Reaction mass of trans-5-hexyldihydro-4-methylfuran-2(3H)-one and cis-5-hexyldihydro-4-methylfuran-2(3H)-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-20 and 2015-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-conform study according to guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of trans-5-hexyldihydro-4-methylfuran-2(3H)-one and cis-5-hexyldihydro-4-methylfuran-2(3H)-one

EC Number:

922-963-4

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of trans-5-hexyldihydro-4-methylfuran-2(3H)-one and cis-5-hexyldihydro-4-methylfuran-2(3H)-one

Test animals

Species:

other: EPISKIN Standard Model

Strain:

other: EPISKIN Standard Model

Test system

Type of coverage:

other: 30 µL (47 µL/cm2) of the undiluted test item was dispensed directly on top the skin surface.

Preparation of test site:

other: The epidemis in the upper wells were preincubated for 60 minutes (37 ± 1.5°C, 5 ± 1% CO2, 95 ± 5% RH). The inserts were transferred from upper wells into the lower wells and preincubation was continued for 4.25 hours (37 ± 1.5°C, 5 ± 1% CO2, 95 ± 5% RH).

Vehicle:

unchanged (no vehicle)

Controls:

other: 5% Sodium Lauryl Sulphate in deionised water (positive control) and Dulbecco's Phosphate Buffered Saline (negative control) were used.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (47 µL/cm2)

Duration of treatment / exposure:

The complete exposure period of the skin equivalents was 60 minutes. According to the protocol of the test kit supplier, the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2 within 60 minutes. Outside the incubator the 6-well plates were placed in a sterile bench at room temperature for 25 minutes in sum.

Observation period:

not applicable

Number of animals:

Three replicates per test, negative control and positive control respectively were used.

Details on study design:

TEST SITE
- Area of exposure: surface of epidermis 0.6 cm2

REMOVAL OF TEST SUBSTANCE
- Washing: Tissues were gently rinsed with DPBS at least 15 times and afterwards submerged in DPBS at least three times. Subsequently, the tissue was once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the tissues and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
- Time after start of exposure: 60 min after exposure

SCORING SYSTEM: Cell viability by using the MTT test

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (test item)

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min treatment + 42 h post exposure. Remarks: 25.5% relative absorbance (to negative control). (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (positive control)

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min treatment + 42 h post exposure. Remarks: 6.8% relative absorbance (to negative control). (migrated information)

In vivo

Irritant / corrosive response data:

Cell viability: negative control
No. 1: 110.3%
No. 2: 98.8%
No. 3: 90.8%
Mean: 100%
Mean absorbance: 1.532

The absorbance value obtained for the positive control was 0.104 and this result corresponds to 6.8% viability when compared to the results obtained from the negative controls.
Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 25.5% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Other effects:

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance is irritant to skin.

Executive summary:

The irritation potential of the test substance was investigated by using the in vitro Human Skin Model Test (OECD 439, EU B.46). Each three tissues of the human skin model EpiDerm were treated with 30 µL of the test item, the negative (Dulbecco's Phosphate Buffered Saline) or the positive control (5% sodium lauryl sulfate) for 60 minutes. Cell viability was determined using the MTT assay. The absorbance value obtained for the positive control was 0.104 and this result corresponds to 6.8% viability when compared to the results obtained from the negative controls. After treatment with the test item Aprifloren the mean relative absorbance value decreased to 25.5% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. Thus, the test substance was considered to be irritant to skin under the test conditions chosen.