3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

EC Number:

204-729-7

EC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Cas Number:

125-20-2

Molecular formula:

C28H30O4

IUPAC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Details on test material:

- Storage conditions: Room Temperature (20 ± 5 °C)
- Expiry date: 16. Dec. 2019

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability : This test method is an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

tissues were pre-wetted with 20 µL DPBS buffer

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50.4 mg, 51.3 mg resp.
- Concentration (if solution): 100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL methyl acetate (CAS 79-20-9)
- Concentration (if solution): 100 %

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL sterile demineralised water
- Concentration (if solution): 100 %

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes post soak, 18 hours and 10 minutes post-treatment incubation

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number : 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: 24. Jul. 2018, batch no.: 27060
- Doses of test chemical and control substances used : 50 µL for controls , ca. 50/51 mg test item
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 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 29 minutes.
After that, 50 µL of the controls and a defined amount of the test item were applied in duplicate in one-minute-intervals. A small amount of test item dropped into the medium of the second replicate, this is why the medium was aspired and replaced by fresh medium.
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 and 10 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Description of any modifications to the test procedure : no relevant modifications
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : colour development of test item and direct reduction of MTT by test item were assessed. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place and no data correction was necessary.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm
- Description of the method used to quantify MTT formazan : measurement of optical density at 570 nm in plate spectrophotometer
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : OD of negative control: > 0.8 - < 2.5, % mean relative viability of positive control: < 50 % of negative control, variation within replicates < 20 %, viability threshold for eye irritation potential (≤ 60%).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Values for negative control and for positive control were within the range of historical data of the test facility
- Complete supporting information for the specific RhCE tissue construct used : The specific tissue construct used passed the quality control
- Reference to historical data of the RhCE tissue construct : test with specific tissue construct lies within the acceptance criteria, which are based on 1996 QC Database
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : The validity of the EpiOcularTM test 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.
- Positive and negative control means and acceptance ranges based on historical data : negative control (OD): historical: 1.618 (mean), study: 1.783; positive control (viability, %): historical: 34.6 (mean), study: 44.3
- Acceptable variability between tissue replicates for positive and negative controls : < 20 %
- Acceptable variability between tissue replicates for the test chemical: < 20 %

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: viability %

Run / experiment:

Tissue 1

Value:

103.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: viability %

Run / experiment:

Tissue 2

Value:

104.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: viability %

Run / experiment:

Mean

Value:

103.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: negative control (OD): 1.047 - 2.340; positive control (viability %): 21.1 - 53.9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation potential of the test substance to the eye in vitro. After treatment with the test item, the mean value of relative tissue viability was 103.6 %. This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Executive summary:

The eye hazard potential of the test item has been determined in the EpiOcularTM Reconstructed human Cornea-like Epithelium (RhCE) test method following OECD 492 (GLP).

One valid experiment was performed.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

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 and 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 1.8. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 44.3 % (< 50%).

Variation within tissue replicates of the controls and test item was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 103.6 %.

This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.