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:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYI/38593-5/2012

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5,8,10,13,15,18,20,23,25,28,30,33,35,38,40,41,43,45,47,51,53,55,57,59,61,63, 65,67,69,71,73-dotriacontazapentacosacyclo [35.3.3.36,7.311, 12.316,17.321,22.326,27.331,32.22,41.236,43.13,40.15,8.110,13.115,18.120,23.125,28.130,33.135,38.145,51.147,73.153,55.157,59.161,63.165,67.169,71] octacontane-44,46,48,49,50,52,54,56,58,60,62,64,66,68,70,72-hexadecone, hydrochloride hydrate
EC Number:
700-970-1
Molecular formula:
C48N32H48O16xH2O
IUPAC Name:
3,5,8,10,13,15,18,20,23,25,28,30,33,35,38,40,41,43,45,47,51,53,55,57,59,61,63, 65,67,69,71,73-dotriacontazapentacosacyclo [35.3.3.36,7.311, 12.316,17.321,22.326,27.331,32.22,41.236,43.13,40.15,8.110,13.115,18.120,23.125,28.130,33.135,38.145,51.147,73.153,55.157,59.161,63.165,67.169,71] octacontane-44,46,48,49,50,52,54,56,58,60,62,64,66,68,70,72-hexadecone, hydrochloride hydrate
Test material form:
solid

Test animals / tissue source

Species:
other: chicken eye
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
CHICKEN HEADS COLLECTION AND TRANSPORT
Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
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 approximately 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 fluorescein solution 2 % (w/v) 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 bentscissors. 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.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of 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 clamp
holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel
tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and
examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly 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, any eye with cornea thickness deviating 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 started and it was 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.
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
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.
Observation period (in vivo):
Four hour observation period.
Number of animals or in vitro replicates:
The negative control eye was treated with 30 μL of physiological saline. One eye was treated with physiological saline, three eyes with the test item and another three with Imidazole.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
after 240 min
Value:
3.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
after 75 min
Value:
3.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Test Item 







































Observation



Value



ICE Class



Mean maximum corneal swelling at up to 75 min



3.3%



I



Mean maximum corneal swelling at up to 240 min



3.3%



I



Mean maximum corneal opacity



0.00



I



Mean fluorescein retention



1.00



II



Other Observations



Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the
post-treatment rinse.



Overall ICE Class



2xI 1xII



 Based on this in vitro eye irritation in the isolated chicken eyes test with CURCURBIT[8]URIL, the test item in not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.


 


Positive Control







































Observation



Value



ICE Class



Mean maximum corneal swelling at up to 75 min



9.6%



II



Mean maximum corneal swelling at up to 240 min



26.7%



III



Mean maximum corneal opacity



4.00



IV



Mean fluorescein retention



2.83



IV



Other Observations



Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the
post-treatment rinse.



Overall ICE Class



1xIII 2xIV



 The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.


 


NEGATIVE Control







































Observation



Value



ICE Class



Mean maximum corneal swelling at up to 75 min



0.0%



I



Mean maximum corneal swelling at up to 240 min



0.0%



I



Mean maximum corneal opacity



0.00



I



Mean fluorescein retention



0.00



I



Other Observations



None



Overall ICE Class



3xI



The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified.


 


 


 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.
Executive summary:

The purpose of this study was to provide detailed information about the effects of CUCURBIT[8]URIL on the cornea to see if a classification is required. Study was performed to OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants). After the zero reference measurements, the eye was held in horizontal position and 30 mg of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of physiological saline

(Salsol solution, 0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

No significant corneal swelling was observed during the four hour observation period. No corneal opacity change was observed on three eyes and fluorescein retention change (severity 1) was noted on all test item treated eyes; particles of test item were stuck to the cornea and could not be washed off during the study. The effects were clearly greater than a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo. In conclusion based on this in vitro eye irritation in the isolated chicken eyes test with CUCURBIT[8]URIL, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for investigation of full classification.