2-ethyl-3-hydroxy-4-pyrone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2016 - 13 January 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

No 761/2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

published 2003, last (6th) revision 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

14 September 2015

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 vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes (NHEK)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: not applicable

Justification for test system used:

According to testing guideline OECD Guideline 439

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™
- Tissue batch number(s): 17-EKIN-002
- Production date/Shipping date/Delivery date: 10 jan 2017
- Date of initiation of testing: 10 jan 2017 (start tissue exposure experiments)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure/ post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: not specified
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- 5 µL of deionised water was topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item was applied and spread to the wetted triplicate tissues.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

122.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

8.1

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

ACCEPTANCE OF RESULTS:
- The relative mean tissue viability for the positive control treated tissues was 8.1% relative to the negative control treated tissues and the standard deviation value of the viability was 122.8%. The positive control acceptance criteria were therefore satisfied.
- The mean OD570 for the negative control treated tissues was between ≥ 0.6 and ≤ 1.5 and the standard deviation value of the viability was 8.2%. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.4%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Results after treatment with Ethyl Maltol and controls

Test Group	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1/ 2/ 3**	Relative Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	1.053	105.5 / 103.9 / 90.6	8.2	100.0
Positive Control	0.086	7.0 / 7.8 / 9.6	15.9	8.1
Test Item	1.293	112.4 / 129.1 / 126.8	7.4	122.8

** relative absorbance per tissue [rounded values]

*** relative absorbance per treatment group [rounded values]

Applicant's summary and conclusion

Interpretation of results:

other: not skin irritant

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this test, Ethyl Maltol is not a skin irritant.

Executive summary:

In an in vitro skin irritation assay in the human epidermal model EpiSkin (1801400), reconstructed human keratinocytes (moistened with 5µL water) was exposed to 10 mg of Ethyl Maltol (>99%) for 15 minutes. Deionised water was used for the negative control and 5% SLS was used for the positive control. After removal of the test substance via washing, tissues were post-incubated for 42 hours. Tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥ 0.6 and ≤ 1.5 (1.053). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (8.1%). Standard deviation of viability of replicate tissues of all dose/negative control/positive control groups was ≤ 18% (7.4%/8.2%/15.9%).

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test item, Ethyl Maltol, was 122.8 % of the negative control average value i.e. viability was > 50 %. According to these results, the test substance is not irritating.