Ethyl 2-ethylhexanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24/10/2016 to 04/05/2017

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-ethylhexanoate

EC Number:

221-043-3

EC Name:

Ethyl 2-ethylhexanoate

Cas Number:

2983-37-1

Molecular formula:

C10H20O2

IUPAC Name:

ethyl 2-ethylhexanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed epidermis of normal human keratinocytes

Cell source:

other: Adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

-Test System
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch: 16EKIN049
Arrived at RTC on: 06 December 2016

-Preparation and pre-treatment incubation period
The test system was shipped onMonday and received on Tuesday. According to the supplier procedure, tissues were prepared as follows:
Alive tissues: at arrival, plates were opened under a sterile airflow and each insert,containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthicMaintenance Medium.
Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

-Media
Maintenance Medium: SkinEthic; batch: 16MAIN3 080
AssayMedium: SkinEthic; batches: 16ESSC 048 and 16ESSC 052

-Preliminary test
Direct MTT reduction test (Step 1)
Non-specific reduction of MTT was evaluated as follows: two mL of MTT ready-to-use solution (0.3 mg/mL) was incubated with 20 µL of test item at 37°C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 20 µL of the test item was added to 180 µL of distilled water (Baxter; batch no. 15I030I) in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. At the end of the incubation time, colouring of the solution/suspension evident to the unaided eye and measured by spectral analysis at 595 nm, was evaluated.

-Main Assay
Treatment
In theMain Assay, alive tissues were treated with the test item, positive and negative controls.

Exposure period
An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.

Washing
At the end of the exposure, each tissue was rinsed with approximately 25mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.

Post-exposure period
A 42 ± 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

MTT staining
Each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution, with the exception of tissues used for the unspecific colouring potential control. Plates were incubated for approximately 3 hours at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range, an MTT formazan calibration curve was performed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 µl for negative control, positive control and test item

Duration of treatment / exposure:

An exposure time of 15±0.5 minutes was allowed

Duration of post-treatment incubation (if applicable):

A 42 ± 1 hour recovery period was allowed

Number of replicates:

3 replicates for negative control, positive control and test item, 2 replicates for only test item without MTT.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: cell viability in % (obtained from optical density)

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.
The blank, negative and positive controls gave acceptable results and the study was accepted as valid.
The mean cell viability of the test item treated tissues, after the blank subtraction, was 74%.
Based on the results obtained, the test item is classified as not irritant to the skin (UN GHS No Category).

Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducingMTT per se. No relevant interaction between the test teim andMTT was observed. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 0.604. This unexpected result indicated that the test item could have a potential interfering ability, thus an additional control was added in theMain Assay.

In theMain Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit, each measuring 0.3 cm2 (treatment level: 53 µL/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.5). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid.

The NSCliving was 5%, thus only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 74% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 14.7 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item is classified as not irritant to the skin.