Reaction products of tetraisopropyl titanate (4+), hydroxyalkaloic acid and substituted alkanol

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 June 2nd, 2020 to July 2nd, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 June 18th, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The commercially available EpiOcularTM kit was used. EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia. Designation of the kit: OCL-212-EIT; Day of delivery: 30. Jun. 2020; Batch no.: 30665.
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 cor-nea. It consists of highly organized basal cells. These cells are not transformed or trans-fected 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

54.1 mg of test material on 0.6 cm2 of tissue 1
53.7 mg of test material on 0.6 cm2 of tissue 2

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

17 hours and 45 minutes

Number of animals or in vitro replicates:

2 in vitro replicates

Details on study design:

CHEMICALS AND MEDIA
- MTT solution: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan, was prepared by the testing facility. A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL at – 20 ± 5 °C. 2 mL of the stock solution were thawed and diluted with 8 mL of as-say medium (resulting in 1 mg/mL). This MTT-solution with the concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium di-rectly before use.
- DPBS-Buffer: “Dulbecco`s Phosphate Buffered Saline” (DPBS) was used for the rinsing of the tissues. A subset was procured; the other subset was prepared by the tetsing facility.
Composition of the subset from MatTek Corporation:
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 L

Composition of the subset prepared by the testing facility:
KCl 0.4 g
KH2PO4 0.4 g
NaCl 16.0 g
Na2HPO4 * 2H2O 2.87 g
H2O ad 2 L
The buffer which was procured from MatTek Corporation was used for rinsing the test item from the tissues. The buffer which was prepared by the tetsing facilitywas only used for preparing the MTT concentrate.
- MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium).
- Assay Medium for Main Test: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)

PRE-TEST
- Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability of direct MTT reduction. To test for this ability, 49.9 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and ≥ 95% relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of H2O demin. was used as negative control. The MTT solution 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
52.3 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. Then, two 200 µL aliquots of the resulting solution and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm.
After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was -0.0025 (≤ 0.08). 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 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 ≥ 95% 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 ≥ 95% relative humidity for 17 hours and 37 minutes.
- Exposure 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 ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and a defined amount of the test item (see table 7.2-a) were applied in duplicate in one- minute- 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 ≥ 95% 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 17 hours and 45 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% 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 solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 165 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, contain-ing 2 mL isopropanol, taking care that no isopropanol was 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-well-plate. The plate was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 µl isopropanol each, serving as blank.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Value:

1.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the EpiOcularTM test at the tetsing facility was demonstrated in a proficiency study. For this purpose, 15 proficiency chemicals (indicated by the
OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized.. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. OD of negative control: 1.7.
- Acceptance criteria met for positive control: Yes. % tissue viability of positive control: 33.3%
- Acceptance criteria met for variation within replicates: Yes, 12.8% variation within replicates for negative control, 7.3% variation within replicates for positive control, 0.3% variation within replicates for test item.
- Range of historical values if different from the ones specified in the test guideline: negative and positive controls are in the range of historical values.

Applicant's summary and conclusion

Interpretation of results:

other: According to OECD 492, the test does not allow discrimination between eye irritation/reversible effects (Cat 2) and serious eye damage/irreversible effects on the eye (Cat1). For this reason further testing with other suitable test methods is required.

Conclusions:

Under the conditions of this test, the substance is considered either eye irritant or inducing serious eye damage.

Executive summary:

Determination of Eye Hazard Potential of Titanium Glycolate Monoethanolamine using the EpiOcularTM Reconstructed human Cornea-like Epithelium (RhCE) test method was performed following OECD 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. One valid experiment was performed. After treatment and rinsing of the test item from the tissue, the cell viability of the tissues was evaluated by MTT test for cell viability. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear eye irritating effects. The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 1.8%. This value is below the threshold for eye irritation potential (≤ 60%). Therefore, under the conditions of the test, Titanium Glycolate Monoethanolamine is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

However, according to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.