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

Endpoint summary

Description of key information

DPRA: Hydroxymethylpentanon shows a moderate chemical reactivity

LuSens: Hydroxymethylpentanon has a keratinocyte activating potential

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

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.

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.

Run / experiment:

other: Reaction with cysteine-peptide

Parameter:

other: Peptide depletion in %

Value:

41.01

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Reaction with lysine-peptide

Parameter:

other: Peptide depletion in %

Value:

4.45

Vehicle controls validity:

valid

Positive controls validity:

valid

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Feb 2016 - 02 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

In addition, the LuSens is based on the following publication: Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D,
Ravenzwaay BV, Landsiedel R.., LuSens: A keratinocyte based ARE reporter gene
assay for use in integrated testing strategies for skin sensitization hazard identification.
Toxicol In Vitro. 2014 Dec;28(8).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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.

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.

Details on the study design:

Cell line:
LuSens (Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen)

Controls for the LuSens
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 450 μg/mL in 1% DMSO in culture medium 3
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA, CAS no. 97-90-5), 18 μg/mL in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). The test-substance preparations were prepared by stirring. Reason for the vehicle: The test substance was soluble in 4% DMSO in culture medium 3. Form of application: Visual inspection of each dilution step was performed.

Experimental procedure:
Preparation of the cells
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. Each test substance preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition a clear plate was treated in parallel for the determination of cell viability.

Luciferase assay
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of Steady-Glo-preparation (= 100 μL Steady-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells
were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectralphotometer.

Positive control results:

The positive control substance ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) led to a >3 fold induction of luciferase, therefore the positive control was considered valid.

Run / experiment:

other: 1st experiment

Parameter:

other: fold induction

Remarks:

0 ug/mL

Value:

1

Vehicle controls validity:

other: increased fold induction was observed in several vehicle controls, potetially from evaporating test substance in the neighboring wells. Therefore, the test was repeated with lower doses in experiments 2 and 3.

Positive controls validity:

valid

Run / experiment:

other: 1st experiment

Parameter:

other: fold induction

Remarks:

59 ug/mL

Value:

11.78

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2nd experiment

Parameter:

other: fold induction

Remarks:

0 ug/mL

Value:

1

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2nd experiment

Parameter:

other: fold induction

Remarks:

59 ug/mL

Value:

2.84

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3rd experiment

Parameter:

other: fold induction

Remarks:

0 ug/mL

Value:

1

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 3rd experiment

Parameter:

other: fold induction

Remarks:

59 ug/mL

Value:

3.03

Vehicle controls validity:

valid

Positive controls validity:

valid

For further information on results, see attached background material.

Executive summary:

The keratinocyte activating potential of test substance Hydroxymethylpentanon was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 experiments were performed. The following results were observed: At concentrations used in the main experiment the test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. In the 1st experiment 5 of 20 vehicle control values were taken from evaluation as they showed increased fold-inductions. The effect was attributed to evaporation from high test substance concentrations in neighboring wells of the test plate. In experiments 2 and 3 lower test substance concentrations were tested and the effect was not observed again. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable as fold inductions above 1.50 were obtained in all evaluable concentrations of Hydroxymethylpentanon.

In summary, after 48 hours of exposure to test substance Hydroxymethylpentanon luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance Hydroxymethylpentanon has a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Experimental data was generated for Hydroxymethylpentanon. The substance was tested in the direct peptide reactivity assay (DPRA) and a keratinocyte activation assay (LuSens).

In the DPRA, peptide depletion following incubation with Hydroxymethylpentanon was 41.04% for cystein-peptides and 4.45% for lysine-peptides. 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.

In the LuSens assay, it was observed that Hydroxymethylpentanon induced luciferase activity in LuSens cells in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance Hydroxymethylpentanon has a keratinocyte activating potential.

Based on the prediction model for in vitro skin sensitisation testing, two out of three tests have to be congruent in order to arrive at a conclusion regarding the skin sensitization potential of a given test substance. Since congruent results were observed in DPRA and LuSens, testing the substance in the h-CLAT test detecting dendritic cell activation was considered not necessary. In accordance with the prediction model, the substance is considered to have a skin sensitizing potential. Currently, sensitization potency cannot be determined using in vitro tests. Therefore, no further information on the potency of Hydroxymethylpentanon can be given.

In addition to experimental data, the classification of Hydroxymethylpentanon is based on an impurity (Formaldehyde; specific concentration limit for skin sensitization according to Regulation (EC) No 1272/2008 Annex VI : >=0,2%).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with Regulation (EC) 1272/2008, Hydroxymethylpentanon fulfills the criteria for classification as skin sensitizer category 1 (H317: May cause an allergic skin reaction).