3-tert-butyladipic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-12 to 2018-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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 09. Oct. 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals, for use with MatTek Corporation's Reconstructed Human EpiOcular™ Model

Version / remarks:

14. July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-tert-butyladipic acid

EC Number:

233-759-3

EC Name:

3-tert-butyladipic acid

Cas Number:

10347-88-3

Molecular formula:

C10H18O4

IUPAC Name:

3-tert-butylhexanedioic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Test animals / tissue source

Species:

other: reconstructed human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Batch No.: 27027
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 ± 5 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium 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.

Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, the controls and a defined amount of the test item were applied in duplicate in 1-min- intervals. 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 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

- RhCE tissue construct used, including batch number

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27027
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 mg (test substance), 50 µL (controls)
- Duration and temperature of exposure; post-exposure incubation periods: 6 hours at 37 ± 1 °C; 25 min at room temperature + 18 hours at 37 ± 1 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary. Furthermore, no colour development was visible. Therefore, the main test was performed without colourant controls.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
The inserts were removed from the 6-well plate and discarded. 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. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a Microtiter plate photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is: < 50 % of negative control
3. Acceptable variability between tissue replicates: < 20 %

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: % viability

Run / experiment:

Tissue 1

Value:

1.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: % viability

Run / experiment:

Tissue 2

Value:

1.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct MTT reduction: No
- Colour interfrence with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD = 1.6
- Acceptance criteria met for positive control: Yes, % mean relative viability of positive control was 38.5 %
- Variation within replicates:
2.9 % (negative control)
4.9 % (positive control)
0.0 % (test item)

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made.

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was ≤ 60%. Thus, no prediction can be made.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD guideline 492 (LAUS, 2018).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours. 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 25 minutes and further 18 hours with fresh medium.

Following treatment with the test item, the tissue viability was ≤ 60% and, thus, according to OECD 492 no prediction can be made for the test item identified. The test substance either needs to be classified in cat 1 or cat 2 with respect to eye irritation.