Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.


Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
In the experiment, 30 μL of the 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.
Three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. One negative control eye was treated with 30 μL of physiological saline.
Duration of treatment / exposure:
10 seconds
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.
Duration of post- treatment incubation (in vitro):
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.
Number of animals or in vitro replicates:
test item: 3 eyes
positive control: 3 eyes
negative control: 1 eye

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 min
Value:
ca. 4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 min
Value:
ca. 11.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
ca. 4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
ca. 3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on this in vitro eye irritation assay in isolated chicken eyes with Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine, the test item was irritant, UN GHS Classification: Category 1. However, the test item contains alcohol, and this test method has been shown to have high false positive rates for alcohols.
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 (25 June 2018).

 

After the zero reference measurements theeyeswere held in horizontal position and 30 µL test item was applied onto the centre of the corneas in such a way that the entire surface of the corneas was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive controleyeswere treated with 30 µL 5% (w/v) Benzalkonium chloride solution. The negative controleyewas treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treatedeyesand one negative control treatedeyewereexamined.

 

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. Thus, the study was considered to be valid.

 

Slight cornea swelling (mean 11.6%) was observed during the four-hour observation period on test item treated eyes. Severe cornea opacity change (severity 4 on all eyes) was observed on all three eyes. Severe fluorescein retention change (severity 3 on all eyes) was noted on the treated eyes.

 

 

Based on thisin vitroeye irritation assay in isolated chicken eyes with Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine, the test item was irritant, UN GHS Classification: Category 1. However, the test item contains alcohol, and this test method has been shown to have high false positive rates for alcohols.