Dipotassium dodecenylsuccinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dodecenylsuccinate

EC Number:

260-612-0

EC Name:

Dipotassium dodecenylsuccinate

Cas Number:

57195-28-5

Molecular formula:

C16H26K2O4*H2O

IUPAC Name:

Dipotassium dodecenylsuccinate

Test material form:

liquid

Details on test material:

30 % aqueous solution
pure substance is solid (bulk/powder), but is only used as aqueous solution and blends it into mixture
Substance dissolves in water at 40wt%, then becomes gel and becomes solid, if mixed 61wt% with water

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No. of test material: 170727
- Purity test date: Content Dipotassium dodecenylsuccinate: 30%, Water: 70%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, stored in a tightly closed container, in a cool, well-ventilated place, protected from light.
- Stability under test conditions: No data on stability were available to LPT
- Solubility and stability of the test substance in the solvent/vehicle: water

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
1. Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.

2. Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 360.57 mg test item were dissolved in 3 mL highly purified water immediately before testing to prepare a 100 mM solution. A correction factor of 3.33 was used due to the content of 30% Dipotassium¬dodecenyl¬succinate and 70% water in the test item. The test item solution was then tested as such without any further dilution by incubating at 1:10 and 1:50 ratio with the cysteine and lysine peptides, respectively.

3. Positive control, reference controls and coelution control:
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A), the stability of the reference controls over time (reference control B) and to verify that the solvent used to dissolve the test item does not impact the percent peptide depletion (reference control C). The appropriate reference control for the test item was used to calculate the percent peptide depletion for the test item. In addition a coelution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible coelution of the test item with either the lysine or the cysteine peptide.

4. Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analyzed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.

5. Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (0.666 mM of cysteine peptide in sodium phosphate or 0.667 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.

I: Standard calibration for cysteine peptide
Sample Identifier Amount of Component
K1 1600 µL stock solution (0.666 mM cysteine peptide or 0.501 mg cysteine peptide/mL) + 400 µL acetonitrile
K2 1000 µL K1 + 1000 µL dilution buffer (80% sodium phosphate buffer + 20% acetonitrile)
K3 1000 µL K2 + 1000 µL dilution buffer
K4 1000 µL K3 + 1000 µL dilution buffer
K5 1000 µL K4 + 1000 µL dilution buffer
K6 1000 µL K5 + 1000 µL dilution buffer
K7 Blank: 2000 µL dilution buffer

II: Standard calibration for lysine peptide
Sample Identifier Amount of Component
K1 1600 µL stock solution (0.666 mM lysine peptide or 0.518 mg lysine peptide/mL) + 400 µL acetonitrile
K2 1000 µL K1 + 1000 µL dilution buffer (80% ammonium acetate buffer + 20% acetonitrile)
K3 1000 µL K2 + 1000 µL dilution buffer
K4 1000 µL K3 + 1000 µL dilution buffer
K5 1000 µL K4 + 1000 µL dilution buffer
K6 1000 µL K5 + 1000 µL dilution buffer
K7 Blank: 2000 µL dilution buffer

6. HPLC preparation and analysis:
I: HPLC equipment:
Pump: Thermo Fisher Scientific, HPG-3200SD
Detector: Thermo Fisher Scientific, VWD-3400RS
Sampler: Thermo Fisher Scientific, WPS-3000 SL
Column Thermostat: Thermo Fisher Scientific, TCC-3000SD
Data system: Chromeleon 7.2, Thermo Fisher Scientific, on a host computer

II: HPLC conditions:
Column / Pre-column: Agilent, Zorbax SB-C-18, 2.1 mm x 100 mm 3.5 µm particle.
Column temperature: 30°C
Sample temperature: 25°C ± 2.5°C
Detection: UV at wavelength = 220 nm
Mobile phase : A - water, 0.1% Trifluoroacetic acid (TFA) (v/v); B - acetonitrile, 0.085% TFA (v/v)
Flow: 0.35 mL/min
Injection volume: 8 µL
Run length: 20 min

III: Gradient program
Time Eluent A Eluent B
0.5 min 90 10
10.0 min 75 25
11.0 min 10 90
12.0 min 10 90
13.0 min 90 10
20.0 min 90 10

If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would be noted as percent peptide depletion would be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class.
HPLC analysis for the cysteine and lysine peptides were performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.

Results and discussion

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 83.44% cysteine and 57.08% lysine. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean (%)

Parameter:

other: cysteine peptide depletion

Value:

0.76

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: mean (%)

Parameter:

other: lysine peptide depletion

Value:

0.78

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No precipitate in the reaction mixture at the end of the incubation time and no coelution were observed.

The linearity of standard calibration curve was r²= 0.9999 forcysteine peptideand for lysine peptide. Hence the requirement of r²> 0.99 was met.

The mean peptide concentrations of reference controls were well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.

All acceptance criteria of validity were fulfilled in this test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Dipotassium dodecenylsuccinate revealed a mean cysteine and lysine peptide depletion of 0.770% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).