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:
14 Feb - 14 Apr 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
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted Feb 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Rheinland-Pfalz, Germany
Type of study:
direct peptide reactivity assay (DPRA)

Test material

In chemico test system

Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST METHOD
The DPRA is an in chemico method, which quantifies the remaining concentration of cysteine or lysine containing peptide, following 22 hours incubation with the test item at 25 ± 2.5 °C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. Cysteine and lysine peptide percent depletion values are calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

TEST SYSTEM
- Supplier of synthetic peptides: Genecust, Dudelange, Luxemburg
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in appropriate buffer solution.
Cysteine-containing peptide: 0.669 mM Cys-Peptide solution was prepared by dissolving 22.6 mg of the peptide in 45.0 mL phosphate buffer, pH 7.5 (batch no. 20180215 and batch no. 20180308).
Lysine-containing peptide: 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. 20180214 and batch no. 20180312) and by dissolving 15.5 mg of the peptide in 30.0 mL ammonium acetate buffer, pH 10.2. (batch no. 20180412).

POSITIVE CONTROL
- Cinnamaldehyde (CAS 104-55-2, 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.

CO-ELUTION CONTROL
- Co-elution control was prepared without peptide, to verify if the test item absorbs at 220 and 258 nm and has a similar retention time as a peptide and/or interfere with the data analysis.

REFERENCE 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.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: cysteine-containing peptide: 1:10; lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: minimum of 22 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for test substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent Technologies with UV Detector
- Analytical Column: ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenomenex Security Guard C18, 4x3 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in water (HPLC grade)
B: 0.085% (v/v) trifluoracetic acid in acetonitrile (HPLC grade)
- Flow: 0.55 mL/min
- Column temperature: 30 ± 2 °C
- Gradient:
Time (min): 0, 10, 10.5, 12, 13, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 7 μL
- peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, and a buffer blank were measured.

Results and discussion

Positive control results:
Mean peptide depletion (%) of the positive control was 31.57, 26.01 and 34.53% in all three experiments for the lysine peptide, respectively, and 77.61 and 81.40% in both experiments for the cysteine peptide, respectively.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Mean peptide depletion (%)
Value:
7.81
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Mean peptide depletion (%)
Value:
7.83
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS
- The mean cysteine peptide depletion and SD of the three replicates of the positive control cinnamaldehyde were 77.61% ± 2.46% and 81.40% ± 2.00% for both experiments, and thus in the acceptable range as defined in the acceptability criteria.
- The mean lysine peptide depletion and SD of the three replicates of the positive control 2,3-butanedione were 31.57% ± 1.48%, 26.01% ± 2.69% and 34.53% ± 2.31% for the three experiments, and thus in the acceptable range as defined in the acceptability criteria.
- The SD for the test item replicates were 1.42 and 3.75%, respectively, and thus < 14.9% in both experiments with the cysteine peptide.
- The SD for the test item replicates were 5.38, 5.06 and 2.40%, respectively, and thus < 11.6% in all three experiments with the lysine peptide.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 7.81% in the first experiment. The value was verified with a mean depletion of 7.83% in the second experiment, therefore the test item was classified with:
DPRA Prediction: positive
Reactivity class: low

Any other information on results incl. tables

Table 1: Calculated peptide depletion values for the Lys-Peptide (Experiment 1)

 

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

30.20

 

31.57

 

1.48

Positive control Rep. 2

31.39

Positive control Rep. 3

33.14

Test item Rep. 1

4.04

 

4.90

 

5.38

Test item Rep. 2

0.00

Test item Rep. 3

10.65

Rep.: Replicate

SD: Standard deviation

Table 2: Calculated peptide depletion values for the Lys-Peptide (Experiment 2)

 

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

24.28

 

26.01

 

2.69

Positive control Rep. 2

24.64

Positive control Rep. 3

29.11

Test item Rep. 1

1.57

 

3.68

 

5.06

Test item Rep. 2

0.00

Test item Rep. 3

9.45

Rep.: Replicate

SD: Standard deviation

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

 

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

32.96

 

34.53

 

2.17

Positive control Rep. 2

33.64

Positive control Rep. 3

37.00

Test item Rep. 1

3.00

 

4.79

 

2.40

Test item Rep. 2

7.51

Test item Rep. 3

3.85

Rep.: Replicate

SD: Standard deviation

Table 4: Calculated peptide depletion values for the Cys-Peptide (Experiment 1)

 

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

74.78

 

77.61

 

2.46

Positive control Rep. 2

78.77

Positive control Rep. 3

79.27

Test item Rep. 1

9.85

 

10.71

 

1.42

Test item Rep. 2

9.94

Test item Rep. 3

12.35

Rep.: Replicate

SD: Standard deviation

Table 5: Calculated peptide depletion values for the Cys-Peptide (Experiment 2)

 

Sample name

Depletion [%]

Single

Mean*

SD

Positive control Rep. 1

79.37

 

81.40

 

2.00

Positive control Rep. 2

81.47

Positive control Rep. 3

83.37

Test item Rep. 1

8.24

 

11.98

 

3.75

Test item Rep. 2

11.98

Test item Rep. 3

15.74

Rep.: Replicate

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: "low" skin sensitising potential based on the key event "protein reactivity"
Conclusions:
Under the conditions of the test, the test substance showed reactivity towards selected proteins. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No. 1272/2008.