Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 29 September 2017 to 01 February 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

Constituent 1

Reference substance name:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

Dimethyl adipate

Constituent 2

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Constituent 3

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl succinate

Test material form:

liquid

Remarks:

Colourless to brown liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not assessed. The formulation was a colorless liquid and was used just after its preparation.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was diluted in the selected vehicle (acetonitrile) at 100 mM.

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 acetonitrile.
As several test items were assayed concurrently with this vehicle, its results were shared.

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 98.9%.

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 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.

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 diluted in the selected vehicle (acetonitrile) at 100 mM. This formulation was a colorless liquid and was used just after its preparation.


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).

2.1.3) 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).

2.1.4) 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).

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 for the lysine peptide or 26 hours and 53 minutes for the cysteine peptide (see § Study plan adherence), 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 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.

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.
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 77.61% which corresponds to Hight reactivity.

In vitro / in chemico

Results

Key result

Parameter:

other: mean of the percent cysteine and percent lysine depletions (%)

Value:

1.62

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no 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

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 was observed in the test item samples incubated with the cysteine peptide, these vials were centrifuged at 400gfor a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive control samples incubated with both peptides and reference control samples prepared in acetonitrile with the cysteine peptide were also centrifuged at the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected into the HPLC/UV system.

For the other samples (i.e.test item samples incubated with the lysine peptide, and reference control samples prepared in acetonitrile with the lysine peptide and co-elution samples prepared with the lysine and cysteine dilution buffer), the vials were directly transferred into the HPLC/UV system.

 

EVALUATION OF THE RESULTS

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 2.61%,

.            for the lysine peptide, the mean depletion value was 0.63%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 1.62%. However,it is to be noted thatprecipitate was observed at the end of the incubation with the cysteine peptide.As a consequence,the corresponding peptide depletion may be underestimated.

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

Applicant's summary and conclusion

Interpretation of results:

other: The DPRA prediction is considered as negative and the test item is likely not to have any potential to cause skin sensitization.

Conclusions:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item, Reaction mass of diethyl adipate diethyl glutarate and diethyl succinate,

to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment. The design of this study was based on the OECD Guideline 442C and the study was performed in compliance with the OECD Principles of Good Laboratory Practice.

Methods

 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-Violetdetection at 220 nm.

Peptide reactivity was reported as percent depletion basedon 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 test item was diluted at 100 mM inacetonitrile.

 

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:

.            for the cysteine peptide, the mean depletion value was 2.61%,

.            for the lysine peptide, the mean depletion value was 0.63%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 1.62%. However, it is to be noted that precipitate was observed at the end of the incubation with the cysteine peptide. As a consequence, the corresponding peptide depletion may be underestimated.

 

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

 

Conclusion

 

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item Reaction mass of diethyl adipate diethyl glutarate and diethyl succinate was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide.