3-Pentanone, 1-hydroxy-2-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 Feb 2016 - 02 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Justification for non-LLNA method:

In agreement with the latest amendment to the information requirements for skin sensitization, an in vitro tests were preferred to an in vivo study for clarification of a sensitization potential of Hydroxymethylpentanon.

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-2-methylpentan-3-one

EC Number:

939-893-5

Cas Number:

27970-79-2

Molecular formula:

C6 H12 O2

IUPAC Name:

1-hydroxy-2-methylpentan-3-one

Test material form:

liquid

Specific details on test material used for the study:

Name of test substance: Hydroxymethylpentanon
Batch identification: J 1812
CAS No. : 27970-79-2
Purity: 90.6 area-% (90.6% was used for calculation of 100 mM preparation for the DPRA)
Homogeneity: The test substance was homogeneous by visual inspection.
Physical state / color: Liquid / yellowish, clear
Molecular weight: 116.16 g/mol
Log KOW: 1.71
Proposed reaction mechanism for protein binding by OECD toolbox: The OECD toolbox proposed the following reaction mechanism for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism): Schiff base formation.

In chemico test system

Details on the study design:

Synthetic peptides:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Controls for the DPRA
Negative control (NC): = vehicle control (VC) = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), 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.

The test substance was prepared as a 100 mM preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.
Vehicle: acetonitrile
Reason for the vehicle: The test substance was soluble in acetonitrile

Three samples of the test substance were incubated with each peptide. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance). Additionally triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours. 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.

Results and discussion

Positive control results:

The positive control substance ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) led to a mean peptide depletion of 47.61% when incubated with cysteine-peptide and 12.06% with lysine-peptide. Therefore, the positive control was valid.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Reaction with cysteine-peptide

Parameter:

other: Peptide depletion in %

Value:

41.01

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: Reaction with lysine-peptide

Parameter:

other: Peptide depletion in %

Value:

4.45

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: Combined mean peptide depletion

Parameter:

other: Peptide depletion in %

Value:

22.73

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Executive summary:

The reactivity of Hydroxymethylpentanon 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 synthetic peptides for ca. 24 hours 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. Three samples of the test substance were incubated with each peptide. Additionally triplicates of the concurrent vehicle control (= VC) 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: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides. No co-elution of test substance and peptides was noticed. The mean C-peptide depletion, caused by the test substance was determined to be 41.01%. The mean K-peptide depletion, caused by the test substance was determined to be 4.45%. Thus, the mean peptide depletion was calculated to be 22.73%.

Based on the observed results it was concluded that Hydroxymethylpentanon shows a moderate chemical reactivity in the DPRA under the test conditions chosen.