Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2018
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:
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)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 44034
Description: Cream to light grey powder
Expiry date: 30 November 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
In each experiment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
Duration of treatment / exposure:
The control eyes and 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.
Duration of post- treatment incubation (in vitro):
The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Details on study design:
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.
The cornea thickness of the eyes should not change by more than 5% within the -45min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.Additional gentle rinsing with at least 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 75 min
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 240 min
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 75 min
Run / experiment:
II
Value:
0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 240 min
Run / experiment:
II
Value:
1.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
II
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
II
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate showed no corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (09 October 2017).

In each experiment after the zero reference measurements, the eye was held in horizontal position and powdered 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the study was considered to be valid.

Experiment I: No corneal swelling was observed during the four-hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change was observed on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on these in vitro eye irritation assays in isolated chicken eyes with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, the test item was non-irritant, UN GHS Classification: No Category.