Registration Dossier

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:
2020
Report date:
2020

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:
(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide
EC Number:
857-673-6
Cas Number:
1879067-61-4
Molecular formula:
C48H52F4N4O4.2Br
IUPAC Name:
(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni u 129., Hungary
Chicken heads were collected from a commercial abattoir after chickens (approximately 7 weeks old) had been slaughtered for human consumption. Heads were collected by a slaughter house technician. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a sealed plastic box (4-5 heads/box). The heads were immediately transported to the test laboratory at ambient temperature. The heads were processed within 2 hours of collection.
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, to avoid 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 still 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.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting 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.
The prepared eye was placed in a steel retainer. The cornea was positioned vertically with the eye in the correct relative position (same position as in the chicken head), taking care to avoid putting too much pressure on the eye by the retainer. Due to the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. 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/minute or 0.1 to 0.15 mL/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes (approximately nine to twelve) were selected, after being placed in the superfusion apparatus they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) 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 for all eyes, or eyes that showed any other signs of damage were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization was started and conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatization and treatment periods.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied as supplied (although it was ground to fine powder). An amount of 30 mg test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.
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:
One eye for negative control (physiological saline), three eyes for treatment with test substance, three eyes for positive control (imidazole)
Details on study design:
The time of application was observed, then after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with at least 20 mL physiological saline (B. Braun Pharmaceuticals SA, Lot number 90352Y05-2) at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum required.
Additional gentle rinsing with 20 mL saline (3 or 4 times) was performed at each time point when the test item or the positive control material was observed to remain on the cornea.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. A slit-lamp microscope was used for the measurements and was set at a 0.095 mm slit-width.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
corneal swelling 
Run / experiment:
Mean of three eyes
Value:
3.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A mean maximum corneal swelling of 3.2% was noted at up to 75 minutes and 240 minutes after application.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean of three eyes
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean of three eyes
Value:
0.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Test substance was stuck on all corneal surfaces after the post-treatment rinse. All surfaces were clear at 75 minutes after the post-treatment rinse. Positive control substance was stuck on the cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. No other morphological effects were observed.
The positive control substance imidazole was classified as severely irritating, based on the observations on corneal swelling, corneal opacity change and fluorescein retention change. The negative control physiological saline (Sasol solution, NaCl 0.9% w/v) was classified as non-irritating.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not irritating to the eye based on the results of the in vitro eye irritation study.
Executive summary:

An in vitro eye irritation study was performed under GLP on isolated chicken eyes to OECD TG 438 (June 2018). Fresh isolated chicken eyes were obtained from a commercial abattoir and used within 2 hours of receipt. Three eyes were used for the treatment with the test substance, alongside one negative (physiological saline) and three positive (imidazole) control eyes. After the preparation of the chicken eyes and the zero reference (baseline) measurements, each eye in the treatment group was held in a horizontal position and 30 mg of powdered, neat test substance applied onto the centre of the cornea such that the entire surface of the cornea was covered. After ten seconds, the surface was rinsed with physiological saline. In the experiment the positive control eyes were treated in a similar way with 30 mg of powdered imidazole. The negative control eye was treated with 30 μL of physiological saline. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects were evaluated over a four hour observation period. No significant corneal swelling (mean 3.2%) and no significant cornea opacity change (severity 0.5) was observed on any of the three eyes treated with the substance. Slight fluorescein retention change (severity 1.0 on two eyes and severity 0.5 on one eye) was noted on the three eyes. Based on these findings, the test substance was considered as non-irritant. The results obtained with negative and positive controls demonstrated the sensitivity of the assay and that the study was valid.