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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:
12 Sep - 30 Sep 2017
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:
4 Feb 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Deutschland
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile
EC Number:
947-727-8
Cas Number:
568591-00-4
Molecular formula:
C9H13N3
IUPAC Name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

In chemico test system

Details on the study design:
TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA
Batch number: 260515HS_DWW1115
- Purity: > 95%
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA
Batch number: 120514HSDW_W0517
Purity: > 95%

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
- Batch number: 1673078, Fisher Chemical
- Purity: ≥ 99.9%
The test substance was soluble in acetonitrile at 100 mM.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBS2662V, Sigma Aldrich
- Purity: ≥ 95%
The stock concentration of the positive control was prepared at 100 mM.

STABILITY AND PRECISION CONTROL
Stability and precision controls were prepared.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: at least 24 ± 2 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent, 1200 Series, with Chemstation, Rev. B.04.01
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with pre-column Phenomenex AJO4286 4.0 x 2.0 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20
% A: 90, 75, 10, 10, 90
% B: 10, 25, 90, 90, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 and 0.000 mM
- Column temperature: 30 °C
- Injection volume: 10 µL

Results and discussion

In vitro / in chemico

Results
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Remarks:
(mean value of 3 replicates)
Value:
0 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Co-elution of the test substance with lysine peptide peak was observed. (Therefore Prediction Model 2 was considered)
- The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.
- For the test substance no turbidity, precipitation or phase separation was observed when diluted with the lysine or cysteine peptide solution.
- Precipitation was observed for the positive control when diluted with the cysteine peptide solution (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.
- Phase seperation was observed for the positive control when diluted with the lysine peptide solution. Whereas turbidity was observed for the samples of the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis.
- Since the acceptance criteria for the depletion range of the positice control were fulfilled, the observed precipitation and phase seperation was regarded as insignificant.
- Co-elution of test item with the lysine peptide peak was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria were met.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.57%.
The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was > 0.99 (0.9992). The mean peptide depletion of the cysteine peptide was between 60.8% and 100% (73.05 %). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5265 mM, RC Cacetonitrile: 0.5170 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.97%, RC Cacetonitrile: 0.87%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 14.9% (PC: 0.61%; test item: 0.00%).
For the lysine run the coefficient of determination for the calibration curve was > 0.99 (0.9999). The mean peptide depletion of the lysine peptide was between 40.2% and 69.0% (58.30%).The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.4996 mM, RC Cacetonitrile: 0.4989 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.24%, RC Cacetonitrile 0.14%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 11.6% (PC: 1.46%; test item: 0.00%).

Any other information on results incl. tables

 RESULTS

Table 5: Depletion of lysine-containing peptide

Sample

Peak area (at 220 nm)

Peptide concentration (mM)

Peptide Depletion (%)

Mean Depletion (%)

SD (%)

CV (%)

Positive control

2198.9507

0.2087

57.98

58.30

1.46

2.50

2249.3828

0.2136

57.02

2099.5659

0.1992

59.88

Test substance*

-

-

-

-

-

-

-

-

-

-

-

-

SD    Standard Deviation

*        A major peak in co-elution control of the test substance was observed at the retention time of the lysine peptide. Therefore, co-elution of test substance occurred and no peak areas could be determined.

 

Table 6: Depletion of cysteine-containing peptide

Sample

Peak area (at 220 nm)

Peptide concentration (mM)

Peptide Depletion (%)

Mean Depletion (%)

SD (%)

CV (%)

Positive control

1268.3999

0.1469

72.35

73.05

0.61

0.83

1222.3235

0.1418

73.35

1217.9059

0.1413

73.45

Test substance*

4613.9087

0.5199

0.00

0.00

0.00

n.a*

4641.1514

0.5230

0.00

4606.9868

0.5192

0.00

n.a.     not applicable

Table 7: Categorization of the test substance

Prediction model

Prediction model 1

Prediction model 2

Test substance

Mean peptide depletion (%)

Reactivity Category

Prediction

Mean peptide depletion (%)

Reactivity Category

Prediction

Test substance*

-

-

-

0.0

Minimal reactivity

no sensitiser

Positive control

65.67

High reactivity

sensitiser

73.05

Moderate reactivity

sensitiser

 

 

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: no skin sensitising potential based on the key event “protein reactivity”.
Conclusions:
Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity. No skin sensitising potential was observed based on the key event “protein reactivity”.