Sodium 4-oxovalerate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 7th to June 9th, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Adopted 28 July 2015

Principles of method if other than guideline:

Additional information was taken from:
- “EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals” by MatTek Corporation, document no. MK-24-007- 0055, dated 14. Jul. 2014
- Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. “Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test”. Toxicology in Vitro 12, 455-461

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01

Molecular formula:

n.a

IUPAC Name:

Similar substance 01

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the model used: 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.
- Commercial Name: EpiOcular™ kit.
- Supplier: MatTek
- Batch: 23713

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μl

Duration of treatment / exposure:

29 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Details on study design:

PRE-TEST
- Assessment of Direct Reduction of MTT by the Test Item: the test item 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 ± 1 % CO2 and 80 –100 % relative humidity 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 a clear and colourless liquid. 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 H2O demin. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After incubation, no colour development was visible and no data correction was necessary.

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 and the solution was stored at 2 - 8 °C in the dark. The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test item, resp. negative control, resp. positive control and filled with 1 ml assay medium in the appropriate wells. All 24 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.
- Exposure 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 29 ± 1 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 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.
- Measurement: the inserts were pierced with an injection needle, taking care that all colour was 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 spectral photometer at 570 nm.

DECISION CRITERIA
Viability > 60 % Non eye irritant - No GHS category for eye irritation
Viability ≤ 60 % Eye irritant -GHS category 1 or 2

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Viability %

Run / experiment:

Tissue 1

Value:

2.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Viability %

Run / experiment:

Tissue 2

Value:

2.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Viability %

Run / experiment:

Mean of Tissue 1 and Tissue 2

Value:

2.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: within the range of historical data of the test facility
- Acceptance criteria met for positive control: within the range of historical data of the test facility
- Validity criteria were met: OD of negative control = 2.2 %, % Formazan production of positive control = 21.8 %, Variation within replicates = 1.3 % (negative control), 1.3 % (positive control), 0.1 % (test item).

Any other information on results incl. tables

Deviations from the Study Plan: in the additional tests, 50 μl instead of 50 mg of test item were used. This can be seen as uncritical, because it was an error in the study plan due to unclear aggregate state of the test item.

Applicant's summary and conclusion

Interpretation of results:

other: classified for eye irritation (Cat.1 or Cat.2) according to the CLP Regulation (EC) No.1272/2008

Conclusions:

Eye irritant or inducing serious eye damage (Cat.1 or Cat.2)

Executive summary:

The potential of the test material to induce eye irritation in a human cornea model in an in-vitro study, performed according to the OECD Guideline 492.The test item 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. 50 μl demineralised water was used as negative control and 50 μl methyl acetate was used as positive control.

The mean tissue viability was found to be 2.5 %. This value is well below the threshold for eye irritation potential (≤ 60 %).

Under the conditions of the test system, the test substance is considered as eye irritant or inducing serious eye damage in the EpiOcular^TM Eye Irritation Test.