Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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)
Version / remarks:
adopted 26 Jul 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Medicines & Healthcare products Regulatory Agency, UK

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Characteristics of donor animals: Chickens were approx. 56 days old and weighed 3 kg prior to slaughter.
- Storage, temperature and transport conditions of ocular tissue: Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 sec
Duration of post- treatment incubation (in vitro):
240 min
Reading time points: 30, 75, 120, 180 and 240 min (± 5 min)

Number of animals or in vitro replicates:
3 eyes for test group and positive control; 2 eyes for negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The integrity of the cornea was measured with a microscope after instillation of sodium fluorescein (2%, w/v). Acceptable values for fluorescein retention and corneal opacity scores are ≤ 0.5. Eyes acceptable for testing were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors.
Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ± 1.5 °C.
Once all eyes were placed in the superfusion apparatus, the eyes were examined again for corneal damage with the microscope. Corneal thickness measurements are taken with a depth measuring device on a microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.


EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for 45 min for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item was then rinsed from the eye using 20 mL of isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BQ 900 slit-lamp microscope
- Damage to epithelium based on fluorescein retention: Haag-Streit BQ 900 slit-lamp microscope
- Swelling: Corneal thickness was measured with a depth measuring device no. 1 on a slit-lamp microscope.
- Macroscopic morphological damage to the surface: Haag-Streit BQ 900 slit-lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
(corneal thickness at time (t) - corneal thickness at time =0) / (corneal thickness at time =0) x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.

- Mean maximum opacity score:
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.

Opacity scores:
0 = No opacity
0.5 = Very faint opacity
1 = Scattered or diffuse areas; details of the iris are clearly visible
2 = Easily discernible translucent area; details of the iris are slightly obscured
3 = Severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible
4 = Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention scores for all test eyes are calculated at the 30 min time interval only. These measurements are used for the overall classification for each test item.

Fluorescein retention scores:
0 = No fluorescein retention
0.5 = Very minor single cell staining
1 = Single cell staining scattered throughout the treated area of the cornea
2 = Focal or confluent dense single cell staining
3 = Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
ICE classification criteria as explained in OECD TG 438 were used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
240 min
Value:
1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
30 min
Value:
0.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
120 min
Value:
15.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, No fluorescein retention was noted in both negative control treated eyes.
- Acceptance criteria met for positive control: Yes, Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes.

Any other information on results incl. tables

Table 2: Ocular reactions in observed eyes

 

Score

ICE class

Test item

 

 

Maximal mean score for corneal opacity

1.0

II

Mean score of fluorescein retention

0.5

I

Maximan mean corneal swelling

15.70%

II

Positive control

 

 

Maximal mean score for corneal opacity

4.0

IV

Mean score of fluorescein retention

3.0

IV

Maximan mean corneal swelling

47.09%

IV

Negative control

 

 

Maximal mean score for corneal opacity

0.3

I

Mean score of fluorescein retention

0.0

I

Maximan mean corneal swelling

0.68%

I

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive according to OECD 438
Conclusions:
The irritation potential of the test substance was assessed in the ICE assay. After treatment with the neat test substance corneal opacity, fluorescein retention and corneal swelling were classed as category II, I and II, respectively. According to OECD 438 the substance can be considered as non-corrosive, but no prediction on the eye irritation potential can be made and additional testing should be conducted for classification and labeling purposes.