Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 April to 20 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Test material

Test material information

1

Reference substance name:

Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

EC Number:

272-897-9

EC Name:

Imidazolium compounds, 1-[2-(2-carboxyethoxy)ethyl]-1(or 3)-(2-carboxyethyl)-4,5-dihydro-2-norcoco alkyl

Cas Number:

68919-40-4

Molecular formula:

R_C8 H14 N2 O4 Na With R=Cn H2n+1 n=8, 10, 12, 14, 16, 18

IUPAC Name:

disodium 3-({2-[N-(2-hydroxyethyl)hexadecanamido]ethyl}amino)propanoate 3-[(2-hexadecanamidoethyl)(2-hydroxyethyl)amino]propanoate

Test material form:

cream / paste

Specific details on test material used for the study:

- Appearance: Clear paste
- Storage condition: At room temperature
- Purity: 99.7%
- Correction factor: No correction factor

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

1. TEST AND CONTROL ITEMS
1.1) Vehicle
Based on solubility results, the retained vehicle was milli-Q water. A sonication of 1 minute was used to improve solubilisation.

1.2) Positive control
The positive control was cinnamaldehyde (CAS No. 104-55-2), batch No. MKBX8146V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 99.1%.
As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was a colorless liquid and was used just after its preparation.

1.3) Co-elution control samples
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were p
repared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

1.4) Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent specified in § Reference control samples preparation.
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.

1.5) 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 (milli-Q water) at 100 mM. The formulation was sonicated for 1 minute. This formulation was an opaque white solution and was used just after its preparation.

1.7) Additional samples to test the neat test item
Since the test item is a mixture, the reactivity of neat test item was evaluated. Neat test item was dissolved to its maximum soluble concentration in the same vehicle used to prepare the apparent 100 mM solution. Then, it was tested as such without any further dilution by incubating it at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively.


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

2.1) 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.

2.1.1) 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).

2.1.2) Reference control samples preparation
2.1.2.1) 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.

2.1.2.2) Reference control C samples
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).

2.1.2.3) 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).

2.1.3) 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

2.1.4) Preparation of additional test item neat samples
- For reactivity of the test item with cysteine peptide: 50 µL of test item formulation prepared at the maximum soluble concentration 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 reactivity of the test item with lysine peptide: In parallel, 250 µL of test item formulation prepared at the maximum soluble concentration was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

2.2) Incubation of the samples
All samples (co-elution controls, reference controls, test item 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 (see § Results).
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


2.3) 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 in cluded 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.

2.4) 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 (replicate 1),
- positive control sample (replicate 1),
- test item study sample (replicate 1),
The injection order of the reference control C, positive control and all test item study samples were reproduced identically for replicate 2 and then replicate 3:
- three reference control B samples.
- additional neat co-elution control sample for the neat test item,
- additional neat test item samples (3 replicates),
- three reference control B samples.

The HPLC/UV method used for the samples analysis is described in CiToxLAB France internal analytical method.

Results and discussion

Positive control results:

The mean of the percent cysteine and percent lysine depletions of Cinnamaldehyde was 75.60% which corresponds to Hight reactivity.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: at 100 mM in milli-Q water with sonication.

Parameter:

other: mean of the percent cysteine and percent lysine depletions

Value:

13.79

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: test item neat

Parameter:

other: mean of the percent cysteine and percent lysine depletions

Value:

21.07

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of 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.

Any other information on results incl. tables

SOLUBILITY RESULTS

Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile and in 1:1 mixture of acetonitrile:milli-Q water even after 1 minute of sonication. A solution was obtained at 100 mM with milli-Q water after 1 minute of sonication. Therefore, the vehicle retained was 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 item and positive control samples) was performed prior to HPLC analysis. As precipitate was observed in the positive samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400g 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

1) at 100 mM in milli-Q water with sonication.

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 23.59%,

- for the lysine peptide, the mean depletion value was 3.98%.

The mean of the percent cysteine and percent lysine depletions was equal to 13.79%. Accordingly, the test item was considered to have low peptide reactivity.

2) test item neat

The test item was also tested neat . 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 35.61%,

- for the lysine peptide, the mean depletion value was 6.53%.

The mean of the percent cysteine and percent lysine depletions with the test item neat was equal to 21.07%.

These results confirm those obtained with test item prepared at 100 mM (low peptide reactivity). Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

This positive 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 sensitization potential of the test item

Applicant's summary and conclusion

Interpretation of results:

other: the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Conclusions:

Under the experimental conditions of this study, the test item Miranol C2M AA, was considered to have low peptide reactivity. The test item is considered positive in the DPRA assay.

Executive summary:

The reactivity of the test item (Miranol C2M AA) to synthetic cysteine and lysine peptides was evaluated was based on the OECD Guideline 442C and the study was performed in compliance with the OECD Principles of Good Laboratory Practice.

This test is part of a tiered strategy for skin sensitization assessment.

 

Method:

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 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)

 

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.

The test item was dissolved at 100 mM in milli-Q water with sonication.

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

- for the cysteine peptide, the mean depletion value was 23.59%,

- for the lysine peptide, the mean depletion value was 3.98%.

The mean of the percent cysteine and percent lysine depletions was equal to 13.79%. Accordingly, the test item was considered to have low peptide reactivity.

The test item was also tested neat. The mean percent depletion values were calculated for each peptide

- for the cysteine peptide, the mean depletion value was 35.61%,

- for the lysine peptide, the mean depletion value was 6.53%.

The mean of the percent cysteine and percent lysine depletions with the test item neat was equal to 21.07%. These results confirm those obtained with test item prepared at 100 mM (low peptide reactivity). Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

This positive 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 sensitization potential of the test item.

 

Conclusion:

Under the experimental conditions of this study, the test item, Miranol C2M AA, was considered to have a low peptide reactivity. The test item is considered positive in the DPRA assay.