2-phenylimidazole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Nov 2017 - 20 Jan 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:

Bayerisches Landesamt für Gesundheit und Lebensmittelrecht, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylimidazole

EC Number:

211-581-7

EC Name:

2-phenylimidazole

Cas Number:

670-96-2

Molecular formula:

C9H8N2

IUPAC Name:

2-phenyl-1H-imidazole

Test material form:

solid: flakes

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 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: 20.02 mg cysteine was dissolved in 38.92 mL phosphate buffer (pH 7.5).
- Concentration: 0.667 mM
Lysine-containing peptide: 20.56 mg lysine was dissolved in 39.09 mL ammonium acetate buffer (pH 10.2).
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: acetonitrile
- Justification for selecting vehicle: the test substance was completely soluble in acetonitrile, therefore acetonitrile was chosen as suitable vehicle for the main experiments.

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).
- Acetonitrile was used to verify that the solvent does 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 69.31%. The mean peptide depletion of the positive control for the lysine peptide was 56.62%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion (%)

Value:

1.69

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

69.31

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

56.62

Other effects / acceptance of 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: Results 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)
STD1	4895.3867	0.5340	4254.6284	0.5340
STD2	2320.1248	0.2670	2161.7087	0.2670
STD3	1141.6589	0.1335	1051.8925	0.1335
STD4	567.6173	0.0667	525.2503	0.067
STD5	292.4744	0.0334	260.5259	0.0334
STD6	148.1837	0.0167	130.8325	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Table 2: Depletion of the 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	1436.3897	0.1610	68.81	69.31	0.45	0.65
	1406.1344	0.1577	69.46
	1396.6488	0.1566	69.67
Test substance	4554.3960	0.5024	1.09	1.69	0.70	41.48
	4535.2725	0.5003	1.51
	4491.5972	0.4955	2.46

Table 3: Depletion of the 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	1675.9452	0.2100	57.93	56.62	1.13	2.00
	1753.5828	0.2197	55.96
	1754.5223	0.2198	55.95
Test substance	4225.5088	0.5290	0.00	0.00	0.00	n/a
	4240.1699	0.5308	0.00
	4214.0840	0.5275	0.00

Table 4: Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model¹

Mean Cysteine and Lysine PPD	Reactivity Class	DPRA Prediction^2
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 Model¹

Cysteine PPD	Reactivity Class	DPRA Prediction^2
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

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.

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event "direct peptide binding"

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is not a sensitiser in the Direct Peptide Binding Assay. 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.