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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2020-11-24 to 2020-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate
EC Number:
848-455-1
Cas Number:
1398610-06-4
Molecular formula:
C25H22Cl2F4N2O4
IUPAC Name:
(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate
Specific details on test material used for the study:
Batch No.: 465884
Purity: 100%

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Selection and preparation of corneas:
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32±1℃. The corneas were incubated for the minimum of 1 hour at 32±1℃.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
- Quality check of the isolated corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
300.1 to 338.0 mg
Duration of treatment / exposure:
240 ± 10 mins
Number of animals or in vitro replicates:
3
Details on study design:
NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: 20% (w/v) Imidazole

APPLICATION DOSE AND EXPOSURE TIME:
The medium from the anterior compartment was removed and 750 μL of the negative control
and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of
the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a
way that the cornea was completely covered (300.1 to 338.0 mg). The holder was slightly
rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of
the solutions over the entire cornea. Corneas were incubated in a horizontal position for
240 ± 10 minutes at 32±1℃.

REMOVAL OF TEST SUBSTANCE:
After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others: Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
The second experiment
Value:
>= -2.5 - <= -0.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, the individual in vitro irritancy scores for the negative controls ranged from -1.4 to 0.3. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 112 to 150. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test item showed opacity values ranging from 0.4 to 6.5 and permeability values ranging from -0.006 to 0.001. The corneas were translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.3 to 6.4 after 240 minutes of treatment with the test item. The mean in vitro irritancy score was 2.5 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 0.3, 6.4 and 0.9, respectively), the test was inconclusive and a repeat experiment was performed.
In the second experiment, the individual in vitro irritancy scores for the negative controls ranged from 0.6 to 2.8. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 106 to 119. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with the test item showed opacity values ranging from -2.8 to -1.1 and permeability values ranging from 0.012 to 0.015. The corneas were clear after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.5 to -0.9 after 240 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since the test item induced an IVIS ≤ 3 in 5 out of 6 corneas, no classification is required for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437. The eye damage of the test item was tested through topical application for approximately 240 minutes.


In the first experiment, the in vitro irritancy scores ranged from 0.3 to 6.4 after 240 minutes of treatment with the test item. The mean in vitro irritancy score was 2.5 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 0.3, 6.4 and 0.9, respectively), the test was inconclusive and a repeat experiment was performed.


In the second experiment, the in vitro irritancy scores ranged from -2.5 to -0.9 after 240 minutes of treatment with the test item.


Since the test item induced an IVIS ≤ 3 in 5 out of 6 corneas, no classification is required for eye irritation or serious eye damage.