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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2017 - 18 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 442C and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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 on 04 February 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Certificate for GLP compliance was not attached to the study report to prove the GLP status.
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
Non-animal testing is the default requirement for skin sensitisation.

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl azelate
EC Number:
217-060-0
EC Name:
Dimethyl azelate
Cas Number:
1732-10-1
Molecular formula:
C11H20O4
IUPAC Name:
1,9-dimethyl nonanedioate
Test material form:
liquid
Details on test material:
Name: Dimethyl Azelate
Batch no.: 1602100006
Appearance: Oily liquid, colorless
CAS No.: 1732-10-1
EINECS-No.: 217-060-0
Molecular formula: C11H20O4
Molecular weight not stated
Purity: 86 %w/w
Production date: 04 October 2016
Expire date: 1 November 2018
Storage: Fridge (2-8°C), keep away from humidity

In chemico test system

Details on the study design:
The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item 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 percent 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 solvent).

Preparation of test item and controls:
- Vehicle: acetonitrile (supplier: CARLO ERBA/batch number: P7E031027F) (without sonication)
- Positive control: Name : Cinnamaldehyde (CAS No.: 104-55-2, Batch No. : MKBX8146V, Supplier : Sigma-Aldrich), purity: 98.9%
- Co-elution control samples: In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.
- Reference control samples: All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent (peptide solution cysteine or lysine). 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 solvent did not impact the percentage of peptide depletion.
- Test item formulation preparation: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile (supplier: CARLO ERBA/batch number: D6N042196N)) at 100 mM. This formulation was colorless limpid solution and was used just after its preparation.

Cysteine peptide:
. Peptide sequence : Ac-RFAACAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH; Ac RFAACAA-COOH
. Molecular weight : 750.88 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 111016HS_MHeW0117
. Storage condition : At -20°C
. Description : White powder
. Purity : 88.78%
. Expiry date : 07 February 2018
The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH; Ac RFAAKAA-COOH
. Molecular weight : 775.91 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 220114HSDWW0117
. Storage condition : At -20°C
. Description : White powder
. Purity : 96.92%
. Expiry date : 25 January 2018
The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

Study design:
The test item was tested in one run per peptide. 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 item 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 item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

- Reference control A and B samples preparation
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 sample preparation
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide: 50 μL of vehicle (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 vehicle (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 100 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 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

- Test item samples preparation
For the reactivity of test item with cysteine peptide: 50 μL of test item 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 item with lysine peptide: In parallel, 250 μL of test item 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 control, reference controls, test item and positive control samples) were incubated during 24 (± 2) hours at room temperature and protected from light before injection onto 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 were centrifuged at 400g 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 both peptides (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 of the peptide signal 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,
. one co-elution control sample,
. three reference control B samples,
. reference control C sample (replicate 1),
. positive control sample (replicate 1),
. test item study samples (replicate 1).
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
. three reference control B samples.

The HPLC/UV method used for the samples analysis according to the recommendations of the OECD guideline No. 442C is described in CiToxLAB France internal analytical method and is summarized in the table below:
. 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)
. Mobil phase: Mobile phase A: acetonitrile + 0.085% TFA / Mobile phase B: milli-Q water + 0.1% TFA
. Flow: 350 µL/minute
. Gradient
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90
. UV Wavelength: 220 nm
. Rinse solution: Acetonitrile
. Oven temperature: 30.0°C
. Autosampler temperature: Nominal temperature of +25°C
. Injection volume: 5µL
. Retention times: Cysteine-peptide: approx. 9.8 minutes / Lysine-peptide: approx. 7.5 minutes
. Total analysis time: 20 minutes

Results and discussion

Positive control results:
Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 77.82% and SD = 0.13%
- Result of positive control samples in lysine assay: Mean % depletion = 50.29% and SD = 0.69%
Mean depletion rate of Cinnamaldehyde: 64.06%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Mean % depletion in Cysteine assay
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion.
Key result
Parameter:
other: Mean % depletion in Lysine assay
Value:
0.69
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other:
Remarks:
Mean depletion rate
Value:
0.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No reactivity / minimal reactivity
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Acceptance criteria met for the calibration curve: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

Solubility results

The test item was found soluble at 100 mM in acetonitrile without sonication step. Therefore, this vehicle was retained.

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 item and positive control samples) was performed prior to HPLC analysis.

As precipitate/micelle were observed in the test item samples incubated with both cysteine or lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate/micelle to the bottom of the vial. Positive control samples incubated with both peptides as well as reference control samples incubated with the cysteine peptide were also centrifuged at the same conditions to force precipitate to the bottom of the vial. Only supernatants were injected into the HPLC/UV system.

For the other samples (i.e. co-elution controls of lysine, reference controls incubated with the lysine peptide and co-elution controls of cysteine), the vials were directly transferred into the HPLC/UV system.

See the attached document "Tables of results DPRA"

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item DIMETHYL AZELATE, was considered to have no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.
The test item is considered negative in the DPRA assay.
Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay (DPRA) in accordance to the OECD Guideline No. 442C and in compliance with GLP. The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item 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 percent 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 solvent).

The test item was dissolved at 100 mM in acetonitrile without sonication.

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 item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was 0.00%,

. for the lysine peptide, the mean depletion value was 0.69%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.34%. Accordingly, the test item was considered have no/minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

Since precipitate/micelles were observed at the end of the incubation with both peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity could not be drawn with sufficient confidence.

However, since the mean of the cysteine percent and lysine percent depletions was < 6.38%, the test item was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item is likely not to have any potential to cause skin sensitization, though with limitations due to its precipitation with the peptides.

This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential of the test item.

Under the experimental conditions of this study, the test item DIMETHYL AZELATE, was considered to have no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.

The test item is considered negative in the DPRA assay.