N,N'-[1,4-phenylenebis(methylene)]bis{N,N-dimethyl-3-[(prop-2-enoyl)amino]propan-1-aminium} dichloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Oct - 09 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

EC Number:

812-548-5

Cas Number:

1621424-03-0

Molecular formula:

C24H40Cl2N4O2

IUPAC Name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity (covalent binding) of the test chemical towards synthetic peptides containing either lysine or cysteine.

TEST METHOD
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure that addresses the molecular initiating event leading to skin sensitization, covalent protein binding, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity. The result may be used to discriminate between skin sensitizers and non-sensitizers, as part of a weight of evidence approach.

TEST SYSTEM
- Supplier of synthetic peptides: RS synthesis, Louisville, USA
- Peptide stock solution preparation: Stock solutions of cysteine and lysine peptides were freshly prepared for each assay.
Cysteine-containing peptide:
- Concentration: 0.667 mM Cys-Peptide stock solution was prepared by dissolving 20.0 mg of the peptide in 40 mL phosphate buffer, pH 7.50.
Lysine-containing peptide:
- Concentration: 0.667 mM Lys-Peptide stock solution was prepared by dissolving 24.8 mg of the peptide in 48 mL ammonium acetate buffer, pH 10.20.

VEHICLE CONTROL
- Substance: Water for chromatography, H2O, Honeywell, HPLC grade (referred to as HPLC grade water)
- Justification for selecting vehicle: In a non-GLP pre-test the solubility of the test item was determined in (in descending order, until it was dissolved completely): 1) Acetonitrile (ACN); 2) HPLC grade water. Based on this non-GLP pre-test, water was found to be a suitable vehicle.

POSITIVE CONTROL
- Substance:
1) Cinnamaldehyde (CAS 104-55-2, food grade ≥ 95%, batch no. MKBT8955V, expiration date: Feb 2020) was used as 100 mM (± 10%) solution in acetonitrile for the Cys-peptide.
2) 2,3-Butanedione (CAS 431-03-8, ≥ 99%, batch no. BCBW1565, expiration date: Oct 2020) was used as 100 mM (± 10%) solution in acetonitrile for the Lys-peptide.
- Preparation: Positive controls were treated identically to the test item. As cinnamaldehyde mixed with the Lys-peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione was used as positive control showing mid-range depletion for the Lys-peptide.

CO-ELUTION CONTROL
Sample prepared from the respective peptide buffer (100 mM Phosphate buffer, pH 7.5 or 100 mM Ammonium acetate buffer, pH 10.2) and the test item, but without peptide. It was included in the assay to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

REFERENCE CONTROLS
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, in total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used to calculate of the peptide depletion. Additionally, a solvent control containing HPLC grade water instead of test item solution was prepared in triplicate and also incubated and analysed together with the samples and was used to calculate the depletion of the test substance.

TEST SUBSTANCE PREPARATION
The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test substance solution). The Lys-peptide samples were prepared in 1:50 molar ratio (0.5 mM peptide and 25 mM test substance solution) using the stock solutions described above. A final volume of 1 mL per sample was prepared for each sample.
The 100 mM test substance solution was prepared by dissolving 146.7 mg (Cys-peptide assay, experiment 2) and 148.4 mg (Lys-peptide assay, experiment 2) test substance in 3 mL HPLC grade water. The solution was vortexed until the test substance was dissolved completely.

INCUBATION CONDITIONS
- Temperature used during incubation: 25.0 °C
- Duration of incubation: 22 h

NUMBER OF REPLICATES
All sample preparations (including controls) were carried out in triplicate in one valid run of the assay.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC components: Solvent Rack SRD3400, Quaternary pump HPG-3400SD, Autosampler WPS-3000SL, Column compartment TCC-3000SD, Photometer VWD-3400RS (Thermo Fisher Scientific)
Column: An ACE Excel SuperC18 150x3 mm column with 3 μm particles and pre-column Phenom-enex SecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the guideline OECD 442C.
Flow rate: 0.55 mL/min
Injection volume: 7 μL
Column temperature: 30 °C
Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution.

Results and discussion

Positive control results:

- The mean peptide depletion with 79.50% and a standard deviation (SD) of 0.52% of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% and SD < 14.9%, respectively, for the Cys-peptide.
- The mean peptide depletion with 23.78% and a SD of 1.60% of three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0% and SD < 11.6%, respectively, for the Lys-peptide.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine/ Lysine

Parameter:

other: mean peptide depletion (%)

Value:

51.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean peptide depletion (%)

Value:

86.39

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: Lysine

Parameter:

other: mean peptide depletion (%)

Value:

16.21

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline OECD 442C were tested.
All ten proficiency chemicals showed the expected DPRA prediction for both peptide assays. For the Cys-peptide assay nine of the ten chemicals and for the Lys-peptide assay all ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean peptide concentration of all solvent controls (Reference A and Reference C) were with 0.52 mM ,0.50 mM and 0.49 mM for the Cys-peptide and 0.51 mM, 0.53 mM, and 0.52 mM for the Lys-peptide, respectively, in the acceptable range of 0.50 ± 0.05 mM.
The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 3.0% for the Cys-peptide and 0.9% for the Lys-peptide, respectively.
Hence, the acceptance criteria were met.

- Acceptance criteria met for positive control:
The mean peptide depletion with 79.50% and a standard deviation (SD) of 0.52% of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% and SD < 14.9% for the Cys-peptide.
The mean peptide depletion with 23.78% and a SD of 1.60% of three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0% and SD < 11.6% for the Lys-peptide.
Hence, the acceptance criteria were met.

- Acceptance criteria met for variability between replicate measurements:
The standard deviation for the test item replicates was 2.87%, which is < 14.9% as required for the percent cysteine depletion for the test item.
The standard deviation for the test item replicates was 1.36%, which is < 11.6% as required for the percent lysine depletion for the test item.
Hence, the acceptance criteria were met.

In the co-elution control of the Lys-peptide assay a small co-elution peak at 220 nm was observed in the test item replicates, but the peak area was < 10% of the mean peptide peak area in the appropriate reference control (HPLC water). Therefore, the peak was considered to be a baseline noise.

- Range of historical values if different from the ones specified in the test guideline:
Positive control Cys-peptide depletion range: 67.09 – 98.37%
Positive control Lys-peptide depletion range: 13.66 – 41.94%

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitising potential based on the key event “protein reactivity”

Conclusions:

Under the experimental conditions reported, the test item FV-1295 shows a reactivity towards the two model synthetic peptides.

There is regulatory acceptance in the EU for the application of the direct peptide reactivity assay to address key event 1: peptide/protein binding in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the present test, the test substance FV-1295 showed reactivity towards selected proteins. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.