Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in chemico
Remarks:
Direct Peptide Reactivity Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 14 June 2016; Experimental completion date: 18 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
This purpose of thestudy (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test substance.

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride
EC Number:
947-079-6
IUPAC Name:
Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride
Test material form:
other: paste
Specific details on test material used for the study:
Sponsor’s identification: Methoxy Cinnamidopropyl Behenyl Dimonium Chloride
Batch No.: INV-1603010
Purity: Solids content, 29.37%
Appearance: Pearly white paste
Expiry/retest date: February 2017
Storage conditions: Room temperature

In chemico test system

Details on the study design:
PEPTIDE AND POSITVE CONTROL:
Synthetic peptide containing Cysteine:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Stated purity: 95% (by HPLC)
Molecular Weight: 751.5 g/mol
Supplier: AnaSpec
Storage conditions: Frozen, -20°C
Expiry/retest date: 5 years.

Synthetic peptide containing Lysine:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Stated purity: 90-95%
Molecular Weight: 776 g/moL
Supplier: AnaSpec
Storage conditions: Frozen, -20°C
Expiry/retest date: 5 years.

Cinnamic Aldehyde (Positive Control):
Batch number: MKBR2427V
Stated purity: >95%
Molecular Weight: 132.16 g/moL
Supplier: SAFC
Storage conditions: Room temperature
Expiry/retest date: February 2019

APPARATUS:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

ANALYTICAL PROCEDURE:
Reagents:
Acetonitrile (ACN): HPLC gradient grade
Propan-2-ol : HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

ASSESSMENT OF TEST ITEM SOLUBILITY:
The solubility of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in acetonitrile was assessed at a concentration of 100 mM and found to be insoluble. Solubility of the test item at 100 mM was achieved using propan-2-ol as a solvent.

PREPARATION OF PEPTIDE STOCK SOLUTIONS:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

PREPARATION OF PEPTIDE CALIBRATION STANDARDS:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

PREPARATION OF STABILITY CONTROLS AND PRECISION CONTROLS:
Stability controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in both acetonitrile and propan-2-ol.

PREPARATION OF POSTIVE CONTROL SOLUTION:
The positive control chemical (Cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile.

PREPARATION OF POSITIVE CONTROLS AND CYSTEINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS:
Propan-2-ol solutions of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

The positive control solutions (prepared in acetonitrile) contained cinnamic aldehyde at a concentration of 5 mM in 0.5 mM Cysteine.

PREPARATION OF POSITIVE CONTROLS AND LYSINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS:
Propan-2-ol solutions of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

The positive control solutions (prepared in acetonitrile) contained cinnamic aldehyde at a concentration of 25 mM in 0.5 mM Lysine.

INCUBATION:
The appearance of the Methoxy Cinnamidopropyl Behenyl Dimonium Chloride and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Just before initiation of the run the appearance of the samples in the vials was assessed and documented again.

ANALYSIS:
The concentration of each respective peptide and the associated peptide positive control was quantified by HPLC using UV detection as detailed in the chromatographic section.

INSTRUMENTAL PARAMETERS:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30°C
Sample temperature: 25°C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient:
Time (min) MP A (%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10

Flow rate: 0.35 mL/minute
Stroke volume@ 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes

CALCULATIONS:
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of propan-2-ol control samples.































Results and discussion

Positive control results:
Cysteine Peptide Depletion: Positive control mean depletion = 72.5%
Lysine Peptide Depletion: Positive control mean depletion = 57.0%

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
mean
Parameter:
other: Cysteine peptide depletion (%)
Value:
-63
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
mean
Parameter:
other: Lysine peptide depletion (%)
Value:
-0.305
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: Overall mean depletion (%)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: DPRA prediction: negative
Other effects / acceptance of results:
Solubility Assessment:
The solubility of the Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in propan-2-ol at a nominal concentration of 100 mM was confirmed.

Acceptance criteria:
All analytical acceptance criteria for each peptide run were met (see overall remarks).

Any other information on results incl. tables

Reactivity Assessment:

The DPRA prediction and the reactivity of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride based on the overall mean and the individual depletion values in the cysteine peptide and the lysine peptide is presented below:

Overall Acheived Depletion Values:

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA Prediction

Methoxy Cinnamidopropyl Behenyl Dimonium Chloride

n/a

-0.305

0.001

No or minimal

Negative

1 Reported as zero

Individual Acheived Depletion Values:

Cysteine Peptide Depletion:

Sample

Peak area (µV.sec)

Peptide concentration1(µg/ml)

Peptide Depletion (%)

Mean Depletion (%)

CV (%)

Propan-2-ol Control

883605

374.91

1.632

1.72

0.35

879343

373.10

2.102

885366

375.66

1.432

Positive control

246753

104.80

72.52

72.5

1.30

244236

103.73

72.82

250620

106.44

72.12

Methoxy Cinnamidopropyl Behenyl Dimonium Chloride

1434259

608.46

-62.53

-63.0

0.40

1437686

609.92

-62.93

1445444

613.21

-63.73

CV: Coefficient of Variation

1: Samples prepared at a concentration of 376 µg/ml (0.5 mM) 

2: Calculated against a mean reference control peak area of 898220 µV. sec (n=6)

3: Calculated against a mean propan-2-ol control peak area of 882770 µV. sec (n=3)

Lysine Peptide Depletion:

Sample

Peak area (µV.sec)

Peptide concentration1(µg/ml)

Peptide Depletion (%)

Mean Depletion (%)

CV (%)

Propan-2-ol Control

768421

385.02

1.262

1.09

0.26

772032

386.83

0.7932

768698

385.16

1.222

Positive control

333680

167.67

57.12

57.0

0.97

338525

170.10

56.52

332343

167.01

57.32

Methoxy Cinnamidopropyl Behenyl Dimonium Chloride

772841

387.23

-0.4053

-0.305

0.19

772959

387.29

-0.4213

770424

386.03

-0.09103

CV: Coefficient of Variation

1: Samples prepared at a concentration of 388 µg/ml (0.5 mM) 

2: Calculated against a mean reference control peak area of 778200 µV. sec (n=6)

3: Calculated against a mean propan-2-ol control peak area of 769720 µV. sec (n=3)

Applicant's summary and conclusion

Interpretation of results:
other: no to minimal reactivity
Remarks:
potential non-skin sensitiser
Conclusions:
Solutions of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. The overall result places Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in the no to minimal reactivity class and therefore it is predicted to be a potential non-skin sensitizer.
Executive summary:

This purpose of the study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride.

Solutions of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. The overall result places Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in the “no or minimal reactivity” class and therefore it is predicted to be a potential non-skin sensitizer.