2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate

Skin sensitisation

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted 05 Feb 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

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

Test material information

Constituent 1

Reference substance name:

2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate

EC Number:

600-039-9

Cas Number:

10023-48-0

Molecular formula:

C12H17N4O4PS

IUPAC Name:

2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate

Test material form:

solid

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: 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

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

52.81

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

74.90

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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 Prediction²
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.