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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:
07 - 22 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
adopted 05 Feb 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Test material

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

In chemico test system

Details on 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 direct peptide reactivity assay (DPRA) is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, the protein reactivity. The reativity of the substance towards model synthetic peptides containing either lysine or cysteine is quantified. In the DPRA the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance is quantified. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 and 258 nm. Cysteine- and lysine peptide depletion values are then calculated and used in the prediction model, which assigns the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier of synthetic peptides: JPT Peptide Technologies GmbH
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the approprate peptide in appropriate buffer solution.
Cysteine-containing peptide: 18.48 mg cysteine was dissolved in 36.584 mL phosphate buffer (pH 7.5).
- Concentration: 0.667 mM
Lysine-containing peptide: 18.82 mg lysine was dissolved in 35.159 mL ammonium acetate buffer (pH 10.2).
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: water
- Justification for selecting vehicle: the test substance was soluble in the vehicle.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile.

CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide.

REFERENCE CONTROLS
- Acetonitrile was used to verify the accuracy of the calibration curve for peptide quantification (Reference contol A) and to verify the stability of the respective peptide over the analysis time (Reference control B)
- Water and acetonitrile were used to verify that the solvents do not impact the percent peptide depletion (Reference controls C)

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 24 ± 2 h

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

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent 1200 Series, with Chemstation, Rev. B.04.01
- Analytical Column: Agilent Zorbax SB-C18, 3.5 µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286, 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
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution
- Injection volume: 10 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 in parallel with the test substance samples

Results and discussion

Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
52.81
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
74.90
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Remarks:
In the lysine run co-elution of the test substance and the lysine peptide due to a shift of the lysine peptide peak was observed.
Other effects / acceptance of results:
OTHER EFFECTS:
In the lysine run a a co-elution of the test substance with the lysine peptide peak was observed. Since the reference control samples allowed clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, it was clear that the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. As all validity criteria were fulfilled the results of the lysine run was not considered having an influence on the quality or validity of the overall results.

ACCEPTANCE CRITERIA

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Table 1: Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD7

0.0000

0.0000

0.0000

0.0000

STD6

153.8068

0.0167

139.6547

0.0167

STD5

321.6256

0.0334

281.4845

0.0334

STD4

642.2243

0.0667

555.3300

0.0667

STD3

1318.5444

0.1335

1100.1805

0.1335

STD2

2593.4377

0.2670

2176.1533

0.2670

STD1

5075.1016

0.5340

4280.0898

0.5340

 

Table 2: Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1193.1283

0.1239

74.99

74.90

0.25

0.34

1187.7434

0.1233

75.10

1210.7732

0.1258

74.62

Test Item

2310.8784

0.2412

51.07

52.81

1.77

3.35

2143.9973

0.2237

54.60

2230.5615

0.2328

52.77

Table 3: Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1861.8433

0.2304

55.78

54.93

0.78

1.42

1926.2322

0.2384

54.25

1904.2300

0.2357

54.77

Test Item

n.a.*

 

 

 

 

 

n.a.*

 

 

n.a.*

 

 

* not applicable due to co-elution

Table 4: Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Table 5: Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Table 6: Categorization of the Test Item

Prediction Model

Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)

Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

n.a.

n.a.

n.a.

52.81

Moderate Reactivity

sensitizer

Positive Control

64.92

High Reactivity

sensitizer

74.90

Moderate Reactivity

sensitizer

 

Applicant's summary and conclusion

Interpretation of results:
other: 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.