2-ethyl-3-hydroxy-4-pyrone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Mar 2017 - 11 Mar 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:

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

1

Reference substance name:

2-ethyl-3-hydroxy-4-pyrone

EC Number:

225-582-5

EC Name:

2-ethyl-3-hydroxy-4-pyrone

Cas Number:

4940-11-8

Molecular formula:

C7H8O3

IUPAC Name:

2-ethyl-3-hydroxy-4H-pyran-4-one

Test material form:

solid

In chemico test system

Details on the study design:

Peptides and positive control
-Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
-Synthetic peptide containing lysine: AC-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
-Positive control: Cinnamic aldehyde, Batch number MKBR2427V purity > 95%, supplied by SAFC, stored at ambient temperature.

Preparation of peptide stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).
Preparation of peptide calibration standards: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls: Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine. In place of test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine. The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine. In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution.

Incubation: The appearance of the Ethyl Maltol, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run.

Analysis: The concentration of both the cysteine and lysine peptides in the presence of Ethyl Maltol and the associated positive controls were quantified by HPLC using UV detection. Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector (Column: Agilent Zorbax SB C18, 3.5 μm, 100 × 2.1 mm, Guard column: Phenomenex AJO4286)

Calculations: The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation: % peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Results and discussion

Positive control results:

69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean n=3

Parameter:

other: cysteine depletion, %

Value:

0.026

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: mean n=3

Parameter:

other: lysine depletion, %

Value:

1.28

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

mean

Parameter:

other: mean cysteine and lysine depletion, %

Value:

0.652

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Reference (stability) controls and precision controls of both peptides were met (CV 0.97%, n = 6 and CV 0.26%, n = 6, for cysteine and lysine, respectively, at 0.50 mM and 0.51 mM).
- Acceptance criteria met for positive control: yes, 69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.25% and SD 0.56%, respectively, for cysteine and lysine depletion by the test item.
 
TEST SUBSTANCE RESULTS:
Mean depletion of 0.0260% and 1.28% was observed for the test substance with cysteine and lysine peptides, respectively. The mean of results depletion by Ethyl Maltol is 0.652% With the test substance not reacting with the cysteine nor lysine peptide it is classed as “no to minimal”, hence the DPRA prediction is negative.

Applicant's summary and conclusion

Interpretation of results:

other: DPRA was negative

Conclusions:

It can be concluded that this DPRA test is valid, and that the test substance was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; FD95MW), Ethyl Maltol (>99%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 hrs at 25°C). Subsequently samples were analysed by HPLC. Reference (stability) controls and precision controls, co-elution controls and a positive control (cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The acceptance criteria for the calibration curve samples, the reference (stability) controls and precision controls and co-elution controls, as well as for the study samples were satisfied. The acceptance criteria for positive control (cinnamic aldehyde) were met; 69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively. The study was therefore considered to be valid.

The test substance caused 0.0260% cysteine peptide depletion and 1.28% lysine peptide depletion. The mean of results depletion by Ethyl Maltol is 0.652% and the test substance was therefore classified as “no to minimal reactivity” based on the Cysteine 1:10 / Lysine 1:50 prediction model and was thus considered to be negative in the DPRA.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 442E were also performed. The data generated with this test will be considered in the context of an integrated approaches such as IATA, combining the result with other complementary information from the other 2 tests.