2(5H)-Furanone, 5-hydroxy-4-methyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: EpiDerm Corrosivity-Test in vitro, similar to OECD 431. However, performed before OECD 431 has come into effect in 2004.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 431

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-hydroxy-4-methyl-2,5-dihydrofuran-2-one

EC Number:

609-869-6

Cas Number:

40834-42-2

Molecular formula:

C5 H6 O3

IUPAC Name:

5-hydroxy-4-methyl-2,5-dihydrofuran-2-one

Constituent 2

Reference substance name:

Hydroxybutenolid

IUPAC Name:

Hydroxybutenolid

Details on test material:

- Name of test material (as cited in study report): Hydroxybutenolid
- Test substance number: 00/0213-1
- Substance type: solid/yellowish-white powder
- Analytical purity: 92.7%
- Purity test date: July 10, 2000
- Date of manufacture: March 6, 2000
- Stability under test conditions: stability in DMSO and water has not been determined analytically.
- Storage condition of test material: room temperature

Test animals

Species:

other: in vitro test

Strain:

other: in vitro test

Test system

Type of coverage:

open

Preparation of test site:

other: in vitro skin model

Vehicle:

not specified

Controls:

other: in vitro test

Amount / concentration applied:

100% of the test substance, at least 25 uL/cm2.

Duration of treatment / exposure:

3 min and 1h

Observation period:

not applicable; Optical density measured at the end of the exposure (3 min; 1 h).

Number of animals:

in vitro test;

Details on study design:

Principles of the test:
The test material is applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability [as determined, for example, by using the MTT
reduction assay] below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

Application of the test and control substances:
Two tissue replicates are used for each treatment (exposure time), including controls. For liquid materials, sufficient test substance must be applied to uniformly cover the skin surface; a minimum of 25 L/cm2 should be used. For solid materials, sufficient test substance must be applied evenly to cover the skin, and it should be moistened with deionisised or distilled water to ensure good contact with the skin. Where appropriate, solids should be ground to a powder before application. The application method should be appropriate for the test substance. At the end of the exposure period, the
test material must be carefully washed from the skin surface with an appropriate buffer, or 0.9% NaCl.

Concurrent positive and negative controls should be used for each study to ensure adequate performance of the experimental model. The suggested positive control substances are glacial acetic acid or 8N KOH. The suggested negative controls are 0.9% NaCl or water.

Cell viability measurements
Only quantitative, validated, methods can be used to measure cell viability. Furthermore, the measure of viability must be compatible with use in a three-dimensional tissue construct. Non-specific dye binding must not interfere with the viability measurement. Protein binding dyes and those, which donot undergo metabolic conversion (e.g. neutral red) are therefore not appropriate. The skin sample is placed in an MTT solution of appropriate concentration (e.g. 0.3 – 1 mg/mL) at appropriate incubation temperature for 3 hours. The precipitated blue formazan product is then extracted using a solvent (isopropanol), and the concentration of the formazan is measured by determining the OD at a wavelength between 540 and 595 nm.

Interpretation of results
The OD values obtained for each test sample can be used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test materials (or discriminating between different corrosive classes), or the statistical procedure(s) used to evaluate the results and identify corrosive materials, must be clearly defined and documented, and be shown to be appropriate. In general, these cut-off values are established during test optimisation, tested during a prevalidation phase, and confirmed in a validation study. As an example, the prediction of corrosivity associated with the EpiDerm model is:

The test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50%, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.

The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: not applicable: in vitro test

Remarks on result:

other:

Any other information on results incl. tables

Exposure: 3 min
Test-Article	OD570 tissue 1	OD570 tissue 2	OD570 KC	mean OD570	mean OD570 KC corrected	viability [% ofNaCl]
NC	1,651	1,736	-	1,693	-	100
00/0213-1	1,343	1,428	-	1,385	-	82
PC_C	0,520	0,430	-	0,475	-	27

Exposure: 1 h
Test-Article	OD570 tissue 1	OD570 tissue 2	OD570 KC	mean OD570	mean OD570 KC corrected	viability [% ofNaCl]
NC	1,842	1,721	-	1,781	-	100
00/0213-1	0,373	0,402	-	0,388	-	22
PC_C	0,174	0,194	-	0,184	-	11

NC       = Negative control

PC_C   = Positive control

OD570 = Optical Density (wavelength 570 nm)

KC       = Killed tissue Control (only included if applicable)

N        = No    

Y          = Yes

Applicant's summary and conclusion

Conclusions:

The test substance was observed to be not corrosive in the EpiDerm Corrosivity Test. However, possible skin irritancy can not be evaluated in this test system.