reaction mass of diethyl (E)-2-methylbut-2-enedioate and diethyl (Z)-2-methylbut-2-enedioate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 September 2016 - 04 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

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)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 2-methylidenebutanedioate

EC Number:

607-321-0

Cas Number:

2409-52-1

Molecular formula:

C9H14O4

IUPAC Name:

diethyl 2-methylidenebutanedioate

Test animals / tissue source

Species:

other: Reconstructed human Cornea-like Epithelium (RhCE)

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step started.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL of the test item

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

12 min + 120 min

Number of animals or in vitro replicates:

Duplicate tissues

Details on study design:

- Details of the test procedure used: MTT Assay

- RhCE tissue construct used, including batch number: EpiOcular Kit, Lot No.: 23737

- Doses of test chemical and control substances used: 50 µL undiluted test chemical / control substances

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 30 min, 37 °C
Post-exposure immersion: 12 min, room temperature
Post-exposure incubation: 120 min, 37 °C

- Number of tissue replicates used per test chemical and controls: 2 per test chemical, 2 per positive control and 2 per negative control

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan: measurement of absorbance at 570 nm (OD570) with a plate reader reader
(Versamax® Molecular Devices, Softmax Pro)

- Description of evaluation criteria used:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Acceptance criteria
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: relative absorbance (%)

Run / experiment:

Tissue 1

Value:

70.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: relative absorbance (%)

Run / experiment:

Tissue 2

Value:

61.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item does not possess any eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test, it is colourless and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

The test met the acceptance criteria. Irritating effects were not observed following incubation with test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (66.0%). Therefore, the test item is not considered to possess an eye irritant potential.