Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 February 2017 - 6 September 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:

04 February 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.59 In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA); Official Journal of the European Union, No. L 112/1

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

EC Number:

916-867-1

Cas Number:

12270-00-7

Molecular formula:

C32H18CoN6O8Na - C32H20CoN8O8Na - C32H22CoN10O8Na

IUPAC Name:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/137

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway,NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Material and technical equipment:
HPLC: Liquid chromatograph Thermo Scientific, Dionex Ultimate 3000 consisting of the following modules:
Pump: HPG-3400RS
Autosampler: WPS-3000TSL
Column oven: TCC-3000
UV-Detector: DAD-3000
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column
SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID (Phenomenex)
HPLC mobile phase A: H2O/ACN/TFA 950/50/1 V/V/V
HPLC mobile phase B: ACN/H2O/TFA 950/50/0.85 V/V/V

Reagents for preparing the buffers for pH 7.5 phosphate buffer (used for solving C-containing peptide):
Sodium phosphate, monobasic monohydrate, CAS no. 10049-21-5
Sodium phosphate, dibasic heptahydrate, CAS no. 7782-85-6 for pH 10.2 ammonium acetate buffer (used for solving K containing peptide):
Ammonium acetate, CAS no. 631-61-8
Ammonium hydroxide, 28% – 30%, CAS no. 1336-21-6

Controls:
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test substance preparation:
The test substance was prepared as a 100 mM (considering a molecular weight of 756 g/mol and a purity of 80%) preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.
Vehicle: acetonitrile
Reason for the vehicle: The test substance was soluble in acetonitrile.

Experimental procedure:
Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm.
In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Preparation of peptide stock solutions:
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

Preparation of calibration samples:
The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution.
(0.534, 0.267, 0.134, 0.067, 0.033, 0.017, 0.000 mM peptide)

Preparation of the test-substance samples:
C-peptide
750 μL C-peptide stock-solution
200 μL solvent (vehicle)
50 μL test-substance (preparation)
(or PC-preparation or solvent (VC))

K-peptide
750 μL K-peptide stock-solution
250 μL test-substance (preparation)
(or PC-preparation or solvent (VC))

The samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Preparation of the co-elution control:
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Data evaluation:
For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples.

Calculation of the peptide concentrations:
For each peptide and test run a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration.The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).

Calculation of the peptide depletion:
The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance concentration (C-containing and K-containing peptide depletion; example calculation for C-containing peptide). When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion.

Acceptance criteria of the DPRA:
The standard calibration curve should have an r² >0.99. The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C should be < 15%. Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion). In addition the positive control should cause depletion of both peptides comparable to historic data.

Evaluation of results: See tables 1 and 2
Chemical reactivity was determined by mean peptide depletion [%] and was rated as high, moderate, low, or minimal.

Limitations of the evaluation by insolubility and gravimetric procedure:
For test substances that are not completely soluble by visual observation in the sample preparations containing the peptides immediately after preparation or after 24 hours, or when a gravimetric procedure is applied (with the exception of application of the undiluted test substance (liquids) or the maximal soluble test-substance concentration (solids)), the result may be under-predictive due to limited availablity of the test substance. In this case mean peptide reactivity ≤ 6.38% (cysteine 1:10 / lysine 1:50 prediction model) or ≤ 13.89% (cysteine 1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion > 6.38% or > 13.89% is considered as “positive”.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion (%)/C-peptide

Value:

100

Vehicle controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

54.36%

Results 2

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion (%)/K-peptide

Vehicle controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

11.34%

Remarks on result:

other: interferance observed (co-elution)

Other effects / acceptance of results:

OTHER EFFECTS:
Co-elution of the test substance and the K-containing peptide occurred as demonstrated by the co-elution control and peak purity. Thus the K-peptide depletion could not be determined. No co-elution of the test substance and C-containing peptide occurred.

ACCEPTANCE OF RESULTS:
The acceptance criteria were met. The positive control caused depletion of both peptides comparable to historic data:

Historic control data of negative control / vehicle control (not including present study)
Acetonitrile
Historic period: Jan 2015 - Jan 2017

C-peptide concentration [mM] K-peptide concentration [mM]
Min 0.451 0.488
Max 0.511 0.536
Mean 0.485 0.510
SD 0.014 0.010
n 35 34

Historic control data of positive control (not including present study):
EGDMA, 50 mM in acetonitrile
Historic period: Jan 2015 - Jan 2017

C-peptide concentration [mM] C-peptide depletion [%] K-peptide concentration [mM] K-peptide depletion [%]
Min 0.144 43.32 0.395 6.79
Max 0.282 70.07 0.491 21.52
Mean 0.217 54.98 0.445 12.99
SD 0.032 6.17 0.020 3.11
n 31 28

Any other information on results incl. tables

Table 3: Mean peptide depletions of Cysteine, Lysine and both peptides

	Cysteine-Peptide mean depletion [%]        SD [%]	Lysine-Peptide mean depletion [%]         SD [%]	mean of both depletions [%]
Eukesolar Red Brown ER Dye powder; dried	100.0          0	interference
PC: EGDMA in ACN	54.36        5.49	11.34          0.67	32.85

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria