2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-10 - 2018-12-13 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

OECD Guideline for the Testing of Chemicals, Part 492, adopted 25. Jun. 2018, “Re-constructed Human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye Irritation or serious eye damage”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

472-110-0

EC Name:

-

Cas Number:

71868-15-0

Molecular formula:

C20H22O5

IUPAC Name:

2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl}-2-methylpropan-1-one

Test material form:

solid: particulate/powder

Remarks:

off white to pale yellow powder

Details on test material:

Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel in the refrigerator (2 – 8 °C); protected from light and humidity.

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemi-cals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcularTM EIT can be used as a replacement of the in vivo Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Specification:
Commercially available EpiOcularTM kit.
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.
Origin:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
The cells used to produce EpiOcular tissues are screened for potential biological contaminants. None of the following potential contaminants were detected:
HIV-1 virus (Oligonucleotide-directed amplification)
Hepatitis B virus (Oligonucleotide-directed amplification)
Hepatitis C virus (Oligonucleotide-directed amplification)
Bacteria, yeast, other fungi (long-term antibiotic, antimycotic free culture)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 52.6 and 50.8 mg / tissue
- Concentration (if solution): undiluted

VEHICLE
none

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number
Commercially available EpiOcularTM kit.
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.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
- Doses of test chemical and control substances used
Test chemical: 52.6 and 50.8 mg/tissue
Controls: 50 µL of the controls
- 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 ≥ 95% relative humidity for 29 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.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- 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)
Spectrophotometer, 570 nm
- Description of the method used to quantify MTT formazan
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 solution. The plate was incubated for 180 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, containing 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.
Measurement
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. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
OD value; > 60% of control
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Parameter Optical Density Negative Control Relative Tissue Viability Positive Control
Demineralised H2O Methyl acetate
Exposure time 6 hours
Mean 1.631 34.8%
Standard deviation 0.271 7.3%
Range 1.047 - 2.340 21.1 - 53.9%
Current study 1.725 42.7%

- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The validity of the EpiOcularTM test at the laboratory 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
Yes
- 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: % tissue viability

Run / experiment:

mean test item

Value:

77.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: % tissue viability

Run / experiment:

mean positive control

Value:

42.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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: not different

Applicant's summary and conclusion

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

Under the test conditions, the test substance was therefore concluded to be non-irritating to eyes.

Executive summary:

A study was performed to assess the potential of the test substance to cause eye irritation in a re-constructed human cornea-like epithelium (RhCE) method according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model, in duplicate, for an exposure time of 6 hours. After treatment, the substance was rinsed from the tissue, then cell viability 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 as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability being 42.7% (< 50%). The variation within tissue replicates of the controls and the test substance was acceptable (< 20%). After treatment with the test substance, mean relative tissue viability was 77.4%. This value is well above the threshold for eye irritation potential (≤ 60%). Under the test conditions, the test substance was therefore concluded to be non-irritating to eyes (Geitlinger, 2019).