Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September 2018 to 01 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

EC Number:

277-429-7

EC Name:

Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Cas Number:

73398-29-5

Molecular formula:

C36H34Br2N2O8S2.2Na

IUPAC Name:

disodium 2-bromo-3-({4-[(2-bromo-4-butyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-6-butyl-4-methylbenzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Ultramarine blue powder
- Storage conditions: The test sample was stored in a dark storage place at room temperature.

In chemico test system

Details on the study design:

MATERIALS
- Vehicle: 1:1 (v/v) mixture of acetonitrile: milli-Q water
- Positive control: Cinnamaldehyde (purity: 99.1%)
- Test material formulation preparation: The test material was pre-weighed and stored under appropriate conditions until ready to perform the testing. It was dissolved in the vehicle at 100 mM. The formulation was a blue liquid solution and was used just after its preparation.
- Co-elution control samples: prepared by incubating the test material formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.
- Reference control samples: For each peptide, the analytical batch included reference control samples (A, B or C). These samples were prepared in triplicate at the nominal concentration of 0.500 mM. These samples were used to:
Reference control A: check the accuracy of the calibration curve for peptide quantification
Reference control B: check the stability of the peptide during analysis
Reference control C: check that the vehicle did not impact the percentage of peptide depletion

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test material was tested in one run. The run was processed as described below.

Preparation of the samples
- The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.
- Co-elution control samples preparation: For the co-elution control with cysteine peptide: 50 μL of test material formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile. For the co-elution control with lysine peptide: In parallel, 250 μL of test material formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).
- Reference control samples preparation: Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM. Reference control C samples: Reference control C samples were prepared for each vehicle used to dissolve the test and positive control materials. For the reference control C prepared with cysteine peptide: 50 μL of each vehicle (1:1 mixture of acetonitrile: milli-Q water or acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reference control C prepared with lysine peptide: In parallel, 250 μL of each vehicle (1:1 mixture of acetonitrile: milli-Q water or acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).
- Cinnamaldehyde (positive control) depletion control samples preparation: For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 98.2 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 98.2 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Test material samples preparation: For the reactivity of test material with cysteine peptide: 50 μL of test material formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile. For the reactivity of test material with lysine peptide: In parallel, 250 μL of test material formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Incubation of the samples
- All samples (co-elution controls, reference controls, test material and positive control samples) were then incubated during 24 (± 2) hours at 25 °C and protected from light before injection into the HPLC/UV system.
- At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.
- Samples presenting precipitate or phase separation (micelles) were centrifuged at 400 g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

Preparation of the calibration curve samples
- One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
- The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least: one blank sample (peptide dilution buffer), one calibration curve injected at the beginning of the analytical batch, three reference control A samples, the co-elution control sample, three reference control B samples, reference control C sample (replicates 1, 2 and 3), positive control sample (replicates 1, 2 and 3), test material study samples (replicates 1, 2 and 3) and three reference control B samples.

The HPLC/UV method used for the samples analysis was as follows:
- Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm, (Waters). In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
- Mobile phase: Mobile phase A: acetonitrile + 0.085 % TFA. Mobile phase B: milli-Q water + 0.1 % TFA
- Flow: 350 μL/minute
- Gradient:
0 minutes: 10 % Mobile phase A, 90 % Mobile phase B
10 minutes: 25 % Mobile phase A, 75 % Mobile phase B
11 minutes: 90 % Mobile phase A, 10 % Mobile phase B
13 minutes: 90 % Mobile phase A, 10 % Mobile phase B
13.5 minutes: 10 % Mobile phase A, 90 % Mobile phase B
20 minutes: 10 % Mobile phase A, 90 % Mobile phase B
- UV Wavelength: 220 nm
- Rinse solution: Acetonitrile
- Oven temperature: 30.0 °C
- Autosampler temperature: Nominal temperature of +25 °C
- Injection volume: 7 μL
- Retention times: Cysteine-peptide: approx. 9.8 minutes, Lysine-peptide: approx. 7.5 minutes
- Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percentage peptide depletion: Each appropriate peak was integrated and the peak area for calibration standards, control and test material samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test material replicate, the percentage depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle) by using the following formula:
% depletion = [ 1 – (Peptide peak area in replicate injection / mean peptide peak area in relevant reference control C samples)] x 100
Then, the mean percentage depletion of the three replicates was calculated for each peptide as well as the mean of the percentage cysteine and percentage lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
- Evaluation of the possible co-elution of the test material with the lysine or cysteine peptides: In order to detect possible co-elution of the test materials with a peptide, chromatograms of the co-elution control samples were analysed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
- the calibration curves should have a coefficient of determination (r²) ≥ 0.99
- the mean peptide concentrations of the reference control A samples should be within ± 10 % of the nominal concentration
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
− for the cysteine peptide, the mean percentage depletion value should be between 60.8 and 100 % with a SD < 14.9 %
− for the lysine peptide, the mean percentage depletion value should be between 40.2 and 69.0 % with a SD < 11.6 %
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0 %.

The test material’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate vehicle should be within ± 10 % of the nominal concentration
- the maximum SD for the test material replicates should be < 14.9 % for the percentage cysteine depletion value and < 11.6 % for the percentage lysine depletion value

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage cysteine and percentage lysine depletion

Value:

80.77 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage cysteine depletion

Value:

61.53 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Percentage lysine depletion

Value:

100 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

SOLUBILITY RESULTS
- During the solubility assay, the test material was found not soluble at 100 mM in acetonitrile or in milli-Q water even after 1 minute of sonication. A solution was obtained at 100 mM with a 1:1 (v/v) mixture of acetonitrile: milli-Q water. Therefore, the vehicle retained in this study was 1:1 (v/v) mixture of acetonitrile: milli-Q water.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES
- At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test material and positive control samples) was performed prior to HPLC analysis. As precipitate and/or phase separation (micelles) were observed in positive samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400 g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Only supernatants were then injected into the HPLC/UV system.
- For the other samples, the vials were directly transferred into the HPLC/UV system.

EVALUATION OF THE RESULTS
- The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.
- Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide:
- for the cysteine peptide, the mean depletion value was 61.53 %
- for the lysine peptide, the mean depletion value was 100 %
- The mean of the percentage cysteine and percentage lysine depletions was equal to 80.77 %. Accordingly, the test material was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.

Any other information on results incl. tables

Table 1: Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked with a Solution at 100 mM of Test Material

Sample Number	Cysteine Peptide		Lysine Peptide		Mean Peptide Depletion Rate (%) of Test Material	Depletion Classification
	Peak Area (µV/sec)	% Depletion	Peak Area (µV/sec)	% Depletion
1	1139767	60.65	0	100.00
2	1106449	61.80	0	100.00
3	1096788	62.14	0	100.00
Mean	-	61.53	-	100.00	80.77	High Reactivity
SD	-	0.78	-	0.00
% CV	-	1.3	-	0.0
Precipitate	No		No
Micelle	No		No

-: not applicable

Table 2: Determination of Cysteine Peptide and Lysine Peptide Depletion Concentration in Reference Control C Samples Prepared in a 1:1 Mixture of Water: Acetonitrile

Sample Number	Cysteine Peptide			Lysine Peptide
	Peak Area (µV/sec)	Concentration (mM)	%Dev	Peak Area (µV/sec)	Concentration (mM)	%Dev
1	2833902	0.497	(-0.6)	2285652	0.485	(-3.0)
2	2959573	0.519	(3.8)	2296643	0.487	(-2.5)
3	2896964	0.508	(1.6)	2286504	0.485	(-3.0)
Mean	2896813	0.508	(1.6)	2289600	0.486	(-2.8)
SD	-	0.011	-	-	0.001	-
% CV	-	2.2	-	-	0.3	-

-: not applicable

 

Table 3: Determination of Interference Due to Co-elution of the Test Material with Cysteine or Lysine Peptides

Sample Number	Peak Detected at the Cysteine Retention Time		Peak Detected at the Lysine Retention Time
	Peak Area (µV/sec)	% Interference	Peak Area (µV/sec)	% Interference
1	0	(0.0)	0	(0.0)
Precipitate	No		No
Micelle	No		No

 

Applicant's summary and conclusion

Interpretation of results:

other: The DPRA prediction is considered as positive

Conclusions:

Under the conditions of this study, the DPRA prediction is considered as positive and the test material was considered to have high peptide reactivity.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to evaluate the reactivity of the test material to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitisation assessment.

The reactivity of the test material was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test material for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle).

The test material was dissolved at 1:1 (v/v) mixture of acetonitrile: milli-Q water. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide: for the cysteine peptide, the mean depletion value was 61.53 % and for the lysine peptide, the mean depletion value was 100 %. The mean of the percentage cysteine and percentage lysine depletions was equal to 80.77 %. Accordingly, the test material was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test material may have potential to cause skin sensitisation.