Applicant's summary and conclusion

Interpretation of results:
other: peptide depletion
Conclusions:
The DPRA prediction is “positive” with moderate reactivity according to the Cysteine 1:10 model. It can be stated that in this study and under the experimental conditions reported, the test item possesses a moderate skin sensitisation potential
Executive summary:

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated 23 h at 25 °C for the Cys peptide and 22.5 h for the Lys-peptide, respectively. The peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously. One valid experiment was performed.

For the Lys-peptide co-elution occurred with the test item. Therefore, the evaluation was carried out with the Cysteine 1:10 prediction model.

The mean peptide depletion in the Cys-peptide assay was 78.11 %. All acceptance criteria were fulfilled; therefore, the test was considered valid. The DPRA prediction for the test item was positive with reactivity class moderate according to the Cysteine 1:10 prediction model.