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Diss Factsheets

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:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide
Molecular formula:
C23H18Cl2F4N2O4
IUPAC Name:
4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide
Test material form:
solid
Details on test material:
Off-white

Test animals / tissue source

Species:
chicken
Strain:
other: COBB 500 and ROSS 308
Details on test animals or tissues and environmental conditions:
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni u 129., Hungary
Collection: heads were collected by a technician from a commercial abattoir after approximately 7 weeks old chicken were slaughtered for human consumption. Heads were inspected for appropriate quality and wrapped with paper moistened with saline and then placed in a sealed plastic box. The heads were then immediately transported to the laboratory at ambient temperature and processed within 2 hours of collection in each experiment.
Eye selection: delivered heads were removed from the plastic box and placed on soft paper. The eyelids were carefully cut away with scissors, avoiding damage of the cornea. One small drop of 2% w/v fluorescin solution was applied onto the cornea surface for a few seconds. The eye was subsequently rinsed with 20 mL of physiological saline. The treated cornea was then examined with a hand-held slit lamp or microscope to ensure that it was undamaged. If the cornea was in a good condition, the eyeball was carefully removed from the orbit.
Preparation of the eye: the eyeball was removed without cutting off the optical nerve too short avoiding pressure on the eye to prevent distortion of the cornea and subsequent corneal opacity. The removed eyeball was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eye was kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Eyes examination and acclimatisation: the prepared eye was placed in a steel retainer. The cornea was positioned vertically with the eye in the correct relative position, by avoiding too much pressure on the eye. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The retainer holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops per minute or 0.1 to 0.15 mL/minute. The chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes (nine to twelve) were selected, placed in the superfusion apparatus, and examined again with the slit lamp microscope. The focus was adjusted to clearly see the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescine staining or corneal opacity score were rejected. The cornea thickness was measured with an optical pachymeter on a slit-lamp microscope, which was set at a 0.095 mm slit-width. Any eye with cornea thickness deviating by more than 10% from the mean value of all eyes, or eyes showing any other signs of damage were rejected and replaced. Once the selected eyes were appropriate for the test, acclimatisation was started for a period of about 45 to 60 minutes. The chambers of the superfusion apparatus were held at a controlled temperature of 32 ± 1.5 °C.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
An aliquot of 30 mg powdered test substance was applied onto the entire surface of the cornea. The positive control eyes were treated in a similar way with 30 mg powdered imidazole. The negative control eye was treated with 30 μL of physiological saline.
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The negative and positive control eyes as well as all test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
Three for test substance, one for negative and three for positive control
Details on study design:
At the end of the acclimatisation period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a base line (t = 0) for each individual eye. The corneal thickness of the eyes should not change by more than 5% between the -45 min and the zero time. No changes in thickness (0.0%) were observed in any eye in each experiment. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment I and II
Value:
>= 12.2 - <= 29.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive control
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment I and II
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive control
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment I and II
Value:
>= 2.5 - <= 3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive control
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment I and II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative control
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment I and II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Netative control
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment I and II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative control
Other effects / acceptance of results:
The results from all eyes used met the quality control standards. The negative control and positive control results were in good correlation with historic data. This experiment was considered to be valid. Historical data for negative control (n = 497) were -3.2 to 3.4% after 75 minutes and -4.8 to 3.4% after 240 minutes for corneal swelling, maximum 0.5 for opacity change and 0.5 for fluorescine retention change. Historical data for positive control (n = 221) were -6.6 to 25% after 75 minutes and -15.9 to 36.7% after 240 minutes for corneal swelling, 3.5 to 4.0 for opacity change and 2.0 to 3.0 for fluorescine retention change.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the in vitro isolated chicken eye assy, the substance is non-irritating to the eye.
Executive summary:

An in vitro eye irritation study was conducted under GLP to OECD TG 438, using the isolated chicken eye test. The study used three eyes for the treatment with test substance, alongside with one negative and three positive control eyes in each of two experiments. After the zero reference (baseline) measurement, each eye in the treatment group was held in a horizontal position and 30 mg of powdered test material was applied onto the centre of teh cornea such that the entire surface was covered. After 10 seconds of exposure, the surface was rinsed with physiological saline. In both experiments, the positive control eyes were treated in a similar way with 30 mg of powdered imidazole and the negative control eye was treated with 30 μL of physiological saline (i.e. 0.9% NaCl solution). Corneal thickness, corneal opacity and fluorescein retention changes were measured and any morphological effects (such as pitting or loosening of the epithelium) were evaluated over a four-hour period. In experiment I, no corneal swelling, no cornea opacity, and no fluorescein retention change was observed during the observation period in any of the three treated eyes. Test substance was stuck on all corneal surfaces after the rinsing, and the surfaces were cleared at 30 minutes after the rinsing. The negative and positive control group results demonstrated the validity of the study and the sensitivity of the test system.