2,4-dimethylpentan-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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))

Version / remarks:

2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Turnkey Testing Strategy

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dimethylpentan-3-one

EC Number:

209-294-7

EC Name:

2,4-dimethylpentan-3-one

Cas Number:

565-80-0

Molecular formula:

C7H14O

IUPAC Name:

2,4-dimethylpentan-3-one

Test material form:

liquid

Details on test material:

Batch 151005
Physical state/appearance: Liquid/ yellowish, clear
Storage conditions: Room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF SE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: After short stirring the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.

OTHER SPECIFICS: The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The test substance was dissolved in a suitable vehicle. Per run three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Prior to the assay the solubilty of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

The test substance samples were prepare din triplicates for each peptide.
C-peptide: 750 µL C-peptide stock-solution, 200 µL solvent (vehicle), 50 µL test substance preparation (or PC preparation or solvent (VC)).
K-peptide: 750 µL K-peptide stock-solution, 250 µL test substance preparation (or PC preparation or solvent (VC)).
The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C ± 2.5 °C in the dark for 24 ± 2 h. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 h after sample preparation and the analysis time itself did not exceed 30 h.

Several vehicle controls were prepared in triplicates in the same way as the test substance samples described above with the vehicle (acetonitrile) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Co-elution control: One sample per peptide was prepared in the same way as the test substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Controls:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate, prepared as a 50 mM solution in acetonitrile.
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean peptide depletion

Value:

8.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: low chemical reactivity

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: mean C-peptide depletion

Value:

15.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 1

Parameter:

other: mean K-peptide depletion

Value:

1.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: Two test runs were performed for the C-containing peptide due to relative high standard deviation of the three values measured in the first test run and as the result was close to the evaluation threshold.

Applicant's summary and conclusion

Interpretation of results:

other: low chemical reactivity

Conclusions:

The mean peptide depletion was calculated to be 8.57 %. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows low chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with the synthetic peptides for ca. 24 h at ca. 25 °C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. One test run was performed for the K-containing peptide and two test runs for the C-containing peptide. Per test run three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24 h incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

Two test runs were performed for the C-containing peptide due to relative high standard deviation of the three values measured in the first test run and as the result was close to the evaluation threshold.

The mean C-peptide depletion, caused by the test substance was determined to be 14.64 % in the first test run and 17.17 % in the second test run. The mean C-peptide depletion of both test runs was determined to be 15.90 %.

The mean K-peptide depletion, caused by the test substance was determined to be 1.24 %.

Thus, the mean peptide depletion was calculated to be 8.57 %.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows low chemical reactivity in the DPRA under the test conditions chosen.