Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 Jan - 26 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 in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

EC Number:

257-479-6

EC Name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

Cas Number:

51858-17-4

Molecular formula:

C25H16N4O12S2.2K

IUPAC Name:

dipotassium dihydrogen 4-{5-[3-carboxylato-5-oxo-1-(4-sulfonatophenyl)-1,5-dihydro-4H-pyrazol-4-ylidene]penta-1,3-dien-1-yl}-5-hydroxy-1-(4-sulfonatophenyl)-1H-pyrazole-3-carboxylate

Test material form:

solid: crystalline

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. The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine.

TEST METHOD
In the DPRA the free concentration of cysteine- or lysine-containing peptide following incubation with the test item 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: 19.16 mg cysteine was dissolved in 37.26 mL phosphate buffer (pH 7.5), concentration: 0.667 mM
Lysine-containing peptide: 18.74 mg lysine was dissolved in 335.63 mL ammonium acetate buffer (pH 10.2), concentration: 0.667 mM

VEHICLE
- Substance: destilled water:acetonitrile (1:1 v/v)
- Justification for selecting vehicle: the test item was completely soluble in destilled water:acetonitrile 1:1 (v/v), therefore, destilled water:acetonitrile 1:1 (v/v) 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
- Reference contol A: acetonitrile was used to verify the accuracy of the calibration curve for peptide quantification
- Reference control B: acetonitrile was used to verify the stability of the respective peptide over the analysis time; its replicates were injected in the beginning and in the end of each HPLC run
- Reference control C (RC C) was set up for the test item and the positive control; RC C for the positive control was prepared using acetonitrile; RC C for the test item was prepared using the test item vehicle (destilled water:acetonitrile (1:1 v/v)); the RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD); additionally, reference control C was used to calculate PPD; the RC C was included in every assay run for both peptides and was injected together with the samples

TEST SUBSTANCE PREPARATION
- test item was prepared as a 100 mM solution in vehicle (dist water:acetonitrile (1:1 v/v))

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: 24 ± 2 h

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

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent 1260 II with Chromeleon 7.2 SR5
- Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm (Agilent Art. Nr. 861753-902)
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; % A: 90, 75, 10, 10, 90; % B: 10, 25, 90, 90, 10
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution
- Injection volume: 4 μL
- Peptide standards: standard calibration curves were generated for both, the cysteine and the lysine peptide; peptide standards were prepared in a solution of 20% acetonitrile:80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 and 0.000 mM

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 74.85%. The mean peptide depletion of the positive control for the lysine peptide was 62.65%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion (%)

Value:

47.14

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

In the cysteine experiment a minor co-elution was observed, corresponding to 0.043 mM (compared to the total elution of 0.273 mM). Due to the co-elution, the obtained value is only an estimation of the peptide depletion, however, as a positive result is obtained, the result can be used for interpretation.

Results 2

Run / experiment:

other: lysine run

Parameter:

other: mean peptide deplation (%)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: see remarks

Remarks:

Co-elution of the test item with the lysine peptide peak in the lysine experiment was observed. Therefore sensitising 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 (acetonitril:water 1:1 (v/v)).

Other effects / acceptance of results:

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples incubated with the peptide. Samples were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Laboratory technical proficiency was demonstrated by correctly obtaining the expected DPRA prediction for the 10 proficiency substances indicated in the guideline (OECD 442C).

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 replicat
es 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 3. 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	4.0850	0.1268	75.22	74.85	0.50	0.67
	4.1130	0.1276	75.05
	4.2401	0.1316	74.28
Test Item*	9.3278	0.2887	44.00	47.14	3.59	7.61
	8.9349	0.2765	46.36
	8.1535	0.2524	51.05

* A minor peak in the co-elution control of the test item was observed at the retention time of the cysteine peptide (peak area = 1.369). Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of cysteine peptide present in the test item samples. The values can only be considered as an estimation of the peptide depletion.

Table 4. 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	5.3694	0.1943	61.31	62.65	1.16	1.85
	5.0902	0.1842	63.32
	5.0901	0.1842	63.32
Test Item*	-	-	-	n/a	n/a	n/a
	-	-	-
	-	-	-

n/a:        not applicable

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

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitising potential based on the key event "direct peptide binding"

Conclusions:

Under the conditions of the test, the test item is a sensitiser in the Direct Peptide Binding Assay. This result alone does not allow for the non-classification or classification according to Regulation (EC) No. 1272/2008 of the test item as skin sensitiser and therefore further data were generated in a Weight of Evidence (WoE) approach.