Ethyl 2-ethylhexanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 16/2/2017 to 26/4/2017

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

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 animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

-Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

-Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 28: Mar. 2017
Batch no.: 23773

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL of the controls and the test item were applied in 1 minute intervals.

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Duplicate

Details on study design:

Pre-Tests
-Assessment of Direct Reduction of MTT by the Test Item
The test item Irotyl was tested for the ability of direct formazan reduction. To test for this ability, 50 μL of the liquid test item were added to 1 mL of MTT reagent in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 for 3 hours. 1 mL of MTT reagent plus 50 μL of H2O demin. was used as negative control. The MTT reagent did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

-Assessment of Coloured or Staining Test Items
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 50 μL of the liquid test item were added to 1 mL of sterile demin. water in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 for 1 hour. After incubation, no colour development was visible. Therefore, the main test was performed without colourant controls.

Main Test
-Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours and 30 minutes.

-Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and the test item were applied in duplicate in 1 minute intervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1 minute intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes resp. 14 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100% relative humidity. After the post-treatment incubation, the MTT Assay was performed.

-MTT Assay and Extraction
A 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The
plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 hours at room temperature, protected from light.

-Measurement
The inserts were pierced with an injection needle, taking care that all colour is extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

27.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: the test substance is considered as producing eye irritation/reversible effects on the eye (CLP Category 2) or serious eye damage/irreversible effects on the eye (CLP Category 1)

Conclusions:

Under the conditions of this test, the test substance is considered as producing eye irritation/reversible effects on the eye (CLP Category 2) or serious eye damage/irreversible effects on the eye (CLP Category 1).

Executive summary:

The test item Irotyl was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 30 minutes. 50 μL of the liquid test item was applied to each tissue. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 26.6 % (< 50%). Variation within tissue replicates was acceptable (≤ 20%). After treatment with the test item, the mean value of tissue viability was 27.7 %. This value is well below the threshold for eye irritation potential (≤ 60%). Under the conditions of this test, Irotyl is considered as producing eye irritation/ reversible effects on the eye (CLP Category 2) or serious eye damage/ irreversible effects on the eye (CLP Category 1).